Sulforaphane increases metabolism to ameliorate obesity and insulin resistance

Study published here: Glucoraphanin ameliorates obesity and insulin resistance through adipose tissue browning and reduction of metabolic endotoxemia in mice

Low-grade sustained inflammation, triggered by chronically high levels of proinflammatory cytokines and gut microbiota-derived circulatory lipopolysaccharide (LPS), links obesity with comorbidities such as insulin resistance and nonalcoholic fatty liver disease (NAFLD) (1,2).

Although a number of pharmacological treatments for obesity and NAFLD have been tested, few drugs are clinically available owing to the lack of long- term efficacy and safety concerns (3,4). Thus, a novel therapeutic approach that would improve energy metabolism and reduce chronic inflammation in obesity is sorely needed.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a basic leucine zipper transcription factor, is widely expressed in human and mouse tissues, and serves as a defense response against extrinsic and intrinsic stressors (5). Upon exposure to electrophilic and oxidative stress, Nrf2 detaches from its repressor, Kelch-like ECH-associated protein 1-nuclear factor (Keap1), and is translocated from the cytoplasm into the nucleus.

This translocation leads to the transcriptional activation of genes encoding phase 2 detoxifying and antioxidant enzymes (6).

In addition to the ubiquitous induction of cytoprotective genes, Nrf2 regulates a large number of genes involved in glucose and lipid metabolism. In the liver, the constitutive activation of Nrf2 via Keap1 knockdown represses the expression of genes involved in gluconeogenesis (7) and lipogenesis (8), thereby alleviating obesity, diabetes, and hepatic steatosis.

Accordingly, synthetic Nrf2 inducers such as synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO)-imidazolide (9), CDDO-methyl ester (known as bardoxolone methyl) (10), and dithiolethione analog, oltipraz (11), have been shown to ameliorate high-fat diet (HFD)-induced obesity and diabetes.

These synthetic Nrf2 inducers also decrease liver and adipose tissue lipogenesis, and enhance glucose uptake in skeletal muscles. However, the mechanisms by which Nrf2 enhances energy metabolism in response to a HFD remain largely unknown.

Although enhanced Nrf2 signaling has shown promising results in several animal studies, the synthetic Nrf2 inducers have caused adverse cardiac events and gastrointestinal toxicities in clinical trials (12,13).

These observations prompted us to explore a safer Nrf2 inducer for the treatment of obesity, insulin resistance, and NAFLD.

Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is one of the most potent naturally occurring Nrf2 inducers; this compound exhibits anticancer activity in cancer cell lines and in carcinogen-induced rodent models (14).

Among the cruciferous vegetables, broccoli sprouts are the best source of glucoraphanin, a stable glucosinolate precursor of sulforaphane (15). In both rodents and humans, glucoraphanin is hydrolyzed by gut microbiota-derived myrosinase into bioactive sulforaphane prior to intestinal absorption (16). A recent clinical study demonstrated the safety of orally administered glucoraphanin (17).

In the present study, we examined the dietary glucoraphanin-mediated modulation of systemic energy balance and the mitigation of chronic inflammation, insulin resistance, and NAFLD in diet-induced obese mice.

Research Design and Methods Glucoraphanin preparation
The sulforaphane precursor, glucoraphanin, was prepared as previously described (17) with minor modifications. Briefly, one day after germination from broccoli seeds (Caudill Seed Company, Louisville, KY), sprouts were boiled in water for 30 min.

The water extract was mixed with dextrinized cornstarch and subsequently spray-dried to yield an extract powder containing 135 mg of glucoraphanin per gram (0.31 mmol/g) (18). The total glucoraphanin titer in the resulting powder was determined by HPLC as previously reported (19).

Mice and diets
Male C57BL/6JSlc mice were purchased from Japan SLC (Hamamatsu, Japan) at 7 weeks of age. Nrf2-knockout (Nrf2-/-) mouse strain (RBRC01390; C57BL/6J background) was provided by RIKEN BRC (Tsukuba, Japan) (6). After a week of acclimation, mice were fed normal chow (NC; containing 2.2% dextrinized cornstarch, 10% kcal from fat, Research Diets, New Brunswick, NJ), NC containing 0.3% glucoraphanin (NC- GR; containing 2.2% extract powder), a high-fat diet (HFD; containing 2.2% dextrinized cornstarch, 60% kcal from fat, #D12492; Research diets), or a HFD containing 0.3% glucoraphanin (HFD-GR; containing 2.2% extract powder) for 14 weeks.

Both the NC and the HFD containing cornstarch or glucoraphanin were prepared by Research Diets. All mice studied were maintained on a 12 h light/dark cycle at 24–26°C with free access to water and food. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kanazawa University, Japan.

Indirect calorimetry
After 3 weeks of feeding, mice were individually housed in an indirect calorimeter chamber at 24–26°C (Oxymax; Columbus Instruments, Columbus, OH). Calorimetry, daily body weight, and daily food intake data were acquired during a 3-day acclimation period, followed by a 2-day experimental period. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in each chamber every 20 minutes. The respiratory exchange ratio (RER = VCO2/VO2) was calculated using Oxymax software. Energy expenditure was calculated as shown below and normalized to the body mass of each subject.
Energy expenditure = VO2 × [3.815 + (1.232 ×RER)]

Metabolic measurements and biochemical analyses
Metabolic parameters, body fat composition, insulin sensitivity, and glucose tolerance were assessed as previously described (20). Plasma LPS levels were analyzed using a Limulus amebocyte lysate assay kit (QCL-1000; Lonza, Allendale, NJ). Plasma LPS binding protein (LBP) levels were determined using an ELISA kit (Enzo Life Sciences, Farmingdale, NY).

Immunoblotting was performed with primary antibodies (Supplementary Table 1) as previously described (20). mRNA expression levels were determined by quantitative real-time PCR using SYBR Green with the primers (Supplementary Table 2) as previously described (20).

Isolation and differentiation of inguinal white adipose tissue-derived primary beige adipocytes
Stromal vascular fractions (SVFs) from inguinal white adipose tissue (WAT) of 7- week-old wild-type and Nrf2-/- mice were prepared as previously reported (21). At confluence, SVF cells were induced for 2 days with differentiation medium containing DMEM/F-12 supplemented with 10% FBS, 20 nM insulin, 1 nM T3, 5 μM dexamethasone, 500 μM isobutylmethylxanthine, 125 μM indomethacin, and 0.5 μM rosiglitazone (all from Sigma-Aldrich, St. Louis, MO).

Induced cells were subsequently cultured in maintenance medium (DMEM/F-12 containing 10% FBS, 20 nM insulin, and 1 nM T3) for 5 days and treated with DMSO or sulforaphane (Toronto Research Chemicals, Toronto, Canada) at the indicated concentrations for 48 h.

Fluorescence-activated cell sorting (FACS)
Cells from the liver and epididymal WAT were prepared as previously described (22). Isolated cells were incubated with Fc-Block (BD Bioscience, San Jose, CA), and subsequently incubated with fluorochrome-conjugated antibodies (Supplementary Table 1).

Flow cytometry was performed using a FACSAria II (BD Bioscience), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Analysis of gut microbiota via pyrosequencing of the 16S rRNA gene
Metagenomic DNA was extracted from mouse cecal content with a QIAamp DNA stool kit (Qiagen, Hilden, Germany). The V1–V2 region of the 16S rRNA gene was amplified using primer sets as previously reported (23).

Mixed samples were prepared by pooling approximately equal amounts of PCR amplicons from each sample and subjected to a GS Junior System (Roche Diagnostics, Basel, Switzerland) for subsequent 454 sequencing.

Pre-processing and taxonomic assignment of sequencing reads were conducted as described previously (23) and separated by unique barcodes. The 16S rRNA sequence database was constructed by retrieving 16S sequences of bacterial isolates (1200–2384 bases in length) from the Ribosomal Database Project Release 10.27.

We used 4000 filter- passed reads of 16S sequences for the operational taxonomic unit (OTU) analysis of each sample. Clustering of 16S sequence reads with identity scores > 96% into OTUs was performed using UCLUST (www.drive5.com).

Representative sequences with identity scores > 96% for each OTU were assigned to bacterial species using BLAST. Principal component analysis using EZR software (http://www.jichi.ac.jp/saitama- sct/SaitamaHP.files/statmedEN.html) was applied for assessment of alterations of cecal bacterial phylum associated with diets.

Statistical analyses
Data were expressed as mean ± SEM. P < 0.05 was considered statistically significant. Statistical differences between pairs of groups were determined by a two-tailed Student’s t-test. An overall difference between more than two groups was determined using a one-way ANOVA.

If one-way ANOVAs were significant, differences between individual groups were estimated using a Bonferroni post hoc test All calculations were performed using SPSS Statistics (v19.0, IBM, Armonk, NY).

Results

Glucoraphanin decreases weight gain and adiposity, and increases energy expenditure in high-fat diet (HFD)-fed mice
To investigate the effects of glucoraphanin on systemic energy balance, we examined the body weight of wild-type mice fed normal chow (NC) or a high-fat diet (HFD), supplemented with glucoraphanin or vehicle (i.e., cornstarch only). Glucoraphanin reduced weight gain only in HFD-fed mice without affecting food intake (Fig. 1A and Supplementary Fig. 1A).

This reduction was not accompanied by evidence of gross toxicity. We determined the plasma concentration of sulforaphane in NC-GR and HFD-GR mice, but not in NC or HFD mice, indicating that glucoraphanin was absorbed as a sulforaphane following food consumption (Supplementary Fig. 1B).

The reduction of weight gain in glucoraphanin-treated HFD-fed mice was largely attributed to the decreased fat mass but not lean mass (Fig. 1B). To assess energy expenditure, we placed the mice in indirect calorimetry cages after 3 weeks of feeding, before an evident change in the body mass of HFD-fed mice was observed (HFD: 29.9 ± 0.5 g vs. HFD-GR: 28.5 ± 0.5 g).

Glucoraphanin-treated mice fed the HFD exhibited consistently higher VO2 and VCO2 than vehicle-treated HFD-fed controls (Fig. 1C and D), leading to increased energy expenditure (Fig. 1E); however, they displayed similar RER (Fig. 1F), suggesting that glucoraphanin supplementation enhanced sugar and fat use under HFD conditions. On NC-fed mice, glucoraphanin did not affect these parameters of energy balance (Fig. 1B–F).

Consistent with increased energy expenditure, glucoraphanin increased the core body temperature of HFD-fed mice by approximately 0.5°C (Fig. 1G).

Glucoraphanin improves diet-induced insulin resistance and glucose tolerance
After 14 weeks of feeding, glucoraphanin supplementation did not affect plasma triglycerides, total cholesterol, and free fatty acid (FFA) levels in either NC- or HFD-fed mice (Table 1). On NC, blood glucose levels were not altered by glucoraphanin, but on the HFD, glucoraphanin-treated mice exhibited significantly lower fasted blood glucose compared with vehicle-treated controls (Table 1).

Additionally, glucoraphanin significantly decreased plasma insulin concentrations in HFD-fed mice under both fasted and fed conditions, resulting in lower homeostatic model assessment-insulin resistance (HOMA-IR; Table 1).

During the insulin tolerance test (ITT), glucoraphanin significantly enhanced the reduction in blood glucose levels in HFD-fed mice, but not in NC-fed mice compared with the vehicle-treated controls (Fig. 2A). Glucoraphanin improved glucose tolerance in HFD- fed mice during the glucose tolerance test (GTT), but had no effect on NC-fed mice (Fig. 2B).

Insulin secretion during the GTT was not affected by glucoraphanin (data not shown). In line with increased insulin sensitivity, insulin-stimulated Akt phosphorylation on Ser473 was enhanced by glucoraphanin in the liver, muscle, and epididymal WAT of mice fed the HFD (Fig. 2C).

Glucoraphanin does not exert anti-obesity and insulin-sensitizing effects in Nrf2-/- mice
Although the Keap1-Nrf2 pathway is a well-known target of sulforaphane, this isothiocyanate has also been reported to modulate different biological pathways independent of the Keap1-Nrf2 pathway (24,25).

To determine whether the anti-obesity and insulin-sensitizing effects of glucoraphanin are mediated through Nrf2, the effects of glucoraphanin on energy balance and glucose metabolism were assessed in NC- and HFD- fed Nrf2-/- mice.

Although food intake and plasma concentration of sulforaphane in NC-GR or HFD-GR diet-fed Nrf2-/- nice were comparable that detected in wild-type mice fed NC- GR or HFD-GR diet (Supplementary Fig. 1C and D), the effects of glucoraphanin following HFD feeding on weight gain (Fig. 3A), VO2 (Fig. 3B), VCO2 (Fig. 3C), energy expenditure (Fig. 3D), RER (Fig. 3E), rectal temperature (Fig. 3F), insulin sensitivity (Fig. 3G), and glucose tolerance (Fig. 3H) were abolished by the Nrf2 deficiency.

These data are consistent with comparable plasma metabolic parameters between glucoraphanin-treated and vehicle-treated mice on the HFD. These metabolic parameters include lipids, blood glucose, insulin, HOMA-IR, and liver enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) (Supplementary Table 3).

Glucoraphanin blocks HFD-induced reduction of Ucp1 expression in WAT of wild- type mice but not in Nrf2-/- mice

The increased energy expenditure and body temperature of glucoraphanin-treated HFD-fed mice suggest an increase in adaptive thermogenesis. However, glucoraphanin supplementation had little effect on the size and number of lipid droplets in the intrascapular brown adipose tissue (BAT) of HFD-fed wild-type mice (Supplementary Fig. 2A).

In addition, the mRNA expression of uncoupling proteins (Ucps), PGC-1α, and deiodinase 2 in BAT and of Ucps in skeletal muscle was not altered by glucoraphanin supplementation in both NC- and HFD-fed wild-type mice (Supplementary Fig. 2B and C). In BAT, HFD increased Ucp1 protein expression, but glucoraphanin did not alter the expression in both wild-type and Nrf2-/- mice (Fig. 4A).

Brown-like adipocytes expressing Ucp1, also known as beige cells, exist in various WAT depots and can contribute to thermogenesis (26). Compared with NC, HFD significantly decreased Ucp1 protein levels in epididymal and inguinal WAT of both wild-type and Nrf2-/- mice (Fig. 4A).

Glucoraphanin supplementation restored HFD-induced reduction in Ucp1 protein levels in epididymal and inguinal WAT of wild-type mice but not those in Nrf2-/- mice.

To examine whether the effects of glucoraphanin were fat cell-autonomous and Nrf2-mediated, we tested the effects of sulforaphane, an active metabolite of glucoraphanin, on the expression of brown fat-selective genes in primary beige adipocytes obtained from inguinal WAT of wild-type and Nrf2-/- mice.

In beige adipocytes derived from wild-type mice, treatment with sulforaphane induced the Nrf2 target gene, NAD(P)H:quinone oxidoreductase 1 (Nqo1; Fig. 4B) and antioxidant genes (Supplementary Fig. 3A).

Concurrently, sulforaphane significantly increased the mRNA expression of brown-fat selective genes, including Ucp1, Prdm16, Cidea, and Elovl3 (Fig. 4B).

In contrast, in Nrf2-deficient beige adipocytes, sulforaphane failed to activate Nrf2 as judged by unaltered mRNA expression of the target genes and to promote the expression of brown-fat selective genes (Supplementary Fig. 3B and Fig. 4C).

Importantly, Nrf2-deficient beige adipocytes exhibited less differentiation levels associated with attenuated lipid accumulation (Supplementary Fig. 3C) and lower mRNA expression of fatty acid binding protein 4 (Supplementary Fig. 3C) and brown-fat selective genes compared with wild-type beige adipocytes (Fig. 4C).

Glucoraphanin reduces hepatic steatosis and oxidative stress in HFD-fed mice
The HFD causes hepatic steatosis and inflammation, eventually leading to steatohepatitis. As shown in Fig. 5A, the increase in liver weight caused by the 14-week- HFD was alleviated by glucoraphanin supplementation.

Glucoraphanin also attenuated HFD-induced hepatic steatosis (Fig. 5B). Additionally, compared with the HFD group, the lower levels of plasma ALT, plasma AST, liver triglycerides, and liver FFAs in the HFD- GR group indicate that glucoraphanin alleviated HFD-induced liver damage (Fig. 5C and D).

The reduction in hepatic steatosis was accompanied by the decreased expression of the following lipogenic genes: sterol regulatory element binding protein-1c (Srebf1), fatty acid synthase (Fasn), and peroxisome proliferator-activated receptor gamma (Pparγ) (Fig. 5E).

Additionally, hepatic levels of malondialdehyde, a marker of lipid peroxidation, were increased by the HFD. Glucoraphanin attenuated lipid peroxidation (Fig. 5F) and decreased gene expression of the NADPH oxidase subunits gp91phox, p22phox, p47phox, and p67phox (Fig. 5E).

The HFD led to a compensatory increase in the expression of genes involved in fatty acid β-oxidation (Ppara and Cpt1a) and anti-oxidative stress (Cat, Gpx1, and Sod1) in the liver. However, glucoraphanin did not increase the expression of these genes in the liver of HFD-fed mice further (Fig. 5E).

Glucoraphanin suppresses HFD-induced proinflammatory activation of macrophages in liver and adipose tissue
In response to the HFD, liver-resident macrophages (Kupffer cells) increase the production of proinflammatory cytokines that promote insulin resistance and NAFLD in mice (27). In particular, chemokine (C-C motif) ligand 2 (Ccl2) promotes the recruitment of chemokine (C-C motif) receptor 2 (Ccr2)-positive monocytic lineages of myeloid cells into the liver (28). These recruited cells produce a large amount of proinflammatory mediators and activate a lipogenic program (28).

Here, we found a prominent induction of tumor necrosis factor-α (Tnf-α), Ccl2, and Ccr2 in the liver of HFD-fed mice, which was markedly reduced in glucoraphanin-treated mice (Fig. 6A).

Glucoraphanin significantly suppressed HFD-induced inflammatory pathways such as c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk) (Fig. 6B).

Glucoraphanin tended to decrease levels of p-NF-κB p65 (Ser536) in HFD-fed mice, although this decrease was not statistically significant (Fig. 6B). Of note, in the liver of Nrf2-/- mice, glucoraphanin failed to suppress HFD-induced inflammatory signal pathways (Supplementary Fig. 4).

In addition, glucoraphanin significantly decreased the HFD-induced hepatic expression of macrophage markers, including F4/80, Cd11b, and Cd68 (Fig. 6C). Tissue macrophages are phenotypically heterogeneous and have been characterized according to their activation/polarization state as M1-like proinflammatory macrophages or M2-like anti- inflammatory macrophages (29).

Consistent with the decreased expression of macrophage markers, glucoraphanin prevented macrophage (F4/80+CD11b+ cell) accumulation in the liver of HFD-fed mice (Fig. 6D).

Additionally, glucoraphanin decreased the number of M1- like liver macrophages expressing surface markers (F4/80+CD11b+CD11c+CD206−) (Fig. 6E).

In contrast, glucoraphanin increased the number of M2-like liver macrophages (F4/80+CD11b+CD11c−CD206+), resulting in a predominantly M2-like macrophage population (Fig. 6E).

Moreover, glucoraphanin decreased the mRNA expression of Tnf-α and NADPH oxidase in the epididymal WAT of HFD-fed mice (Supplementary Fig. 5A).

Although the HFD-induced expression of macrophage markers and macrophage accumulation in epididymal WAT were not altered by glucoraphanin (Supplementary Fig. 5B and C), the number of M1-like macrophages was significantly decreased in the epididymal WAT of glucoraphanin-treated HFD-fed mice (Supplementary Fig. 5D).

Glucoraphanin decreases circulating LPS and the relative abundance of Proteobacteria in the gut microbiomes of HFD-fed mice

Gut microbiota-derived LPS induces chronic inflammation that eventually leads to insulin resistance in obesity, termed metabolic endotoxemia (1,2).

Based on our observation that glucoraphanin alleviates inflammation in the liver and epididymal WAT of HFD-fed mice, we subsequently investigated the effects of glucoraphanin on metabolic endotoxemia and gut microbiota.

In accordance with previous studies (1,2), the HFD induced a 2-fold increase in circulatory LPS levels, which was reduced by glucoraphanin supplementation (Fig. 7A). Furthermore, plasma and hepatic levels of the LPS marker, LBP, were significantly elevated by the HFD and were reduced by glucoraphanin supplementation (Fig. 7B).

A principal component analysis distinguished cecal microbial communities based on diet and treatment, revealing that the metagenomes of HFD-fed mice formed a cluster distinct from the cluster formed by NC-fed mice (Fig. 7C). However, samples from glucoraphanin-treated HFD-fed mice formed a cluster that was indistinguishable from that of vehicle- or glucoraphanin-treated NC-fed mice (Fig. 7C).

Importantly, consistent with previous reports (30,31), further analysis at the phylum level demonstrated that the proportion of Gram-negative Proteobacteria was significantly elevated in the gut microbiomes of HFD-fed mice, which was suppressed by glucoraphanin supplementation (Fig. 7D and E).

The increase in the relative abundance of Proteobacteria in HFD-fed mice is mostly explained by an increase in the relative abundance of bacteria from the family Desulfovibrionaceae (Fig. 7F), key producers of endotoxins in animal models of obesity (30).

In fact, the relative abundance of Desulfovibrionaceae was positively correlated with plasma LPS levels (Fig. 7G). Furthermore, plasma LPS levels were significantly and positively correlated with the hepatic mRNA levels of Tnf-α, gp91phox, and F4/80 (Fig. 7H).

Similarly, the liver expression of other marker genes was both significantly and positively correlated with plasma LPS levels and with one another (Supplementary Table 4).

Discussion
In the present study, we demonstrated that glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, mitigated HFD-induced weight gain, insulin resistance, hepatic steatosis, oxidative stress, and chronic inflammation in mice.

The weight-reducing and insulin-sensitizing effects of glucoraphanin were abolished in Nrf2-/- mice. Additionally, glucoraphanin lowered plasma LPS levels in HFD-fed mice, and decreased the relative abundance of Desulfovibrionaceae.

At the molecular level, glucoraphanin increased Ucp1 protein expression in WAT depots, while suppressing the hepatic mRNA expression of genes involved in lipogenesis, NADPH oxidase, and inflammatory cytokines.

Our data suggest that in diet-induced obese mice, glucoraphanin restores energy expenditure and limits gut-derived metabolic endotoxemia, thereby preventing hepatic steatosis, insulin resistance, and chronic inflammation.

Consistent with previous reports demonstrating the anti-obesity effects of synthetic Nrf2 inducers (9–11), we show that the oral administration of glucoraphanin mitigates HFD-induced weight gain (Fig. 1A). The dose of glucoraphanin used in the current study (about 12 μmol/mouse/day) is similar to that used in other experiments investigating its antitumor effects in mice (14,32,33).

Here, we show that the effect of glucoraphanin on whole-body energy expenditure and the protein expression of Ucp1 in WAT were abolished in Nrf2-/- mice (Fig. 3D and 4A).

A recent study using adipocyte-specific PRDM16- deficient mice indicated that adaptive thermogenesis in beige fat also contributes to systemic energy expenditure (26). The mutant mice in the aforementioned study, which exhibited markedly reduced Ucp1 mRNA expression in inguinal WAT and minimal effects on BAT, developed obesity and insulin resistance in response to a HFD.

Thus, we believe that the increased energy expenditure in glucoraphanin-treated HFD-fed mice stems from, at least in part, an increase in beige fat, even though the expression of Ucps in BAT and skeletal muscle is not altered. Further analysis using Ucp1-knockout mice will elucidate the relative contribution of beige fat to the Nrf2-mediated metabolic effects elicited by glucoraphanin.

Several studies indicated that Nrf2-/- mice are partially protected from HFD-induced obesity and associated with milder insulin resistance compared with wild-type counterparts (9,34,35). Recently, Schneider et al. demonstrated that mitigation of HFD-induced obesity in Nrf2-/- mice, which was 25% less body weight than that of wild-type mice after 6 weeks of feeding (35).

They also found that HFD-fed Nrf2-/- mice exhibit a 20–30% increase in energy expenditure that is associated with an approximately 3-fold up-regulation of Ucp1 protein expression in abdominal WAT (35). In the present study, Nrf2-/- mice gained less weight after 6 weeks of HFD feeding compared with HFD-fed wild-type mice (Figure 1A and 3A; Nrf2-/-: 35.9 ± 0.9 g vs. wild-type: 39.0 ± 0.8 g, P < 0.05).

The lower body mass of Nrf2-/- mice raises the possibility that anti-obesity effect of glucoraphanin was completely phenocopied by Nrf2 gene deficiency. However, several observations suggest that Nrf2-/- mice only partially phenocopy the effect of glucoraphanin on weight gain reduction. 1)

In the present study, the weight difference between Nrf2-/- and wild-type mice was only 8%, which is much less than that in the previous study (35). 2) In Nrf2-/- mice, compared with NC, HFD induced significant weight gain (Fig. 3A), glucose intolerance (Fig. 3H), and insulin resistance as judged by increased HOMA-IR (Supplementary Table 3). 3)

Metabolic rate and energy expenditure of Nrf2-/- mice were comparable with those in wild-type mice (Fig. 1C–E and 3B–D). 4) Ucp1 protein levels in both epididymal WAT and inguinal WAT of HFD-fed Nrf2-/- mice were lower than those in HFD-GR-fed wild-type mice (Fig. 4A).

Taken together, these findings suggest that Nrf2 gene deficiency is not sufficient to block HFD-induced obesity by increasing energy expenditure and Ucp1 expression in WAT depots, and to mask the effect of glucoraphanin.

However, we cannot fully exclude the possibility that the effects of glucoraphanin are mediated by Nrf2 independent-mechanisms. Possible reasons for the discordance in metabolic phenotypes of Nrf2-/- between the previous study (35) and ours may be due to differences in knockout mouse lines and experimental conditions (e.g. age of mice at beginning of HFD feeding, composition of HFD, and temperature in the metabolic chamber).

Our in vitro study of primary beige adipocytes revealed that sulforaphane promotes the expression of brown-fat selective genes (Fig. 4B). Importantly, the concentration of sulforaphane used in cell culture (0.2–5 μM) is comparable with that detected in mice fed NC-GR and HFD-GR diet (Supplementary Fig. 1B). Moreover, we determined that Nrf2 acts as a positive regulator of beige adipocyte differentiation (Fig. 4C and Supplementary Fig. 3C).

The less differentiation levels in Nrf2-deficient beige adipocytes are in agreement with previous reports demonstrating Nrf2 induces white adipocyte differentiation through increasing the gene expression of Pparγ (34) and Cebpβ (36), common transcription factors regulating the differentiation of brown, beige, and white adipocytes.

Furthermore, it is noteworthy that Nrf2 has been reported to bind NF-E2-binding sites in the 5′-flanking region of the human and rodent Ucp1 genes (37).

However, we cannot exclude the possibilities that glucoraphanin affects sympathetic nervous activity or that hormonal factors are regulating fat browning (38). In addition, mitochondrial reactive oxidative species facilitate Ucp1-dependent respiration in BAT and whole-body energy expenditure by promoting the sulfenylation of a specific cysteine residue (Cys253) in Ucp1 (39).

The molecular mechanism by which Nrf2 regulates the expression and thermogenic activity of Ucp1 in beige adipocytes requires further investigation.

Glucoraphanin supplementation improved the systemic glucose tolerance and insulin sensitivity of HFD-fed mice. Although the molecular mechanism by which synthetic Nrf2 inducers enhance glucose uptake is unclear, AMP-activated protein kinase (AMPK) activation may mediate this enhancement in mouse skeletal muscle and adipose tissue (9– 11).

The phosphorylation levels of AMPK (Thr172) and acetyl-CoA carboxylase (Ser79) in peripheral insulin target tissues were comparable between glucoraphanin-treated NC- and HFD-fed mice and vehicle-treated controls (Supplementary Fig. 6).

These data suggest that AMPK activation is not necessary for glucoraphanin to exert its insulin-sensitizing effect on HFD-fed mice. Additional studies using the hyperinsulinemic-euglycemic clamp technique are needed to determine which tissues contribute to the insulin-sensitizing effects of glucoraphanin.

The beneficial effects of glucoraphanin on hepatic lipid metabolism were not accompanied by AMPK activation or the increased expression of fatty acid β-oxidation genes (Fig. 5E).

Instead, glucoraphanin mitigated HFD-induced oxidative stress and inflammation in the liver. In obesity, hepatic inflammation mediated by macrophage/monocyte-derived proinflammatory cytokines promotes lipogenesis through the inhibition of insulin signaling and SREBP activation (40,41). In fact, the depletion of Kupffer cells by clodronate liposomes ameliorates hepatic steatosis and insulin sensitivity in HFD-fed mice (27).

Furthermore, Ccl2- or Ccr2-deficient mice are protected from diet- induced hepatic steatosis even though they still become obese (42,43). Moreover, the specific ablation of M1-like macrophages restores insulin sensitivity in diet-induced obese mice (44), while the deletion of Pparδ, which promotes M2 activation, predisposes lean mice to develop insulin resistance (45).

Therefore, decreased hepatic macrophage accumulation and M2-dominant polarization of hepatic and adipose macrophages account, at least in part, for the protection from hepatic steatosis and insulin resistance in glucoraphanin-treated HFD-fed mice.

One of the most important findings of this study is that glucoraphanin decreases the relative abundance of Gram-negative Proteobacteria, particularly family Desulfovibrionaceae, while reducing circulatory LPS levels (Fig. 7).

Recent studies demonstrated that a significant increase in Desulfovibrionaceae, potential endotoxin producers, in the gut microbiomes of both HFD-induced obese mice and obese human subjects compared to lean individuals (30,31,46). We cannot exclude the possibility that other microbiota-derived products, such as bile acids and short chain fatty acids, also mediate the metabolic action of glucoraphanin.

Whether the interaction between sulforaphane and gut microbiota is affected directly or indirectly by altered host physiology remains to be determined.

However, several studies have suggested that sulforaphane can alter the gut microbiota directly, as isothiocyanates (including sulforaphane) have been shown to exhibit antibacterial activity against Proteobacteria (47,48).

This activity may proceed via redox disruption and enzyme denaturation reactions involving the isothiocyanate reactive group, -N=C=S, the thiol group (-SH) of glutathione and proteobacterial proteins (47,48). Additionally, sulforaphane exhibits antibacterial activity against Helicobacter pylori, a member of the phylum Proteobacteria (49).

We are unaware of any previous reports demonstrating that isothiocyanates inhibit the proliferation of Desulfovibrionaceae. The mechanistic underpinnings of this antibacterial activity require elucidation.

In conclusion, the results of the present study indicate that glucoraphanin may be effective in preventing obesity and related metabolic disorders such as NAFLD and type 2 diabetes.

A recent clinical study demonstrated that supplementation with a dietary dose of glucoraphanin (69 μmol/day) for two months significantly decreased the plasma liver enzymes, ALT and AST, although body mass did not change (18).

Long-term treatment with a higher dose of glucoraphanin (800 μmol/day), which can be safely administered without any harmful side effects (17), may be required to achieve an anti-obesity effect in humans.

Cardamom supplementation improves inflammatory and oxidative stress biomarkers in hyperlipidemic, overweight, and obese pre-diabetic women

This study is published here

Insulin resistance, which is the key feature of pre-diabetes and T2DM, results mainly from low physical activity and obesity, which are associated with the repletion of lipid into adipocytes and the accumulation of adipose tissue.3-5 An increase in insulin, free fatty acid (FFA), and/or glucose levels can increase reactive oxygen species (ROS) production and oxidative stress.6

Oxidative stress is a direct outcome of hyperglycaemia and may be involved in metabolic complications in these subjects.7
 The release of additional acute phase reactants, including TNF-α, IL-6, and CRP, by white adipose tissue has been shown in obese subjects.4 By diminishing insulin receptor signalling and increasing insulin resistance, IL-6 and TNF-α can cause chronic hyperglycaemia in these subjects, leading to the development of diabetes.8
An increased oxidative stress is present in pre-diabetes stages9, which may result in endothelial damage in these subjects.10 Therefore, it can be assumed that interventions to reduce oxidative stress and inflammation could improve the condition of pre-diabetes and prevent its complications and development to T2DM.

Lately, herbal remedies like spices have been considered because of their phytochemical content, which has a beneficial potent.11 Spices may somewhat have the same effects as functional foods to improve health or reduce risks of diseases. 12,13 Like vegetables and fruits, spices also have antioxidant effects.14 Several studies have shown that some spices have great potential to inhibit chronic inflammation and oxidative stress because of their phytochemicals and free radical scavengers like polyphenols, flavonoids, and phenolic compounds.15,16

It has been suggested that by increasing the consumption of spices, chronic diseases morbidity can be prevented.17
Green cardamom, also known as the Queen of Spices consists of the whole or ground dried fruit of Elettaria Cardamomum (Linn.) Maton, which belongs to the ginger family (Zingiberaceae).18,19

So far, several investigations have shown some advantages of cardamom for teeth, gum and throat infection, lung congestion, pulmonary tuberculosis, and gastrointestinal disorders.20 Various studies suggest that cardamom extracts display antimicrobial, anti-cancer, anti-inflammatory, anti-proliferative, pro-apoptotic and anti-oxidative activities.18,20,21-25 Several animal and cellular model studies have shown anti-inflammatory and anti-oxidative activities of cardamom.18,24-30 A few human studies have been conducted to investigate the effects of cardamom on inflammation and oxidative stress status, which have shown conflicting results.11,25,31In one study, supplementation with Greater cardamom or E. Cardamomum improved the antioxidant status in patients with ischemic heart disease or hypertension.25,31 However, the results of another study did not show any significant effect of cardamom on blood oxidative stress and inflammation in T2DM patients.11

According to the results of an in-vitro study, the key components of the essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol), by binding with TNF, IL-1 β; IL-4 and IL-5, show anti-inflammatory activities.18In an animal study, cardamom supplementation improved oxidative stress markers and ameliorated the inflammatory cell infiltration and fibrosis in the liver of rats fed on a high-carbohydrate high-fat (HCHF) diet.27

In a human study, 3g cardamom powder intake for 12 weeks in individuals with primary hypertension significantly increased the total blood antioxidant capacity.25 However, no study has been done yet to evaluate the effects of cardamom supplementation in pre-diabetic subjects who are at risk of oxidative stress and inflammation. Previous studies have suggested the need for well-controlled clinical trials of spices.17 Therefore, there is a hypothesis that cardamom may have beneficial effects on pre-diabetes. So, the present study was designed to investigate the effects of cardamom on blood inflammatory and oxidative stress biomarkers in pre-diabetic subjects.
EXPERIMENTAL STATE
Study design and subjects

This double-blind, placebo-controlled trial study was conducted on 79 newly diagnosed pre- diabetic women from two health care centres of Karaj city in Iran from February to April 2014. The aim of the study was to determine the effect of cardamom supplementation on serum lipids, glycemic indices, blood pressure, oxidative stress, and inflammatory biomarkers in overweight and obese pre-diabetic women. Since the majority of these individuals were female, the investigation was limited to female subjects. This study was performed based on the guidelines laid down in the Declaration of Helsinki. All procedures involving human subjects were approved by the Ethics Committee of Tehran University of Medical Sciences. A written informed consent form was signed and dated by the subjects and investigators. This study was registered on the Iranian Registry of Clinical Trials website (http://www.irct.ir/, IRCT2014060817254N2).
A sample sizeof at least 36 in each group was calculated according to the standard deviation of hs-CRP in a similar study30 in the following order: (α=0.05, power=80%)
= 2.18 / 
d=μ −μ =1.47 =0.47
σ√2 3.07
[(z ∝/ ) + (z )] (1.96 + 0.84)
= d = (0.47) = 36

Considering the loss to follow-up, 40 subjects in each group joined the study. The age of the subjects was in the range of 30 to 70 years with a body mass index (BMI) of 25–39.9 kgm-2and had at least one of the following criteria: Fasting blood sugar (FBS) 100-125 mgdl-1, HbA1c 5.7– 6.4%, two-hour blood glucose 140–199 mgdl-1 identified in the last two months. In addition, they had at least one of these risk factors: 300>triglyceride>150 mgdl-1, total cholesterol>200 mgdl-1, 160>low-density lipoprotein-cholesterol (LDL-c) >100 mgdl-1, high-density lipoprotein- cholesterol (HDL-c)<50 mgdl-1. Exclusion criteria were BMI<25 or ≥40 kgm-2, pregnancy or lactation, professional athletes, allergy to cardamom, smoking, clinical history of peptic ulcers, gall or kidney stones, clinical history of inflammatory diseases like diabetes, cardiovascular diseases, multiple sclerosis (MS), rheumatoid arthritis, cancer, inflammatory diseases of the gastrointestinal tract and respiratory (e.g. asthma, allergies), multivitamin or antioxidant supplement consumption at least two days a week in the past month, taking medications for dyslipidemia, blood glucose disturbance, hypertension, psychiatric disease, thyroid and hormonal disease, following a specific diet over the last three months, having LDL-c≥160 mgdl-1 or triglyceride ≥300 mgdl-1, blood pressure>130/80 mmHg, developing diabetes during the study, and not taking the prescribed supplements more than 10%.

Randomization and intervention
The participants were randomized into two groups using block randomization. The stratified randomization method was used to control the age and BMI (age in the range of ≤40 and 41–70 years, BMI in the range of 25–29.9 and 30–39.9 kgm-2).
Forty subjects were classified into each group to receive one capsule of either cardamom or placebo powder three times a day with meal for eight weeks.

The dose of 3.0 g used in the study was chosen based on the two previous human studies investigating the effects of cardamom supplementation 25, 31. Also, 3.0 g of cardamom powder can be a reasonably large amount to be consumed through a diet. Fruits of E. Cardamom were provided by the Traditional Medicine and Research Centre (TMRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Fruits of cardamom and dried breadcrumbs are transferred to this centre. After grinding and sifting the whole green cardamom and breadcrumbs, the capsules were filled with these materials.
Each capsule contained one gram of green cardamom powder or breadcrumbs. They were exactly similar in shape, size, and appearance. All placebo capsules were placed near cardamom so that they could take its smell. Sixty capsules were placed in similar jars, which were labelled A and B to be delivered to the individuals. Each person in the study got three jars every 20 days. To confirm the compliance of subjects, they were called up every week. To evaluate compliance, the remaining capsules in the jars were counted.

Dietary analysis
A 24-hour food recall questionnaire of a typical day was completed by interview to get information on food intake at the beginning and the end of the study. Then, the N4 software was used to estimate the daily dietary intake of nutrients.
Anthropometry and physical activity assessments
Anthropometric characteristics, including height and weight, were measured at the beginning and end of the study. Body weight was measured with minimal clothing and without shoes by using a digital scale with a measurement accuracy of 100g. Standing height was measured without shoes to the nearest 0.5cm using a tape measure. BMI was calculated by dividing the weight by the square of height. Physical activity was measured by a short form of the International Physical Activity Questionnaire (IPAQ).33

Laboratory measurements
After 12 hours of fasting, 10ml of venous blood was drawn by a trained nurse before and after the intervention period. Blood samples were centrifuged at 3000 rpm for 10 minutes to separate blood cells and serum. Blood cells were washed three times with 0.9 gl-1NaCl solution. Cell membranes were removed by centrifugation at 1200g for five minutes at 4°C. The haemolysates were then used to determine erythrocyte antioxidant enzyme activity. All samples were then stored at -80°C. Serum levels of hs-CRP (Diagnostics Biochem Canada Inc., Canada), IL-6, and TNF-α (Diaclone, France) and PC (ZellBio GmbH Ulm, Germany) were measured using ELISA kits. The inter- and intra-assay coefficients of variation for hs-CRP, IL-6, TNF-α, and PC were 9.1% and 9.5%, 7.7% and 4.2%, 9% and 3.3%, and <12% and <10%, respectively.

Serum TAC and MDA levels and erythrocyte activities ofSOD and GR were measured using assay kits (ZellBio GmbH Ulm, Germany). The inter- and intra-assay coefficients of variation for TAC, MDA, SOD, and GR were <4.2% and <3.4%, 7.6% and 5.8%, 7.2% and 5.8%, and 6.6% and 5.2%, respectively. At the time of laboratory assessment, the serum of one subject in the cardamom group was not available. In addition, the number of available haemolysates for measuring erythrocyte SOD and GR activity in the cardamom group was 39 and in the placebo group, 40.
Statistical analysis Analysis was done by intention to treat (ITT) using SPSS version 16 (SPSS Inc., Chicago, IL, USA). The ITT population included all the enrolled and randomized participants. The missing observations were accounted for by using the Last Observation Carried Forward (LOCF) and Last Observation Carried Backward (LOCB) methods. Normality distribution of data was evaluated using the Kolmogorov-Smirnov test. Non-parametric tests were used for TNF-α, TAC, and PC, which were non-normally distributed after transformation. The hs-CRPIL-6-1 ratio was calculated by dividing hs-CRP to IL-6 levels. To compare the data between the two groups, the independent sample t-test and Mann-Whitney was used for normal and non-normal data respectively, considering the normality of data. The analysis of covariance (ANCOVA) was used to identify any differences between the two groups after intervention, adjusting for baseline measurements and covariates. Differences of P<0.05 were considered to be statistically significant.

RESULTS
To evaluate the effects of cardamom supplementation on blood oxidative stress and inflammation status,80 participants were enrolled in the study. All participants consumed 90– 100% of the prescribed supplements and all of them completed the study.

Demographic and anthropometric measurements and dietary intakes
The distribution of weight, BMI, and mean duration of pre-diabetes and physical activity were almost similar between the intervention and the control groups (Table 1). In addition, no significant difference was observed in weight, BMI and physical activity between the two groups at the end of the study. The mean±SD ages of the participants in the cardamom and placebo groups were 48.3±10.4 and 47.5±10.3 years, respectively, while no significant differences were found between the two groups. The mean ± SD energy for the cardamom group was 2107.5 ± 317.0 and 2153.4 ± 198.9 kcald-1 before and after the study respectively. These figures for the placebo group were 2157.7±242.2 and 2181.1±212.2 kcald-1, respectively. Comparison between the two groups showed that after intervention, saturated fatty acid (SFA) intake was significantly higher (p=0.005) and poly unsaturated fatty acid (PUFA) intake was lower (p=0.02) in the cardamom group (Table 2). No significant difference was found between the two groups in daily energy, vitamin A, C, E, and selenium intake.

Effect of cardamom supplementation on inflammatory and oxidative stress biomarkers
After intervention, between-group comparisons showed that in the cardamom group, the mean TNF-α (p<0.001) was significantly higher, and hs-CRP (p=0.04), hs-CRP IL-6-1 ratio (p=0.01), PC(p<0.001) and MDA (p=0.003) were lower than the placebo group. However, after adjustment for SFA and PUFA intake changes and baseline values, between-group differences of hs-CRP (p=0.02), hs-CRP IL-6-1 ratio (p=0.008), and MDA (p=0.009) remained significant (Table 3). DISCUSSION
This randomized clinical trial is the first investigation of green cardamom effects on inflammatory and oxidative stress indices in overweight and obese pre-diabetic women who are at risk of cardiovascular diseases. After eight weeks, cardamom supplementation reduced serum hs-CRP, and hs-CRP IL-6-1 ratio. In addition, cardamom supplementation reduced serum MDA levels. However, a complete improvement of inflammatory and oxidative stress parameters was not achieved. Different results may be obtained with a higher supplement dose and duration of intervention.

Several animal studies have shown beneficial effects of cardamom on blood inflammation and oxidative stress indices. However, a few studies have investigated its effect on humans. In atherosclerotic rats, cardamom-rhizome-ethanolic-extract significantly increased SOD levels and decreased MDA, CRP and IL-6.30 By reducing the synthesis of eicosanoid mediators of inflammation, a dose-dependent anti-inflammatory effect of cardamom oil on rats has been shown.26 In addition, by decreasing cyclooxygenase-2 (COX-2) and inducible nitric oxidesynthase (iNOS) expression, the anti-inflammatory activity of cardamom has been reported.24 In another study, cardamom supplementation for eight weeks prevented oxidative stress and ameliorated the infiltration of inflammatory cell and fibrosis in the liver of rats fed on a HCHF-diet.27 A cellular study by Bhattacharjee et al. has shown that the key components of essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol) provide anti- inflammatory activity by binding with TNF-a, IL-1 beta; IL- 4; and IL-5.18

In individuals with stage-1 hypertension, Verma et al. found that the intake of 3g cardamom powder for 12 weeks significantly reduced blood pressure, enhanced fibrinolysis, and improved antioxidant status.25 Among the reasons that could lead to different results between Verma’s study and the present study is the lack of a control group in Verma’s study, a different intervention period (12 weeks vs. 8 weeks) and different target groups (hypertensive vs. pre- diabetic individuals).

In another human study by Verma et al., on patients with ischemic heart disease, the intake of G. Cardamom (Amomum subulatum Roxb.) fruit powder for 12 weeks showed an enhancement in the serum total antioxidant status.31 The conflicting results between the current study and that one may be, again, due to differences in the type of cardamom supplemented (E. Cardamom vs. G. Cardamom), different durations of intervention, and different target groups. However, in a study with 204 T2DM patients, intake of 3g cardamom with tea had no significant effect on serum hs-CRP and F2-isoprostan.11 Negative results observed in the study was explained by the combined effects of cardamom and black tea or the dominant effect of black tea.35

Weight gain and obesity, which increase oxidative stress and inflammatory mediators, are major risk factors for insulin resistance, pre-diabetes, and T2DM.4So, any agent with anti-inflammatory and antioxidant effects, such as cardamom, might interrupt this correlation. Cardamom presumably exerts its anti-inflammatory effect by reducing the synthesis of eicosanoid mediators of inflammation.26The antioxidant effect of cardamom also comes from the fact that it is a potent blocker of lipid peroxide formation and scavenger of superoxide anions and hydroxyl radicals.35 Recent studies have shown that the serum levels of CRP in IGT or IFG patients—in other words, pre-diabetic patients—is higher than that in normoglycaemic people.36-38 CRP is an indicator of systemic inflammation, and increased CRP during obesity is thought to be caused by IL-6 derived from adipose tissue.39Recent studies have shown that lower hs-CRP to IL-6 may reflect decreased inflammation.

So, it can be considered as a marker of the inflammation status.40-42Our results showed that cardamom significantly decreased the serum hs-CRP and hs-CRP IL-6-1 ratio. So, cardamom may have a function in decreasing hs-CRP in pre-diabetic individuals.

Tip:

In the present study, cardamom decreased MDA levels. However, our results did not show a significant influence of cardamom on PC, TAC, SOD, and GR levels. Overweight and obese subjects have more oxidative stress than normal-weight pre-diabetic subjects and may need a greater amount of cardamom or a longer duration of supplementation. So, this is perhaps the reason why, in our findings, cardamom consumption in overweight and obese subjects did not have the profound result on oxidative stress.

This study had some limitations. First, the sample size was small. Second, the intervention duration was too short to understand the real effects of cardamom supplementation. Third, a single 24-hour food recall questionnaire can result in measurement error. Fourth, as the population of the present study comprised only women, the results cannot be generalized to male pre-diabetic subjects. However, this study is one of the first investigations of green cardamom supplementation effects on inflammatory indices and oxidative stress in overweight and obese pre-diabetic subjects. The control group in the present study was another strength of this study.

Synephrine and HCA evaluated for health risks

Phytochemical compounds in sport nutrition: Synephrine and hydroxycitric acid (HCA) as examples for evaluation of possible health risks.

1 Introduction
Nutritional supplements containing phytochemical preparations represent a popular group of products intended for use by professional and amateur sportspeople. The use of such products is based on expectations with respect to promotion of desired effects related to e. g. energy increase, athletic performance, weight loss, muscle growth, hastening of recovery or induction of other physiological or metabolic responses [1].

Highly heterogeneous preparations from many different botanical species are currently being used in sports supplements. For example, so called “pre-workout” supplements which are claimed to stimulate fat-burning metabolism and physical performance when taken shortly before training, often contain multiple phytochemicals/herbals together with natural or synthetic caffeine sources [2].

Many consumers tend to consider such products as risk-free, because they are of the opinion that “natural” equals “safe”.. However, depending on the individual phytochemicals, their doses and the combination of compounds within the supplements, certain phytochemical products may potentially induce unwanted side effects, interfere with medications, or even cause serious health damage. For example, sport supplements containing preparations of Ephedra herb (e.g. Ephedra sinica) were widely used for performance-enhancing by athletes.

However, the safety of food supplements containing Ephedra herbs, their preparations or their alkaloids such as ephedrine and its congeners was questioned by several safety authorities, as cases of severe health damage such as sudden cardiac death or stroke resulting from Ephedra herb use were reported, many of which occurred in young adults using Ephedra herb at the dosages recommended on the product labels [3-5].

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It was considered that caffeine was likely to enhance the cardiovascular and central nervous system effects of ephedrine [6]. As a result, the US Food and Drug Administration (FDA) considered food supplements containing ephedrine alkaloids illegal for marketing [4, 7].

In 2013 the European Food Safety Authority (EFSA) concluded, following the “Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements” [8], that Ephedra herb and its preparations containing Ephedra alkaloids used as food supplements were of significant safety concern at the estimated use levels [9].

Unlike drugs, which must be approved by the competent Authorities before they can be marketed, food supplement products do not require pre-market review or approval within the EU. The responsibility for the safety of food supplements in the EU and compliance with food law provisions lies with manufacturers and distributors. In the case of concerns regarding potential public health risks in association with the use of particular products or a type of botanical preparation, the safety assessment is conducted by member states Authorities.

The aim of the present review was to analyse the risks of typical natural compounds used in sports food supplements as well as to discuss the current challenges regarding the risk assessment of such phytochemical compounds. For this purpose, two examples of phytochemicals – synephrine and hydroxycitric acid – were chosen.

The currently available toxicological animal data and relevant human studies on these compounds have been summarized and discussed with regard to possible health risks. To this end, the databases PubMed/Medline and Embase were searched with different strategies to identify relevant publications, the last search update being in November 2016. Numerous search strategies were used, including the phytochemicals of interest (e. g. “synephrine” or “hydroxycitric acid”) in connection with the endpoints of interest e.g. “toxicity”, “adverse effects”, “adverse events” or “safety”. In addition, currently published reviews, evaluations by scientific bodies or health authorities regarding further relevant original studies/publications were checked.

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2 Synephrine
Synephrine, also referred to as p-synephrine, is an alkaloid with adrenergic activity which is naturally found in bitter oranges and other citrus fruits. Synephrine may be used as the active ingredient in sports supplements with the intention to promote weight loss as well as to improve athletic performance and to increase energy. It is usually added to the supplements in form of bitter orange (Citrus aurantium) extract. Synephrine is commonly found in “pre-workout” supplements marketed as having “thermogenic properties”, where it has replaced ephedrine, after the latter was prohibited because of its strong association with serious adverse cardiac and cerebrovascular effects [10]. Synephrine is frequently present in products in combination with caffeine and/or multiple herbal ingredients. Because of its known sympathomimetic properties and especially due to possible adrenergic effects on the cardiovascular system, the use of synephrine in food supplements has raised concern for consumer safety. The toxicologically relevant data for synephrine are summarized below.
2.1 Chemical characteristics, dietary occurrence, exposure, medical use
Synephrine or p-synephrine is chemically known as 1[4-hydroxyphenyl]-2-methyl-aminoethanol (CAS No. 94-07-5, chemical formula C9H13NO2, molar mass 167.21 g/mol). Structurally, synephrine is closely related to ephedrine and to the catecholamines epinephrine and norepinephrine (Fig. 1). The p-synephrine, which is found naturally in citrus fruits and used in supplements in form of bitter orange extracts, should not be confused with m-synephrine (synonyms: phenylephrine, neo-synephrine), which is a sympathomimetic drug used primarily as a decongestant. Synephrine exists in two enantiomeric forms: the l-form or (-)-synephrine, which is found in bitter orange and other citrus fruits, and the d-form or (+)-synephrine, which is usually not found in nature [11]. The biological activity of (-)-synephrine was shown to be roughly twice that of the racemate of (±)-synephrine [11]. Hereinafter, the designations “(-)-synephrine”, “(+)-synephrine“, or “(+)-synephrine” are used, if it is possible to distinguish between the individual enantiomers or the racemate, otherwise the term “synephrine” will be used. In humans, synephrine is found in trace amounts in the adrenal gland and is considered a trace bioamine [10].
Synephrine is present in most Citrus fruits, both in their fruits and juice. The dietary exposure to synephrine occurs primarily via ingestion of citrus fruits and their products (juices, marmalade, etc). The total daily intake of synephrine via conventional food, estimated for the German population under consideration of maximum concentrations of synephrine, amounts to 6.7 mg/day for average consumers and to 25.7 mg/day for high consumers [12]. Estimations for the French population considering the maximum levels in citrus fruits yielded an average synephrine intake of 4.3 mg/day and 17.7 mg/day at the 95th percentile [13].
Synephrine is usually added to food supplements in form of extracts prepared from the fruit rinds of bitter oranges (Citrus aurantium) with 6-10% synephrine content or as purified phytochemical (up to 95% purity) [14, 15]. The different food supplements provide highly variable synephrine daily doses which typically range between 5 and 100 mg.
Synthetic synephrine has been medically used worldwide as a sympathomimetic drug under the names Oxedrine or Sympatol®. It consisted of a racemic mixture of (-)- and (+)-synephrine in the form of tartrate and was used for treatment of hypotensive states at oral doses of 100 to 150 mg three times daily [16]. For the German drug Sympatol®, which is no longer available, hypertension, coronary heart disease, tachycardia and arrhythmia were mentioned as contraindications [17].

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2.2 Kinetics and Metabolism
From the plasma concentration data, it appears that synephrine has a low bioavailability when taken orally. After dosing of ten healthy subjects with 46.9 mg synephrine, the measured Cmax was about

2.85 ng/ml, the Tmax about 75 min and the half-life about 3 h [18]. After ingestion of 21 mg synephrine by adults engaging in moderate physical activity, the measured plasma synephrine levels were below 2 ng/ml [19]. Likewise, a pharmacokinetic study of the pharmaceutical Sympatol® showed that the time to peak plasma concentration for orally taken synephrine was 1 to 2 hours, and the elimination half-life was about 2 hours [20]. Synephrine was shown to be a substrate for monoamine oxidase (MAO) enzymes in rat brain mitochondria preparations, displaying Km and Vmax values of 250 μM and of 32.6 nM/mg protein/30 min, respectively [21].
2.3 Toxicological data from animal studies
Rodent studies involving oral administration of synephrine (whether in form of Citrus aurantium extracts or as purified phytochemical) showed that it has the potential to induce cardiovascular toxicity, ranging from an increase in blood pressure and heart rate to ventricular arrhythmias and death. Published animal studies which have addressed the potential toxicity of synephrine/Citrus aurantium when administrated by gavage are summarized in Table 1.
In mice, acute dosing with Citrus aurantium extract (2.5% synephrine) at doses ranging from 1000 to 5000 mg/kg bw produced a reduction of locomotor activity, whereas administration of purified synephrine at doses of ≥300 mg/kg bw induced a reduction of locomotor activity as well as gasping, salivation, piloerection and exophthalmia [22]. These effects were reversible and resolved within 3-4 hours. The authors suggested that the effects may have been due to adrenergic stimulation [22]. In a subsequent subchronic study by the same group [23] mice were treated daily for 28 days with either a methanolic extract of C. aurantium (7.5% synephrine, 400, 2000, or 4000 mg/kg bw) or purified synephrine (30 or 300 mg/kg bw). No clinical signs of toxicity and no deaths were observed in any of the treatment groups.

In the rat, daily treatment for 15 days with hydroethanolic Citrus aurantium extracts (doses ranging from 2.5 to 20 mg/kg bw and day) standardized to either 4 or 6 % synephrine resulted in a dose- dependent decrease of food consumption and body weight gain, as well as an increased mortality in all treatment groups. Furthermore, rats given the highest extract dosage (20 mg/kg bw per day) developed significant electrocardiogram alterations (ventricular arrhythmias and “enlargement of QRS complex”) from 10 to 15 days of treatment [24].

In a 28-days-gavage study rats were treated daily with two different Citrus aurantium extracts (7.25 or 95.0% synephrine) [25]. Synephrine doses were 10 or 50 mg/kg bw per day from each extract. Additionally, caffeine (25 mg/kg bw per day) was added to these doses, since many food sports supplements also contain caffeine. An increase in blood pressure and heart rate was observed in all synephrine-treated groups, whereas more significant effects were observed with less purified (7.25% synephrine) Citrus aurantium extract, suggesting that other components in the botanical preparation may additionally alter these physiological parameters. The increases in blood pressure and heart rate were more pronounced when caffeine was added [25].

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In a subsequent 28-days-gavage study conducted by the same research group [26] the cardiovascular effects of Citrus aurantium extracts containing synephrine (7.25 or 95.0%) were studied in exercising animals (running on a treadmill for 30 min/day, 3 days/week). Similar to the previous study, the rats were dosed daily with synephrine doses of 10 or 50 mg/kg bw per day from each extract with and without addition of caffeine (25 mg/kg bw). Both tested synephrine doses of both extracts significantly increased systolic and diastolic blood pressure for up to 8 hours after dosing. Addition of caffeine to the extract treatments further increased blood pressure parameters, and these combined effects were significantly different compared to the effects of the caffeine alone. Authors concluded that the combination of synephrine, caffeine and exercise can have significant effects on blood pressure and heart rate [26].

2.5 Human data
Studies on acute effects
Only a few human studies have been published in peer-reviewed journals, in which isolated synephrine administration was assessed for cardiovascular effects. The majority of studies have been conducted with products containing not only synephrine (present as extracts of C. aurantium), but also other ingredients such as caffeine, green tea, ginseng extract, guarana extract, etc. Some of these studies reported that synephrine taken alone as C. aurantium extract [29] or in combination with caffeine [18, 19] acutely increased systolic and diastolic blood pressure and heart rate in otherwise healthy normotensive adults. Other studies reported no such acute effects when comparable synephrine doses were tested in healthy young normotensive individuals [30, 31]. The relevant human studies evaluating acute effects of synephrine ingestion on cardiovascular parameters are briefly summarized in the following:

Ingestion of a single synephrine dose of 54 mg by healthy adult subjects (n=15) led to an increase in systolic blood pressure between 1 and 5 hours after ingestion, and an increase in diastolic pressure between 4 and 5 hours after ingestion compared to the placebo group. The maximum difference in systolic pressure was 7.3 ± 4.6 mmHg, observed 3 hours after ingestion, while the maximum difference in diastolic pressure was 2.6 ± 3.8 mmHg, observed 5 hours after ingestion. Heart rate acceleration was observed 2 to 5 hours after ingestion with a maximum deviation from the control group of 4.2 ± 4.5 beats per minute (bpm), observed 4 hours after ingestion [29]. By contrast, providing a lower dose of synephrine did not seem to significantly affect the cardiovascular parameters. A single dose of 27 mg synephrine (ingested as a standardised 6% extract of C. aurantium) showed no effect on systolic or diastolic blood pressure or on the length of the QT interval in 18 healthy volunteers [32]. Also a single dose of 26 mg synephrine (C. aurantium extract) administered to 22 healthy weight-stable subjects had no effect on blood pressure and heart rate [33].

In another study, conducted in healthy adult subjects (n=10 per group), no significant variations in blood pressure or heart rate were observed up to 75 minutes after ingestion of a single oral dose of 50 mg of synephrine alone or combined with the flavonoids hesperidin and naringin [34]. According to a further study in 18 healthy young adults (proven to be free of cardiovascular disease prior to onset of study), consumption of 49 mg synephrine did not result in any significant changes in electrocardiograms, heart rates, blood pressure, blood chemistries, or blood cell counts at any time point (measurements conducted at baseline, 30, 60, 90 min, 2, 4, 6, and 8 h after ingestion) in either control or synephrine-treated group [30].

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In another study, a combination of a synephrine dose of 5.5 mg (C. aurantium extract) with 239 mg caffeine significantly elevated blood pressure and heart rate in healthy normotensive adults (n=10). In the same study, when the synephrine preparation was ingested alone at a dose of 46.9 mg synephrine, no effect on blood pressure, but a significant increase in heart rate was observed [18]. In a further study by the same group [19], healthy, normal-weight adults (n=10) ingested a single dose of a supplement containing 21 mg synephrine in combination with 304 mg caffeine under resting conditions or 1 h prior to moderately intense exercise. A significant increase in diastolic blood pressure (peak at 3 h) was observed after supplement ingestion vs. placebo. Systolic blood pressure was likewise increased between 2 and 3 h in the supplement group, although the difference was not significant in comparison to placebo. According to another study [35], conducted in 23 healthy subjects, ingestion of a food supplement whose composition included, among others, 13 mg of synephrine and 176 mg caffeine did not result in any quantifiable effect on blood pressure or on heart rate, compared to a placebo group.

In a recently published study, aimed to investigate the metabolic, lipolytic and cardiovascular responses to supplementation with synephrine alone and in combination with caffeine during resistance exercise in 12 young healthy men (20-24 years), no changes in heart rate were reported after ingestion of 100 mg of synephrine alone, but a significant increase was observed when taken together with 100 mg caffeine [36]. The blood pressure changes were not assessed in this study.

Studies involving repeated application
In a double-blinded, placebo-controlled study conducted in healthy subjects (25 per group), 49 mg synephrine per single dose (C. aurantium extract) were given alone or in combination with naringin (approx. 101 mg) and hesperidin (approx. 576 mg) twice daily for 60 days. No clinically significant treatment-related changes in systolic or diastolic blood pressures, blood chemistry or blood cell counts were observed at the end of the study in comparison to baseline measurements [37]. However, acute hemodynamic effects could have been missed in this study, since subjects were evaluated only at baseline, day 30 and day 60. Thus, it is not clear at which time point after the last dosing the
measurements were collected. Moreover, given the large within subject variability regarding heart rate 11 and blood pressure throughout a typical day, it may be difficult to detect changes based on a single time measurement over a several week period. The study authors ́ statement “no adverse events were reported by any of the subjects” is difficult to interpret as the used “quality of life questionnaires” were not designed to detect negative treatment-related symptoms [38].

It May Block Fat Production And Reduce Belly Fat

Case reports observed in conjunction with use of synephrine-containing supplements.
It is important to mention that a number of published case reports have associated products containing synephrine with various adverse effects, such as myocardial infarction [39, 40], ischemic stroke [41], acute arterial hypertension [42], tachycardia [43], bradycardia [44], syncope and QT prolongation [45], ischemic colitis [46], vasospasm and stroke [47], ventricular fibrillation [48] or apical ballooning cardiomyopathy [49]. Health Canada reported about the collection of a series of cases of serious adverse reactions suspected of being associated with synephrine-containing natural health products [50, 51]. In 2014, the French Agency for Food, Enviromental and Occupational Health (ANSES) compiled about 18 reports of adverse reactions collected from 2009 to 2014, which were mainly related to cardiovascular health and were linked to the consumption of food supplements containing synephrine [52]. In general, the majority of reported cases concern people who took these food supplements in a sports context and/or for weight-loss purposes. Despite the fact that most of the mentioned clinical reports contain insufficient information and causality with respect to synephrine is not easy to prove, since the products in question usually also contained caffeine and/or other herbal preparations, these reports indicate that synephrine in particular in combination with caffeine may potentially cause severe cardiovascular effects.

2.6 Mode of action
The available mechanistic data indicate that effects of synephrine on the cardiovascular system are attributable to adrenergic stimulation. As shown in Fig. 1, synephrine is structurally similar to adrenergic agonists such as adrenaline, noradrenaline and ephedrine. Synephrine is supposed to act at α- and (to a lesser extent) β- adrenergic receptors, resulting in vasoconstriction and increased blood pressure. In vitro studies in human and mammal cells and tissues have reported the activity of synephrine on adrenergic receptors of different classes, though the receptor-binding affinity of synephrine is considered to be within orders of magnitude lower than that of endogenous adrenergic agents as norepinephrine [53].

It has been reported, for example, that synephrine interacts directly with α1-adrenergic receptors and causes contraction of the rat aorta, anococcygeus (a type of smooth muscle in rectum), as well as the guinea pig atria and trachea [54]. Synephrine showed vasoconstrictive effects in rat aorta, and these effects were sensitive to α1-adrenergic receptor antagonists but not to β-adrenergic antagonists [55]. Synephrine was reported to reduce portal pressure and elevate mean arterial pressure in sham- operated and portal hypertensive rats [56].

SIDE EFFECTS – Generally Recognized As Safe (GRAS)

Synephrine-containing C. aurantium extracts as well as purified synephrine increased glycogenolysis, glycolysis and perfusion pressure in the isolated perfused rat liver, and these effects were sensitive to several adrenergic antagonists [57]. Synephrine induced hemodynamic and metabolic effects in isolated perfused rat liver, which were strongly inhibited by α-adrenergic antagonists and moderately affected by β-adrenergic antagonists [58]. Moreover, synephrine induced an antidepressant-like activity in mouse models of immobility tests, which have been attributed to stimulation of adrenergic receptors [59]. The claimed effect of synephrine as a weight-loss stimulant is also attributed to adrenergic properties, in this case to the activation of β3-adrenergic receptors and consequent induction of thermogenesis [53].

2.7 Health risks of synephrine: Assessment and discussion
The main health concern about synephrine use in food and sports supplements refers to its potential as a sympathomimetic to induce adrenergic effects on the cardiovascular system. Synephrine displays a proven sympathomimetic activity and was used as an active ingredient in medicinal products. For the German drug Sympatol® which is no longer available on the German market, hypertension, sclerotic vessels, coronary heart disease and tachycardic arrhythmia were mentioned as contraindications [17].
Animal studies involving oral administration clearly showed that supplementation with synephrine (in form of C. aurantium extract or as purified phytochemical) can lead to elevated blood pressure; and that ingestion of synephrine in combination with caffeine can induce considerable cardiovascular effects with additional alteration of heart rate [25, 26].

Some human studies showed that certain synephrine-containing food supplements raised blood pressure in healthy, normotensive adults. Such effects were typically observed between 2 and 4 hours post dosing. Though the results from human studies with oral ingestion of synephrine-containing products were not consistent, it should be taken into account that observed effects of synephrine on the cardiovascular system appear to depend on the dose, time point of measurements and the presence of other additional stimulants, e. g. caffeine. Moreover, for many of the available studies it is unclear to which extent potential bias, due to funding by food supplement industry, may exist. In addition, it should be considered that most of the studies were small and tested generally only healthy and normotensive volunteers. Individuals with underlying cardiovascular diseases, that might be unrecognized, or other conditions as described as contraindications for medical uses of synephrine may be vulnerable subgroups of the population. Another limitation is that human studies investigating the acute effect of synephrine on blood pressure were done at rest, thus effects the stimulant ingredients may have had under strenuous physical exercise conditions (i. e. in states with highly increased heart rate and blood pressure) would not have been seen.

WHO SHOULD NOT TAKE GARCINIA CAMBOGIA

A role of supplements containing synephrine and caffeine has been implicated in many serious adverse events documented in case reports. Caffeine is a central nervous system stimulant and increases systolic blood pressure through interaction with adenosine receptors [60]. It is also known to enhance the effects of other sympathomimetics such as ephedrine [61]. Though these case reports usually do not demonstrate causation or association, considering the repeated co-occurrences, the nature of the observed adverse reactions together with available evidence from animal studies, they amplify the potential safety concerns regarding the use of synephrine-containing supplements.

In its Scientific Opinion on the application of the Qualified Presumption of Safety approach for the safety assessment of botanicals and botanical preparations [62] EFSA regarded synephrine as a principal substance of concern and concluded that: “…the use of extracts of Citrus aurantium in food supplements would have to be restricted to levels where no significant increase of synephrine exposure compared to historical intake levels with traditional foods is to be expected“.

The German Federal Institute for Risk Assessment (BfR) recently issued a risk assessment of sports and weight loss products containing synephrine and caffeine [12], noting that the available toxicity data were not sufficient to allow the derivation of a safe intake level. Since synephrine is naturally present in the human diet (see Section 2.1), the BfR used the “presumption of safety” approach in accordance with the EFSA-guidance document for the safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements to propose a safe intake level of synephrine from food supplements[8]. The BfR concluded that the quantities of synephrine provided by the food supplements should not exceed 6.7 mg/day, which corresponds to the median value of dietary synephrine intake observed from conventional foods [12].

The French food safety authority ANSES in its assessment of synephrine from 2014 [13] concluded that intake levels of synephrine through food supplements must remain below 20 mg/day and recommended not taking synephrine in combination with caffeine. It also recommended to avoid the use of products containing synephrine during physical exercise and discouraged its use by sensitive individuals (people taking certain medications, pregnant or breastfeeding women, children and adolescents)”.

3 Hydroxycitric Acid (HCA)
Hydroxycitric acid (HCA) is a fruit acid naturally occurring in fruits of the tropical plant Garcinia cambogia. HCA is used as the active ingredient in a variety of food supplements intended for weight loss. In sports supplements HCA or Garcinia cambogia extract is often a component of so called “fat burner” mixtures. The phytochemical is claimed to suppress the appetite, inhibit the synthesis of lipids, or burn fat via thermogenesis.

The use of HCA as a slimming supplement is mainly based on its ability to inhibit the ATP citrate lyase, the enzyme which plays an important role in fatty acid synthesis [63-65]. HCA is generally added to the supplements in form of Garcinia cambogia extracts. However, the composition of such commercially available extracts is often not clearly specified. Health concerns regarding safety of the HCA-containing supplements have been raised, based on results from animal studies which have suggested that high doses of certain Garcinia cambogia extracts or of HCA itself may be toxic to the testis. The relevant toxicological data on HCA as food supplement are summarized below.

3.1 Chemical characteristics, dietary occurrence and exposure
HCA is chemically known as 1,2-dihydroxypropane-1,2,3-tricarboxylic acid (CAS No. 27750-10-3, chemical formula: C6H8O8, molar mass: 208.1 g/mol). Due to the presence of two chiral centers in the molecule HCA may exist in form of four stereoisomers: (-)-HCA (2S, 3S), (+)-HCA (2R, 3R), (-)- allo-HCA (2S, 3R) or (+)-allo-HCA (2R, 3S). Only the (-)-HCA stereoisomer is known to be naturally present in fruit rinds of Garcinia species (G. cambogia, G. indica, G. atroviridis) [63, 64, 66]. Commercially available HCA products are usually prepared by water or methanol extraction from the fruit rind of Garcinia cambogia – a small- or medium-sized tree, native to Southeast Asia. In the dried pericarp of Garcinia cambogia fruits HCA is present at levels of up to 30% by weight. In aqueous solution, the free HCA is unstable and is converted to its more stable lactone form (see Fig. 2). In food supplements the salts of HCA with sodium, calcium-potassium or magnesium are usually used, because this modification increases the stability of the acid and prevents it from being converted into its lactone form, which is thought to be less biologically active [64, 66-68].

In some countries specifications for Garcinia cambogia preparations are published [69]. However, within the worldwide market the specification of HCA preparations/Garcinia cambogia extracts used in individual products is often unclear and in addition, available specifications leave significant portions of HCA- preparations/Garcinia cambogia extracts unaccounted for. Different HCA products are available, which contain varied quantities of HCA (usually 50% to 60%) and are generally marketed as HCA or as a “Garcinica cambogia extract” [63, 70]. Detailed information concerning specifications is available only for individual products, such as for example a calcium-potassium HCA double salt with a HCA content of 60 % (Ca/K-60 % HCA-salt) and, among other specified constituents, 16 % potassium and 11 % calcium, prepared from the Garcinica cambogia fruit rinds [68].

DOSAGE AND WHEN TO TAKE IT

Garcinia cambogia fruit rind is used in Asian countries for culinary purposes, primarily as a condiment or flavoring agent [70, 71]. There is presently no reliable information available concerning the daily intake of HCA via conventional food in these countries. It can be assumed that consumer exposure to HCA in Germany occurs primarily through its use as a food supplement. The daily dosage of HCA recommended according to product labelling ranges in different commercially available products between 500 and 3000 mg [63].

3.2 Kinetics and Metabolism
From the plasma concentration data, it appears that HCA has a limited bioavailability in humans when taken orally. After ingestion of 2 g of calcium-potassium-HCA salt with a HCA content of 60 % (Ca/K-60 % HCA-salt) (corresponding to 1200 mg HCA) by healthy adult volunteers (n=4) the measured peak plasma HCA concentrations ranged between 4.7 and 8.4 μg/ml (reached 90-120 min after dosing), suggesting a limited efficiency of HCA absorption. According to the author’s estimations, in case of complete absorption the plasma HCA concentrations should have reached approximately 46 μg/ml [72]. Calcium and magnesium salts of HCA are slightly soluble in aqueous media and are supposed to be poorly absorbed in the gastrointestinal tract [73].
3.3 Toxicological data from animal studies
HCA showed relatively low acute toxicity in rodents. The determined oral LD50 of a calcium- potassium-HCA-salt with a HCA-content of 60% HCA (Ca/K-60 % HCA-salt) in rats was higher than 5000 mg/kg, which corresponds to 3000 mg/kg HCA [71]. Several repeated-dose studies (28 days and more) reported that high doses of HCA preparations induced testicular toxicity when administered orally to rats. However, in other animal studies these effects were not observed. Studies which addressed the question of potential testicular toxicity of HCA/Garcinia cambogia preparations are summarized in Table 2.
In a subchronic toxicity study, an extract from Garcinia cambogia containing 41.2 % HCA (63.4% of which was present in the lactone form) was tested in developing male Zucker obese rats [74]. The animals were fed for 92 or 93 days with diets containing different HCA levels of 0, 10, 51, 102 and 154 mmol/kg diets, which corresponded to an average intake of HCA doses of 0, 78, 389, 788 and 1244 mg/kg bw. Each diet group was pair-fed to the highest HCA group (154 mmol/kg diet).

Testicular atrophy and toxicity were observed in animals of the highest and second highest HCA 18 groups (778 and 1244 mg HCA/kg bw and day), whereas no significant treatment-induced effects were detected in other organs. A dose-dependent atrophy and degeneration of germ cells was seen in histopathological examination of the testes in the two highest dose HCA groups. Furthermore, significant plasma hormone changes indicating disturbed spermatogenesis (decrease in levels of Inhibin B, a hormone which is produced exclusively by the testis; increase in the follicle-stimulating hormone- (FSH-) plasma concentrations) were observed in these two HCA groups. No changes were observed in plasma concentrations of testosterone and LH in any of the treatment groups.

Additionally, severe diarrhea was observed in animals fed the highest HCA dose. The authors of this study identified a no-observed-adverse-effect-level (NOAEL) of 51 mmol HCA per kg diet, which corresponded to 389 mg HCA/kg bw and day [74]. A lowest-observed-effect-level (LOAEL) was 102 mmol HCA per kg diet, which was equivalent to 778 mg HCA/kg bw and day, respectively.
Comparable effects on the testes were also observed in another rat strain, when the same HCA- containing product (Garcinia cambogia extract containing 41.2 % HCA) was tested in male Fischer 344 rats [75]. Reduced testis weights, degeneration of the germ cells in testis and impaired spermatogenesis were observed in animals ingesting a Garcinia cambogia extract containing a HCA dose of 102 mmol/kg diet (the lowest dose which induced adverse effects in the previous study [74]) for 4 weeks.

Similar to the previous study [74], significant changes in serum hormone levels related to spermatogenesis (decrease in inhibin B and increase of FSH concentrations) were reported in HCA- treated animals compared to the control group. When the same Garcinia cambogia extract (HCA content 41.2 %) was tested in female rats, no changes in sex hormones (FSH, luteinizing hormone, estradiol and progesterone) and no morphological signs of toxicity in the follicle and corpus luteum were observed after feeding the animals a diet containing 154 mmol HCA/kg diet (a dose which induced marked testicular toxicity in the subchronic toxicity study [74]) for a period of 28 days [76].

Another animal study, which is only available as a conference abstract [77], aimed to clarify whether HCA itself or other ingredients of Garcinia cambogia extract caused testicular toxicity. Fischer 344 rats were fed diets containing 0, 0.13, 0.66 and 3.31 % of HCA-calcium-salt (99.5% purity) or 5% Garcinia extract powder containing 66.2% HCA (dose equivalent to 3.31% HCA-calcium-salt) for 28 days. A decrease in epididymal weights, an increase in cell debris in epididymal ducts, a significant decrease of spermatocytes as well as degenerative changes of Sertoli cells and elongated spermatids were reported in rats fed 3.31 % of HCA-calcium-salt and 5% Garcinia extract powder diets. Since the observed effects were almost identical in both former groups with respect to quality and extent of effects, the authors concluded that the observed testicular toxicity might be attributable to HCA as the principle ingredient of Garcinia cambogia preparations [77].

A more recent study, which is only available as a scientific correspondence [78], reported that no histopathological changes were found in testes of adult male rats after dosing with 212-1063 mg/kg bw/day of HCA lactone (98% purity) or equimolar concentrations of HCA (300-1500 mg/kg bw/day; unknown purity) for 8 weeks. No detailed information is available concerning the study findings.
When the Ca/K-60 % HCA-salt was tested in a 90-day subchronic toxicity study in rats, no signs of testicular toxicity were reported [79, 80]. Sprague-Dawley rats received the test product by gavage at dose levels of 0.2, 2.0, and 5.0% of feed intake, which was approximately equivalent to HCA doses of 60, 600, and 1500 mg/kg and day, respectively [68].

After 90 days of administration a significant reduction of food intake (26% in males and 23% in females receiving the highest HCA dose) as well as body weight (16% in males and 13% in females of the highest HCA dose group) was found in HCA-treated animals. No significant effects on organ weights, hepatic DNA fragmentation and lipid peroxidation, no hematological or biochemical alterations and no significant histopathological changes in the different investigated organs (including kidney, liver, prostate and testes) were reported in HCA-treated animals. Notably, testicular atrophy and aspermatogenesis were seen in one animal, but unfortunately no information was provided as to which group this animal belonged to (i.e. control or HCA group, or affected HCA-dose group, respectively). Overall the study authors concluded that the Ca/K-60 % HCA-salt did not cause any changes in major organs or in hematology, clinical chemistry, and histopathology. [79].

The authors identified a NOAEL for the tested HCA-preparation of 2500 mg Ca/K-60 % HCA-salt/kg bw and day, which corresponds to 1500 mg HCA/kg bw and day [68, 79, 80].
The same HCA product (Ca/K-60 % HCA-salt) was further tested in a two-generation reproduction toxicity study in Sprague-Dawley rats [81]. Male and female animals were fed a diet containing 0, 1000, 3000, or 10000 ppm of the HCA-preparation for a period of 10 weeks prior to mating, during mating, and across two generations. No treatment-related adverse effects on reproductive performance in terms of fertility and mating, gestation, parturition, litter properties, lactation, sexual maturity, and development of offspring were observed during HCA-exposure of male and female rats of the F0 and F1 generations. No treatment-related changes in sperm quality parameters (sperm count and sperm motility), no changes in male fertility indices and no histopathological alterations in testes were observed in male rats of the F0 and F1 generations. The authors identified a NOAEL for the tested Ca/K-60 % HCA-salt of 10000 ppm in feed, which was equivalent to a HCA intake of 610.8 and 914.4 mg/kg bw and day, in male and female rats, respectively [81].

Following this two-generation study, a developmental toxicity study with male and female rats of the F2 generation was performed [82]. Animals received the same dietary exposure levels of HCA as those employed for the two-generation study (0, 1000, 3000 and 10000 ppm of the Ca/K- 60 % HCA- salt) until mating, and the female animals up to the 20 day of pregnancy. No evidence of maternal toxicity and no external, soft tissue or skeletal abnormalities in the fetuses were observed on the 20th day of gestation. At the highest HCA dose group a 13% lowering of maternal body weight gain, a non-significant reduction of corpora lutea numbers, a non-significant increase of early resorption numbers as well as a significant reduction of mean litter size were observed. The authors concluded that the tested HCA product was not found to be teratogenic at any of the dietary dose levels tested and identified a NOAEL of 10000 ppm, equivalent to a Ca/K-60 % HCA-salt dose of 1249 mg/kg bw/day, corresponding to a HCA dose of 744 mg/kg bw/day [82].

3.4 Information on genotoxic effects
No genotoxic activity was observed for the Ca/K-60 % HCA-salt when tested in an Ames bacterial reverse mutation assay at concentrations up to 25000 μg per plate or in a chromosomal aberration test in CHO cells at concentrations up to 125000 μM/ml [68, 83]. In a mouse micronucleus assay, the same HCA preparation (Ca/K-60 % HCA-salt) caused a significant increase in the number of micronucleated polychromatic erythrocytes at the dose of 12500 μmol/kg (i. p. application) [83]. However, there are several methodological questions to the validity of the latter study (the use of DMSO as a solvent in test groups, no clear dose-dependency of observed effect, etc.) which complicates its interpretation [84].
No genotoxic activity was observed for a “Ca-type” Garcinia cambogia extract (65% HCA content) when tested in a chromosomal aberrations test in Chinese hamster lung cells (up to 4000 μg/ml) or in an in vivo micronucleus induction test in male Slc mice (up to 2000 mg/kg) [85]. Taken together, the available data indicate that HCA is unlikely to have a mutagenic potential.

3.5 Human data
Clinical studies
The majority of the human studies with HCA-preparations published so far were conducted with slightly to moderately overweight persons to examine the claimed effects of HCA on weight loss, promotion of appetite suppression and other beneficial effects on body composition (reduction of body fat, improvement of plasma cholesterol or fat oxidation parameters, etc.) and were not designed to detect possible adverse effects of tested HCA products (studies are reviewed elsewhere [63, 86, 87]). Table 3 summarizes human studies involving HCA ingestion that are relevant for the risk assessment and which provide detailed information on observed adverse effects and/or safety-relevant clinical and laboratory parameters. In general, no severe or serious adverse effects were reported in any of these studies, and the observed symptoms were mild, infrequent and included headache, nausea, diarrhea, and other gastrointestinal symptoms (see Table 3).
A HCA dose of 3000 mg/day (in form of Garcinia cambogia extract containing 60% HCA, no further specifications) was administered to 10 healthy male volunteers (26 – 56 years) for 30 days [88]. No treatment-relevant changes were observed in hematological and biochemical blood parameters. No changes in serum testosterone levels were observed in comparison to the initial values and no serious adverse effects were reported. In a further study with the same Garcinia cambogia extract, a HCA dose of 3000 mg/day was ingested by 48 healthy subjects (24 female, 24 male, normal weights) for a period of 12 weeks with [89]. This study included a 2-week “run-in” placebo treatment period.

No treatment-related changes in hematology, blood biochemistry or urinalysis were detected and no serious adverse effects were observed. To address the question of possible effects of the tested HCA- preparation on male testicular function, the levels of serum inhibin B and FSH (markers of spermatogenesis) were investigated in male test subjects. Slight increases of serum inhibin B and FSH levels (in comparison to those at week 0) observed at week 12 were interpreted by the study authors as being of no physiological significance [89]. No significant differences were observed for serum LH and testosterone levels in male as well as for serum FSH, LH, and progesterone in female test subjects. The identical Garcinia cambogia extract was further tested in a randomized, placebo- controlled, double-blind study on 18 overweight volunteers (placebo group n=21) for 12 weeks with subsequent 4 weeks follow-up [90, 91]. No significant alteration of serum testosterone, estrone, and estradiol levels were observed in the treatment group when compared to the placebo group. Similarly, hematology and serum clinical pathology parameters did not reveal any significant adverse effects.

Another randomized, placebo-controlled, double-blind study tested a dose of 1200 mg HCA (given as Garcinia cambogia extract containing 60% HCA, no further specifications) in 29 overweight volunteers (placebo group n=29) over 10 weeks [92]. Analysis of blood biochemistry revealed no treatment-related changes in plasma liver enzymes (ALT, AST), erythrocyte antioxidant status or plasma adipocytokines. Two 8-week randomized, double-blind, placebo-controlled trials were conducted in overweight subjects under calorie-restricted diet (< 2000 kcal per day) that received the Ca/K-60 % HCA-salt with a HCA dose of 2800 mg per day [93, 94]. The observed adverse effects are presented in a summarizing publication [95]. An increased incidence of headache was observed in the group receiving HCA in comparison to the placebo group, whereas other mild symptoms were comparable between the groups (see Table 2). Notably, a statistically significant increase in serum serotonin levels was observed under HCA treatment compared to the placebo group [95]. Case reports observed in conjunction with use of HCA-containing supplements

A series of recently published cases of (hypo-)mania (typical symptoms include irritability, aggressive behaviour, decreased sleep and delusions) were reported in association with the use of HCA- containing supplements [96-98]. In each case, the affected individuals developed manic symptoms while taking a supplement containing HCA in form of Garcinia cambogia extract over several weeks prior to onset of symptoms. According to these reports, two persons had no history of psychiatric illness [96, 98] whereas three other individuals [96, 97] had a diagnosis of bipolar disorder, but were stable (symptom-free) without or under treatment with mood stabilizers or neuroleptics until they started supplement ingestion. Though, due to the multifactorial mechanisms of mania and in some
cases concomitant use of prescribed drugs, it was not possible to establish HCA as causative principle
in most of these cases, it has been discussed that HCA may trigger mania in predisposed individuals due to its known serotonergic activity [96, 97]. In one case [97] the causal association between consumption of the Garcinia cambogia supplement and occurrence of hypomania was evaluated as probable/likely, as an improvement of manic symptoms was seen when the HCA-containing supplement was withdrawn.
In another recent report [99], a patient experienced two episodes of serotonin syndrome, each time after having taken a HCA-containing product (Garcinia cambogia extract with 60% HCA, 1000 mg daily) in addition to a prescribed antidepressant drug. The authors suggested that combining HCA- containing supplements with serotonergic drugs might increase the risk of serotonergic side effects.

3.6 Mode of action
Serotonin increase in brain due to ingestion of HCA was supposed as a possible mechanism for adverse psychiatric effects. Notably, HCA was suggested to influence appetite by promoting release and synaptic availability of serotonin [64]. Indeed, HCA was shown in vitro to induce increased serotonin release from the isolated rat brain cortex [100]. Elevated serotonin levels were reported in the brain tissues of male and female rats ingesting HCA [67]. Furthermore, a significant increase in serum serotonin levels was observed under HCA treatment in human clinical trials [95].

Possible mechanisms underlying testicular toxicity of HCA-preparation were addressed in one study, which investigated effects of zinc supplementation (0.01% and 0.05% in feed) on testicular toxicity of “Ca-type” Garcinia extract in F344 male rats (n=10) [101]. While a slight atrophy of spermatids and decreased inhibin-B plasma levels were observed in animals fed a diet with 5% extract, no signs of testicular toxicity were observed when 0.05% zinc was simultaneously supplemented to the extract- treated animals. The authors concluded, that zinc deficiency in diet or zinc depletion may be a possible mechanism of testicular toxicity of Garcinia cambogia extract [101].

3.7 Health risks of HCA: Assessment and discussion
Based on the available toxicological data, a main health concern associated with use of HCA preparations in food and sports supplements refers to male reproductive endpoints. However, rodent studies involving oral administration of various HCA preparations at high doses reported heterogeneous results (see Table 3).
At least three different HCA products (Garcinia cambogia extracts with 41% or 66% HCA and a preparation of a calcium salt of HCA) caused testicular atrophy and toxicity in male rats. The testicular toxicity included atrophy of seminiferous tubules, degeneration of germ cells, degeneration of Sertoli cells and impaired spermatogenesis.

These effects were reproducible and were observed in two different rat strains [74, 75, 77]. From animal data, obtained by testing the Garcinia cambogia preparation containing 41.2% HCA (no further specifications) a LOAEL of 778 mg HCA/kg/day and a NOAEL of 389 mg HCA/kg/day were identified. On the other hand, no testicular adverse effects were reported in animal studies testing another HCA preparation comprising a Ca/K- 60 % HCA-salt.

With this preparation, a NOAEL of 610.8 mg HCA/kg/day was identified for male rats in a reproduction toxicity study, which was the highest HCA dose tested [81]. In addition, in a 90-days toxicity study testing the Ca/K-60 % HCA- salt in rats, a NOAEL of 1500 mg HCA/kg/day was identified [79, 80]. It should be noted however, that in this study testicular atrophy and aspermatogenesis were reported in one animal (no information provided as to which group this animal belonged to; i.e. control or HCA group, or affected HCA-dose group, respectively) [79].

Taken together, results of the animal studies are not uniform and partly contradictory, and may depend on the Garcinia cambogia product or HCA preparation tested. Currently, it needs to be further elucidated to which extent observed adverse effects on the male reproductive system are to be ascribed to specific Garcinia cambogia extracts and constituents thereof or to HCA itself. However, adverse effects on the male reproductive system observed with high doses of certain Garcinia cambogia extracts/HCA preparations should be taken seriously and adequately addressed in risk assessment of such products.

Although the results obtained from several animal tests suggest serious adverse effects on the male gonadal system at high doses of certain Garcinia cambogia extracts/HCA preparations, the question of possible effects on human male reproduction associated with long-term oral ingestion of HCA- containing supplements has been addressed solely in one human study involving 24 male subjects. In this study [89], no physiologically relevant changes in plasma spermatogenesis markers (plasma levels of inhibin B and FSH) were detected following 12-weeks administration of 3000 mg HCA per day.

However, no determinations of classical markers of spermatogenesis, such as sperm concentration, total sperm count and morphology, were performed in male subjects following ingestion of HCA supplements. Thus, human data is currently insufficient to conclude on the safety of HCA with regard to the human male reproductive system. In addition, although no serious adverse effects were observed in clinical trials testing different HCA preparations at the daily doses up to 3000 mg HCA, no human data is available on the health effects of HCA ingestion over a period exceeding 12 weeks (see Table 3).

Garcinia cambogia is known to be used in India and Southeast Asia as a condiment. However, the “presumption of safety” approach as proposed by EFSA could not be applied for the derivation of the acceptable intake levels of HCA from food supplements, since adequate data on HCA intakes via traditional/conventional food that are not associated with adverse effects is lacking. Hence, the use of NOAEL values determined from animal toxicity studies as the “point of departure” is the only possible alternative for quantitative assessment/estimation of safe HCA levels in food supplements. In this case, the comparability of the given HCA preparation with that tested in animal studies would be of importance for choosing the appropriate NOAEL.

For HCA preparations that have not been further specified, rather the lowest determined NOAEL of 389 mg HCA/kg/day as a “worst-case scenario” should be applied. (For other HCA-preparations for which adequate animal data is available, other NOAELs may be applicable.) The use of daily HCA doses of 1000-3000 mg, which are commonly present in sports food supplements, corresponds to the ingestion of approx. 14-42 mg HCA/kg bw/day for a 70-kg person, resulting in intake levels that are only by a factor of 9 to 27 lower than the lowest NOAEL of 389 mg HCA/kg/day from animal studies.

It may be questioned whether such a margin to the NOAEL of only up to 27 is sufficient to ensure human safety, in view of the lack of adequate human data on the safety of HCA-preparations, particularly with respect to the human male reproductive system. It is noted that, in the context of risk assessment of chemicals, a safety factor of 100 is usually applied to derive human health-based guidance values from NOAELs observed in animal studies, to account for interspecies differences and variability in humans.

In conclusion, knowledge gaps and substantial uncertainties exist regarding the safety of HCA preparations found in commercially available food supplements, particularly with regard to the human male reproductive system. Considering the serious adverse effects on the testes observed in several animal studies with high doses of certain Garcinia cambogia extracts/HCA-preparations as well as in view of lack of the adequate human data on the safety of the long-term use of HCA-preparations, there are open questions regarding toxic effects on the male reproductive system of HCA-containing food supplements. In addition, in view of the recently published cases of mania/hypomania suspected of having been induced by the use of the supplements containing HCA in predisposed individuals or patients taking certain antidepressants, further research is needed to clarify the potential psychiatric effects of HCA- or Garcinia cambogia-preparations.

Concluding remarks
Many botanical supplements contain active ingredients that have the potential to elicit effects in the body, including unwanted or adverse effects. Thus, depending on the substances involved, their dose and possible combinations of substances within the product, such supplements may pose a risk to human health under certain conditions. Regarding products that contain phytochemical ingredients that possess sympathomimetic activity, severe health risks have been described for Ephedra herb- containing food supplements. There is concern that sports food supplements containing other sympathomimetics, such as synephrine, may also pose cardiovascular health risks, particularly when such phytochemicals are ingested in combination with caffeine. In the case of Garcinia cambogia extracts and HCA preparations, current safety concerns regarding possible testicular toxicity exist, which are based on toxicological data obtained for certain HCA-preparations in animals. Furthermore, as recent human psychiatric case reports suggest, use of HCA may pose a potential health risk to patients with bipolar disorder or those taking certain antidepressants.

A number of problems may be encountered in the context of risk assessment for supplements containing phytochemical preparations. Relevant information regarding the used botanical preparation(s), including the botanical origin, extraction procedure and the chemical composition is often not provided. Botanical preparations may contain a number of different biologically active substances which are often not sufficiently toxicologically characterized, neither individually nor with respect to possible combined effects and interactions. In addition, many products contain a number of botanical preparations of different species. For products containing mixtures of plant extracts, risks due to combined effects are difficult to assess.

Available toxicological data on the active ingredients or on the whole product are often scarce, and in many cases do not provide sufficient information on toxicokinetics or on relevant toxicological endpoints. In addition, studies, if available, often do not meet the requirements of existing guidelines (e.g. OECD test guidelines). Human studies involving such preparations often have shortcomings with respect to the number of participants and duration of the study and/or reporting of safety related data. Although case reports may provide indications for adverse effects due to consumption of supplements containing phytochemicals, the causality between the use of the product and the adverse effects is often difficult to establish, even though scoring systems are in place (e.g. [102]).

It should also be considered, that adverse effects resulting from use of botanical supplements may be under-reported [103]. Although implementation of post-marketing surveillance or nutrivigilance systems might be expected to contribute to improvement of risk assessments, a formal post-marketing surveillance system for food supplements is currently not in place in many countries.

Finally, in view of the present lack of data for risk assessment for many phytochemicals or botanical preparations used in food supplements, the importance of comprehensive systematic safety testing, preferably prior to product marketing, is emphasised, to improve the basis for risk assessment, and thus to ensure a high level of consumer protection.

The effect of Garcinia Cambogia as coadjuvant in the weight loss process

The World Health Organization (WHO) classifies obesity as an epidemic resulting from the imbalance between intake and energy expenditure, as a consequence of a sedentary lifestyle and poor eating habits1,2,3, as well as the socio demographic conditions4. This disease constitutes a significant global health problem that attacks adults and children and affects a worldwide, growing number of people5.

It is characterized as a chronic disease caused by excessive accumulation of body fat resulting in significant loss of quality of life and longevity. It occurs in developed and developing countries3,6, and it has the involvement of a complex interaction of environmental and genetic factors, besides being associated to morbidity and mortality related to heart attack, high blood pressure, diabetes and even cancer7,8.

Consequently, obesity is a public health problem that requires attention and multisectoral actions to promote a healthy lifestyle and improve the prevention and its control among population4,9.

The Ministry of Health (MoH), through Vigitel (Telephone-based Surveillance of Risk and Protective Factors for Chronic Diseases)10, in 2013, revealed overweight in 51% of the Brazilian population; men are the majority. In 2006, this percentage was 43%, a fact that shows that the obesity rate has been growing in the country. In 2012, ABESO (Brazilian Association for the Study of Obesity and Metabolic Syndrome)11 released the report “World Health Statistics 2012” of WHO revealing that obesity is the death cause of 2.8 million of people per year and that 12% of the world population is considered obese, and the American continent has the highest incidence of the disease.

Since obesity is reaching epidemic proportions, its effective management is an important clinical problem. Despite scientific efforts to understand the mechanisms that lead to overconsumption of food and overweight, at the moment, few weight management approaches are effective in a long-term12.

Therapeutic strategies to treat obesity include synthetic drugs and surgery, which can result in high costs and serious complications. Medicinal agents, herbal products, offer an alternative approach to manage weight, in combating obesity and co-morbidities associated resulting in a safer and more efficient treatment without risks to health2,13, due to their fewer side effects compared to synthetic drugs3. Thus, the phytotherapic represent a new potential coadjuvant or alternative therapy for the treatment of obesity14 and have frequently been used to promote weight loss15.

These agents act through five basic mechanisms, including the stimulation of thermogenesis, reduction of lipogenesis, increase of lipolysis, appetite suppression and decrease of lipids absorption13. Currently, the term “thermogenic fat burner” has been used to describe nutritional supplements that promote, somehow, the fat metabolism by increasing energy expenditure, fat burning, weight loss, lipid oxidation during the exercise16.

The probable reasons for obese people prefer herbal products for weight control include healthy weight loss without any side effects; lower demanding in lifestyle changes such as diet and exercise; ease of acquisition, available without a prescription; more easily accepted than a professional consultation with a doctor or a nutritionist; 100% natural with the perception that natural means safe7.

For these reasons, people in all countries have been using herbal medicines for weight control and treatment of obesity1,6,8. But despite these substances promise to improve or prevent the obesity, costs, effectiveness and side effects have to be considered and, for these reasons, they must be studied intensively1,2.

Among the natural supplements, herbals for the treatment of obesity, stands out the natural extract obtained from dried fruit of the tree Garcinia cambogia (GC), which is found in the forests of South India and South Asia, has been studied extensively and used as a supplement for weight loss1,2,6,8,17.

The hydroxyl citric acid or hydroxycitric acid (HCA) is extracted from the rind of the fruit. It is an organic acid considered the main active ingredient, which acts as a potential supplement to the weight control3,17,18,19 by causing the appetite suppression and reducing the body’s ability to form adipose tissue3. Besides acting as a phytotherapic coadjuvant for the treatment of obesity, the GC extract and its active component HCA have also been used to reduce cholesterol and triglycerides.

It is also available with addition of calcium, magnesium, potassium and mixtures thereof due to other effects, such as improvement of glucose tolerance and of blood pressure20,21.

Unlike chemical stimulants used for weight loss, it does not act on the central nervous system and does not cause insomnia, nervousness, changes in blood pressure or heart rate and its efficiency does not decrease along the time17,21. This way, the GC extract is quickly becoming a popular ingredient among many supplements for weight loss and has been used routinely for many centuries for not showing toxic effects3.

The evaluation of its toxicity to the weight control is extremely important, as it requires a continuous consumption in the long term in order to maintain its effects8. Currently, the GC is being released by the National Health Surveillance Agency (ANVISA) and has been indicated as a coadjunct of overweight for participating in the regulation of appetite22, but the sale can only be made under a medical prescription.
The Resolution of the Federal Council of Nutritionists (FCN) of Brazil nr.390/200623 regulates the dietary prescription of nutritional supplements by the nutritionist and determines that it can be done based on the nutritional diagnosis in the following cases: I. specific physiological states; II. pathological conditions; III. metabolic changes, and the prescription of nutritional supplements should still be based on the following premises: I. food consumption adequacy; II. definition of the period of supplementation use; III. systematic re-evaluation of the nutritional status and of the dietary plan.

Due to the large supply in the market, marketing and popular use of herbal medicines, such as GC to act as a coadjuvant in treatments for obesity, this study aimed to deepen the scientific evidence on the effects of GC administration as a coadjuvant factor in the treatment of obesity regarding to its effectiveness, form of action, recommended daily dosage, side effects and contraindications, as a way of food and nutritional security for the population.

Methodology
Literature review study for which there were consulted the LILACS-BIREME, MEDLINE and SciELO databases and selected scientific articles published in English, Portuguese and Spanish, between the period of 2007 and 2014 that conducted studies involving the administration of GC as a way of treatment for obesity. The descriptors used for research articles in the databases were the following: Garcínia Cambogia in Portuguese, and in English the terms used were “Garcinia cambogia”, “weight loss and obesity” and “Hydroxycitric Acid (HCA)”; this latter is not an indexed descriptor in the Health Science Descriptors (DeCS), but given the importance of this term for the search, it was adopted as a keyword. Thirty-four articles were identified, but only 21 were related to the objectives of this study. The first analysis of the articles was conducted by the title and then by the summary. In addition, 20 references were included because of their relevance to the study.
Results
Table I shows the main results of non-randomized studies with supplementation of GC/HCA and table II presents the main results of randomized studies with supplementation of GC/HCA.
Discussion
Due to the dramatic increase of the number of obesity cases in recent years, this disease has become one of the most important problems that public health has faced in several countries around the world, nowadays. In view of this, several methods to assist weight loss have arisen, like miracle diets and dietary supplements, but, often, little is known about the regular use and long-term of certain substances.
According to Kimet et al.24, GC is a popular supplement for weight loss. Studies suggest that the use of GC stimulates the burning of body fat, helps to inhibit appetite by reducing the desire to eat with a consequent reduction in food intake, and promotes satiety, and also acts in weight maintenance.

Being a study that aims the GC administration as a coadjuvant in the treatment of weight reduction, it can provide important information for both scientific uses as for the general population about the effects of its administration as a way to help in the treatment of obesity.

Table I

Treatment

Result

Effectiveness and way of acting
In the analysis of the effectiveness of GC, the study of Kovacs and Westertep-Plantenga28 concluded, in an experimental condition in humans, that the treatment with HCA during overfeeding with CHO can reduce the DNL. In rats, Kim et al.24 verified that the supplementation of this herbal medicine helped to reduce the body fat, but not to decrease weight or appetite.

Although, in humans, Kim et al.15 observed that the GC supplementation did not reduce the percentage of body fat, as it did not act on the decrease of appetite, BW, BMI and waist-hip ratio (WHR). Murer2 concluded that individuals with and without GC supplementation showed reduction of body fat and emphasized that the combination of diet and physical activity still remains the most suitable for positive changes of body composition.

The experiment of Anton et al.14 in humans also did not observe significant effects with the administration of GC dosages on food intake, satiety, weight loss, and oxidative stress levels. They highlighted that further research is needed to explore the promising effects of herbal medicines on food intake and satiety levels. However, they chose the compound derived from GC for their study because they noted in the literature its potential in acting in the reduction of food intake, in BW and in the levels of oxidative stress with safety, affecting the neuroendocrine pathways related to satiety.

Lira-García et al.29, in their review study with among sixteen assessed studies, found only one study that demonstrated significant weight loss between the control group and the experimental group with dosage of 1200 mg/day of GC and concluded that it was not possible to prove the effectiveness of the alternative products for weight loss because there is not enough GC protected against obesity induced [24] by HFD through the modulation of the synthesis of fatty acids and β-oxidation, but induced hepatic fibrosis, inflammation and oxidative stress.

The HFD and HSD groups showed [25] a significant increase in feed intake,
BW, BMI, TG, LDL, oxidative stress and renal disorder, while the groups supplemented with GC showed improvement of the harmful effects of HFD and HSD diets, with consequent reduction of feed intake, increase of the MDA level and decreased oxidative stress in renal tissue.

The supplementation of the GC HFD [26] failed to reduce the rising levels of serum lipids.
High intakes of HCA alone did not [20] lead to signs of inflammation or hepatotoxicity.

Table I

Result
References
EGML and GCE did not promote [15] BW loss neither decreased the TC in overweight individuals consuming
usual diet. EGML increased levels of HDL-C. There were no serious adverse effects reported by the intake of EGML, GCE or placebo (starch).
No significant effects were observed [14] with the administration of the GC dosages or adverse effect level
(NOAEL) in humans at doses of
4000mg/day.

The supplementation of HCA [27] enhances the rate of glycogen synthesis in human skeletal muscle
and improves the postprandial insulin sensitivity.

The combination of diet and physical [2] activity remains the most suitable for positive changes in body composition.

The treatment with HCA during [28] overfeeding with carbohydrates can reduce DNL.

The study of Onakpoya et al.30, also of review, using data from randomized clinical trials (RCTs) in order to examine the effectiveness of GC/HCA extract as weight reducing agent, observed a small significant difference in loss weight, favoring the HCA over the placebo and they concluded that the ECR suggest that the GC/ HCA extracts can cause short term weight loss, confirming the review study of Astell et al.31, who concluded that the results of the RCT showed that the GC extract is effective in reducing body weight by suppressing appetite. The study of Amin, Kamel and Eltawab25, with rats, also concluded that the GC supplementation decreased the feed intake.

The theory behind the GC/HCA is that it works as anti-obesity agent by acting in the neuroendocrine pathways, related to the satiety, producing an anorectic effect, by promoting the inhibition of citrate lyase
enzyme that suppresses the appetite and increases the burning of body fat. Thus, it assists in regulating appetite, with consequent reduction of food intake, caloric restriction and weight loss. By inhibiting this enzyme, the body increases the oxidation of carbohydrates and inhibits the lipogenesis3,14,17,18,32,33. According to the ANVISA34, the register situation of GC in Brazil is classified in the category of appetite modulators and products for special diets, and it is indicated as a coadjuvant of overweight to participate in the regulation of appetite22.

Recently, there was found that the GC supplementation can be used as a metabolic regulator of obesity and lipid abnormalities in the system of mammals.19 Pandya et al.21, Sethi3, and Krishnamoorthy17 claim that GC/HCA reduces the lipid levels in blood, such as triglycerides and cholesterol, besides of increasing the thermogenesis. The HCA inhibits competitively the extramitochondrial citrate lyase enzyme that catalyzes the cleavage of citrate to acetyl-CoA and oxaloacetate, a key step in lipogenesis, necessary for the synthesis of fatty acids and cholesterol35,36.

However, in some studies, such as by Kim et al.24, in rats, there was found that the GC supplementation did not cause significant differences in the levels of TG, TC, HDL-c, phospholipids and free fatty acids. Similarly, the study of Ates et al.26 observed that the GC supplementation coupled with the high fat diet failed to reduce the increased serum levels of lipids in rats. In humans, Kim et al.15 also observed that after the supplementation of GC there were no significant differences in lipid levels (triglycerides and low-density lipoprotein LDL-c), or of adipocytokines (hormones of high adipocytes in obesity). A laboratory study performed by Simon et al.6 also concluded that there were not observed significant effects for the treatment of obesity and of other dyslipidemia with GC.

In contrast, the study of Amin et al.25 in rats, concluded that the GC supplementation improved the harmful effects caused by HFD or HSD, such as hypertriglyceridemia, increase of production of LDL, and of oxidative stress. Santos et al.36 reported in their review study that the administration GC showed inhibition of lipogenesis in the liver of rodents, adipose tissue, and small intestine but without confirmation in humans and claim that the only applicability of the HCA as anti-obesity agent seems to be the reduction of appetite, due to its anorectic effect.

Pandya et al.21 and Krishnamoorthy17 claim that the GC/HCA acts in the suppression of appetite by making glycogen synthesis in the liver and in other body tissues, increasing the energy levels.
The study done by Cheng et al.27 found that the HCA supplementation reinforced the glycogen synthesis rate in human skeletal muscle, improved the postmeal insulin sensitivity, and demanded higher energy expenditure in fat oxidation. Kim et al.2 observed that in rats the GC supplementation caused lower glucose levels, assuming that this could improve glucose tolerance by contributing to the reduction of visceral fat, since it is responsible for insulin resistance even in hyperlipidic diets.

Several in vivo studies have contributed to the understanding of the anti-obesity effects of GC/HCA via the release of serotonin in the brain, which has been considered as the main mechanism to decrease appetite and absorption of glucose and also in the increase of oxidation of fat, reducing DNL29,33. In recent studies in female mouse, there has been observed the effect of HCA on the regulator genes of obesity29. However, studies related to the presence of enzymatic inhibitors in extracts of these plants that participate or are responsible for its anti-obesity properties are scarce in the literature. Since the research conducted to evaluate the effective and safe use of herbal medicines is incipient, the notifications of events help in the generation of new information, promoting its rational use32.

Santos et al.36 still claim that in order to qualify the HCA as an anti-obesity effective metabolic agent it should produce a stimulating effect on the skeletal muscle, on the total fat oxidation or on the calorie consumption, but this has not been proved yet. Studies with this herbal demonstrated its effectiveness in combating obesity; however, there miss further studies on its mechanism of action to generate more security in its therapeutic use.

Tucci12 also states that the GC can contribute to the appetite suppression, but it still should be better demonstrated. In his review study, he noted that some phytochemicals show promising effects on weight control, however, more data is needed to define the real magnitude of effects and ideal doses. For Chandrasekaran7, an ideal anti-obesity herbal has to reduce the weight by 10% in relation to the placebo during the period of treatment, showing evidence of improvement of biochemical tests, like in the levels of lipids and glucose, without any side effects.

Daily Recommended Amount
The study of Amin et al.25 observed positive effects, such as decreased appetite and improve of the harmful effects caused by high fat and sucrose diets in rats that received supplementation of 50 mg/day of GC. The study of Cheng et al. 27 highlighted that the HCA supplementation enhanced the rate of glycogen synthesis in human skeletal muscle and improved the post-meal insulin sensitivity in the amount of 500 mg/day combined with a meal high in CHO (80% CHO, 8% LIP, 12% PROT) after 60 minutes of bicycle. Kovacs and Westertep-plantenga28 observed that the treatment with three capsules of 500 mg/day (1500mg/day) of HCA during the overfeeding with carbohydrate can reduce the DNL.

Ates et al.26 observed in rats that the GC supplementation combined with the hyperlipidic diet was not able to reduce the increase of the serum lipid levels in a dose of 2.390 mg/day of GC and suggest that higher dosages of GC extract should be investigated. Kim et al.15, noticed that for humans the intake of 2000 mg/ day of GC did not promote weight loss neither decreased the total cholesterol in overweight individuals consuming their usual diet. Anton et al.14 also did not observe significant effects in individuals with the administration of higher dosages of GC, 2800 mg/day and 5600 mg/day.

Murer2 concluded in his study that the combination of diet and physical activity remain the most suitable for positive changes in body composition by observing the combination of normocaloric diet associated with the intake of two capsules of 500 mg/day of GC (1000 mg/day), since the supplemented group and the unsupplemented group, fed only with normocaloric diet reduced body fat.
Although Onakpoya et al.30, in their review article with ECR have concluded that the dosage of HCA

The effect of Garcinia Cambogia as Nutr Hosp. 2015;32(6):2400-2408 2405 coadjuvant in the weight loss process used among the studies ranged from 1000 mg/day to 2800 mg/day, which resulted in a small weight loss, and they said that the magnitude of the effect is small and the clinical relevance is uncertain; so, future clinical trials should be stricter and better reported and that the ideal dose of HCA is currently unknown.
GC supplements are available in various forms, including pills, capsules and powders. The herbal medicine is usually standardized to contain fixed percentage of HCA, and the usual dosage from 300 mg to 500 mg should be administered three times a day and ingested half an hour before meals with water3.

Side effects and contraindications
About the side effects and contraindications of the GC usage, there was observed in the study of Kim et al.24 that the prolonged use of GC in the administered dose in rats can cause hepatotoxic effects and even develop non-alcoholic hepatic steatosis because of the accumulation of collagen in the liver, independently of being caused by hyperlipidic diet.

Lobb37 claims that there are a growing number of reports of hepatotoxicity caused by supplements containing HCA. In his study, he approached six case reports: two women, of 33 and 40 years old, and four men of 19, 27, 28, and 30 years old; underestimating the incidence of hepatotoxicity associated with weight loss with the HCA. Each report showed similarities; in the screening of hepatic abnormalities and in the symptoms presented by patients, who were healthy and with normal hepatic functions. Among the laboratory findings and symptoms there were reported fatigue, nausea, vomiting, colic, fever, chills, anorexia, abdominal pain, jaundice in a period ranging from three days to three weeks; deregulated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase and bilirubin. Onakpoya et al.30, in their review study, observed, gastrointestinal adverse events twice more common in the HCA group in comparison to the placebo group.

In contrast, Clouatre and Preuss20 claim that the results obtained about the HCA safety and efficacy are different from a variety of studies performed with animals and humans, since they observed in rats that the HCA produced protective effect against the hepatic toxicity associated with ethanol and administration of dexamethasone, and kept levels of SGOT, SGPT, and alkaline phosphatase at almost normal levels. The compound was found to reduce the inflammatory response in the brain, intestine, kidney and serum, and they highlight that high intakes of HCA, by itself, did not lead to signs of inflammation or hepatotoxicity.

Sethi3 also affirms the protection capacity of the GC against external hepatotoxins such as alcohol, and a recent study showed its effect preventing liver cells becoming fibrocytes. Shivashankara et al.38, observed in their review study that pre-clinical studies conducted recently have shown that some herbal medicines, such as GC, protect against ethanol-induced hepatotoxicity, through the mechanism mediated by antioxidant action, elimination of free radicals, anti-inflammatory and antifibrotic, but they emphasized that future studies would be required to establish its applicability in humans.

In humans, Kim et al. 15 did not observe reports of serious adverse effects by the individuals who consumed GC supplements, corroborating the study of Anton et al.14 who also did not observe adverse effect (NOAEL) in humans with doses higher than 4000 mg/ dia. Lira-García et al.29, concluded in their review study that among the eight studies that were evaluated, in none of them were observed adverse effects on the use of GC.
The review studies conducted by Chuah et al.8, and Chuah et al.33, concluded that there was not observed adverse effect (NOAEL) in dosages of GC/HCA of up to 2800 mg/day, suggesting its safety for use. Most of the reports demonstrated the efficacy of GC/HCA, and there wasn’t found any toxicity.

Sethi3 concluded in his review study that the herbal medicines are more beneficial in the treatment of obesity due to its fewer side effects and also act on the prevention of diseases such as type 2 diabetes, heart disease, high blood pressure. Until now, there is no case study or report showing the direct adverse effect of HCA8, as well as there is no evidence that demonstrates hepatotoxicity associated with the HCA, and the true agents need to be firmly identified, along with the dose to which the negative effects are induced39.

Pandya et al.21 also claim that there aren’t any known side effects for the usage of this herb. However, it is not recommended for people diagnosed with diabetes or people suffering from any kind of dementia or syndrome, including Alzheimer disease as well as pregnant and lactating women and has contraindications regarding the concomitant use of certain drugs.

Egras et al.40 concluded in their review study that many obese people use food supplements for weight loss and that, so far, there is little clinical evidence to support their findings, but it is necessary to determine their efficacy and safety. Health professionals should be aware of the products available for weight loss to help their patients and to determine the risks and benefits of the supplement used to loss of weight. Yuliana et al.41 still claim that despite insufficient data regarding to its safety and efficacy, many herbal medicines are available for sale without a prescription, such as the GC, which reduces the appetite, and that the quality control of these herbal medicines also becomes important.
This review study has several limitations.

Although the research has involved studies in electronic media, there may not have identified all those available involving the use of GC/HCA as a supplement for weight loss. Furthermore, the methodological quality of most studies identified from this study is short. These factors hinder conclusive findings about the effects of GC/HCA on body weight.

Final considerations
Studies suggest positive results concerning the effectiveness of the GC in weight loss process, by reducing the appetite, the percentage of fat, the lipogenesis process, as well as the improvement of biochemical levels, such as triglycerides, cholesterol and glucose, muscle glycogen synthesis, and postmeal insulin sensitivity.

However, the ideal dose has not been well established yet; however the GC supplements are available in 300 mg and 500 mg dosages, with the intake direction of three times a day, with water, half an hour before the meals.

There is little evidence of adverse effects and signs of protective effect against the hepatotoxicity induced by ethanol. Therefore, it is necessary to carry out more randomized, controlled studies, clinical trials to evidence the efficacy of this herbal in the weight loss process, as well as the set of the posology, dosages, indications and contraindications.

Sulforaphane a powerful tool to fight cancer, aging, and other inflammatory health issues

Sulforaphane has been shown to be an effective antioxidant, antimicrobial, anticancer, anti-inflammatory, anti-aging, neuroprotective, and anti-diabetic (R). It also protects against cardiovascular and neurodegenerative diseases (R).

Many test-tube and animal studies have found sulforaphane to be particularly helpful for suppressing cancer development by inhibiting enzymes that are involved in cancer and tumor growth (R, R, R).

According to some studies, sulforaphane may also have the potential to stop cancer growth by destroying cells that are already damaged (R, R).

Sulforaphane appears to be most protective against colon and prostate cancer but has also been studied for its effects on many other cancers, such as breast, leukemia, pancreatic and melanoma (R).

Sulforaphane may also help reduce high blood pressure and keep arteries healthy — both major factors in preventing heart disease (R).

Recent research shows that Sulforaphane can help control blood glucose levels in type 2 diabetic patients as effectively as the most commonly used prescription medicine Metformin (R).

Sulforaphane and Broccoli Sprouts

Sulforaphane (SFN) is an isothiocyanate. It is derived from glucoraphanin, found in cruciferous vegetables such as broccoli, cabbage, cauliflower, brussels sprouts and kale (R, R).

Glucoraphanin is stable, but when the vegetables are cut or chewed, it comes in contact with the enzyme myrosinase, that also occurs naturally in these vegetables, and Sulforaphane is formed(RR).

Unlike the glucoraphanin, sulforaphane degrades quickly (R).

The quantity of glucoraphanin varies greatly in different plants. In general, levels of glucoraphanin and sulforaphane are highest in broccoli sprouts (R), but 3 day-old sprouts can contain up to 100 times more glucoraphanin than in mature plants (R).

Sulforaphane  is rapidly absorbed, reaching peak concentration after 1-3 hours. (RR). Levels are back to baseline within 72 hours (RRR).

Daily consumption of cruciferous vegetables can maintain levels of Sulforaphane in the body, if properly prepared (R), however most people find  it is easier to take a daily supplement.

Sulforaphane activation of  AMPK pathway is key to wide range of benefits

There are many hundreds of studies of Sulforaphane’s  effect in fighting various disease and metabolic problems.  Activation or Inhibition of several different genes are described in how it manages to impact such a wide range of health problems.

As with vitamin antioxidants the notion that supplements act as “antioxidants” in human cells is called into question []. Emerging evidence suggests that the most effective supplement exert their intracellular effects not as direct “antioxidants” per se but as modulators of signaling pathways.

Compared with widely used phytochemical-based supplements like curcumin, silymarin, and resveratrol, sulforaphane more potently activates Nrf2 – which researchers call the “Master Regulator” of Cell Defense (R).

A list of genes and enzymes Sulforaphane influences is at the bottom of this page.

Perhaps even more meaningful is that like Metformin and Berberine (R), Sulforaphane strongly activates AMPK, which raises the intracellular NAD+ concentrations and activates SIRT1 which has been shown to have numerous disease fighting and anti-aging potential(R).

It’s possible many of the health benefits are at least partially related to this AMPK/NAD+/SIRT activity.

Here is a  short list of studies showing Sulforaphane activating AMPK to fight Cancer(R),Diabetes(R),Obesity(R),Neurological disease (R),Heart disease (R),HIV (R),Colitis(R).

Sulforaphane helps prevent and can even kill cancer

3-5 servings per week of Cruciferous vegetables decrease the risk of cancer by 30-40% (R).

Even ONE serving of cruciferous vegetables per week significantly reduced the risk of pharynx, colorectal, esophageal, kidney and breast cancer (R).

In vitro, Sulforaphane has been demonstrated to kill breast cancer cells (R),oral squamous cell carcinoma cells (R), colorectal cancer cells (R),  cervical, liver, prostate, and  leukemia cancer cells (RR), while having little to no effect on healthy cells (R)

Sulforaphane combats cancer by multiple mechanisms:

  • Sulforaphane reduces inflammation by inhibiting the NF-κB pathway(R).
  • Sulforaphane induces cancer cell death (R).
  • SFN inhibits Phase I enzymes that enable cancer cell growth (R).
  • SFN induces Phase II enzymes that clear  DNA damaging chemicals (R).
  • Sulforaphane thereby inhibits cancer cell proliferation  (R)

In addition to the numerous cancer fighting mechanism of Sulforaphane, it is also very effective at enhancing commonly used anti-cancer drugs such as    cisplatin, gemcitabine, doxorubicin, and 5-fluorouracil    , allowing for smaller dosages and limiting toxicity to healthy cells (R).

Sulforaphane helps lower Cholesterol


Clinical studies with humans has shown eating broccoli reduces LDL cholesterol.

Twelve healthy subjects that consumed 100 grams per day of broccoli sprouts lowered LDL cholesterol, increased HDL cholesterol, and improved maarkers for oxidative stress  (R).

Sulforaphane May Help Parkinson’s, Alzheimers, Huntingtons

In mouse models of Parkinson’s disease, Sulforaphane increased dopamine levels in the brain to alleviate loss of motor coordination(RRRR).

A buildup of amyloid beta ( Aβ ) peptides are thought to be the cause of Alzheimer’s disease.  Broccoli sprouts were shown to prevent amyloid beta buildup and cell death (RR).

Sulforaphane has also bee shown to reduce Aβ plaque, and lessens cognitive impairment in mouse models of Alzheimer’s disease (RRR).

Sulforaphane activates a protein that slows huntingtins disease in mice (R).

Sulforaphane Prevents and Combats Heart & Cardiovascular Disease


Observational studies in humans has shown those who eat 3-5 servings of cruciferous vegetables a week have a significantly decreased risk of cardiovascular disease (R).

Research with mice shows Sulforaphane decreases blood pressure (RR).

Rats that were given Sulforaphane after heart attack exhibited  reduced heart damage  (R).

Sulforaphane helps prevent atherosclerosis (R) and minimizes inflammation caused by hardening of arteries in mice (R).

Sulforaphane reduces formation of  blood clots and platelet aggregation in humans (R).

Lastly, Sulforaphane has proven beneficial in minimizing damage from strokes, with decreased brain tissue damage (R), and loss of neurological function (R).

Sulforaphane helps control Diabetes and fight  Obesity


In humans, eating Broccoli sprouts  increased  HDL cholesterol, and lowered  triglycerides, insulin, insulin resistance,oxidative stress, and C-Reactive Proteins (RRR).

Sulforaphane decreases incidence and severity of the following diabetes complications in mice (RR)

  • vascular complications .
  • diabetes-induced heart dysfunction
  • heart damage in mice
  • nephropathy
  • tissue damage

Mice fed a high fat diet to induce obesity that were subsequently treated with sulforaphane for 3 weeks had significantly less weight gain, and improved insulin resistance, glucose and cholesterol levels (R,R).

Sulforaphane is Antiviral

Eating Broccoli sprouts increase the bodies natural anti-virus response and reduce influenza (R, R).

In vitro, Sulforaphane  combats  influenza, HIV, Epstein-Barr virus (R) and hepatitis C virus (R).

Sulforaphane Combats Bacterial and Fungal Infections

Human β-defensin-2 (HBD-2) is a key part of our defense against bacterial invasion. Sulforaphane increases HBD-2 in response to  to 23 of 28 bacterial species (R).

Cystic fibrosis patients have increased levels of Mycobacterium abscessus.  Treatment with Sulforphane of such macrophages  significantly decreased bacterial burden (R).

Sulforaphane Combats Inflammation

Nuclear Factor Kappa-B (NF-kB) is a well known driver of inflammation. Sulforaphane greatly decreases NF-kB activity (R).

As previously mention, Sulforaphane very strongly activates Nrf2, which lowers inflammation (RR).

Sulforaphane May Combat Depression and Anxiety


Inflammation has been recognized as one of the causes of depression. By reducing inflammation, sulforaphane can help combat depression.

Repeated SFN administration reverses depression– and anxiety-like behaviors in chronically stressed mice, likely by inhibiting the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory responses to stress (RR).

In another study, it was shown that Nrf2 deficiency in mice results in depressive-like behavior, while the induction of Nrf2 by sulforaphane has antidepressant-like effects (R).

Also, dietary intake of glucoraphanin during the juvenile and adolescent periods in mice prevents the onset of depression-like behaviors at adulthood (R).

Sulforaphane Protects the Brain and Restores Cognitive Function

Sulforaphane increases neuronal BDNF in mice, a factor that supports the survival of existing neurons and encourages the formation of new neurons and synapses (R).

SFN reduces brain inflammation in various animal models of pathogen-induced neuroinflammation and neurodegenerative disease (RRRR).

Sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 state. This reduces brain inflammation and restores spatial learning and coordination in rats (R).

Sulforaphane is beneficial in various pathological conditions:

  • SFN improves cognitive performance and reduces working memory dysfunction in rats after traumatic brain injury (R).
  • SFN attenuates cognitive deficits in mouse models of psychiatric disease. Also, the intake of glucoraphanin during the juvenile and adolescent periods prevents the onset of cognitive deficits at adulthood (R).
  • SFN alleviates brain swelling in rats, by attenuating the blood-brain barrier disruption, decreasing the levels of pro-inflammatory cytokines, and inhibiting NF-κB (R). SFN also increases AQP4 (a water channel protein) levels, thereby reducing brain swelling (R).
  • SFN prevents memory impairment and increases the survival of hippocampal neurons in diabetic rats (R).

Sulforaphane recovers memory in mice and rats with chemically induced memory impairment (RR R).

SFN exerts positive effects against brain damage induced by acute COpoisoning in rats (R).

Sulforaphane protects human neurons against prion-mediated neurotoxicity (R).

Insufficient NRF2 activation in humans has been linked to neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (R). SFN, as a potent Nrf2 activator, may help in the treatment of these diseases.

Sulforaphane Improves Symptoms of  Autism

Sulforaphane activates several genes that lower inflammation and protect cells from oxidative stress and DNA damage, which are much higher in those with Autism (R).

In a clinical trial of 29 young men with  moderate to severe autism (age 13-27), Sulforphane treatment over 13 weeks resulted in a 35% improvement in disruptive behavior(R).

Sulforaphane relieves Gastrointestinal inflammation, colitis, and ulcers

Aspirin and NSAID’s are very effective at relieving pain, but can damage stomach lining and even cause ulcers.  Sulforaphane has been shown to protect agains such damage in mice (RR,R).

Sulforaphane has also been shown to increase Nrf2 and decrease inflammation in mice with colitis (R,R).

Sulforaphane May be Beneficial in Airway Inflammation and Asthma

Sulforaphane received airway inflammation and asthma symptoms in mice  (R)

Broccoli sprout extract  relieved airway inflammation in humans exposed to vehicle exhaust levels similar to those on a Los Angeles freeway (R).

But in other studies,  broccoli sprouts did not alleviate symptoms of asthma (R),  COPD (R) or ozone-induced airway inflammation (R).

Sulforaphane Can Be Beneficial in Arthritis

We previously pointed out that Sulforaphane strongly activates Nrf2, which relieves inflammation in many conditions.  In additions, sulforaphane was found to inhibit metalloproteinases that cause osteoarthritis and cartilage destruction (R).

Sulforaphane also decreases inflammatory cytokines, reducing symptoms of arthritis in mice (R).

Sulforaphane Protects the Eyes

Sulforaphane protects photoreceptor cells from excessive light exposure damage  (R) and  degeneration (R) in humans, and light-induced retinal damage in mice (R).

Sulforaphane protects human retinal cells and delays onset of cataracts (R), and helps prevent complications after cataract surgery (R).

In mice, sulforaphane helped maintain vitamin A and C levels in retinal cells to prevent damage from oxidative stress (RRR).

Negative Side Effects

Possible liver Toxicity at extreme dosages

There has been a single report of liver toxicity in one individual that consumed over 800 ml per day of broccoli juice for 4 weeks, but function returned to normal wishing 15 days or discontinuing the juice (R).

Note this individual was making juice from mature broccoli plants which have many different active substance and are not recommended for source of glucoraphanin/sulforaphane as the young sprouts have up to 100 times higher glucoraphanin levels.

Maximizing Bioavailability

Glucoraphanin Sources

Broccoli has the highest amounts of glucoraphanin of any vegetable, but it is also found in Brussel sprouts, Kale, Cabbage, Bok Choy, and several others (R).

As previously mentioned, young broccoli sprouts have up to 100 times more glucoraphanin than mature broccoli, making them ideal sources if you want to grow or purchase them (RR).

Myrosinase also required

Remember, Sulforaphane is only formed when it comes in contact with the enzyme myrosinase, by chewing or chopping or other processing.

Myrosinase is a fragile enzyme that is quickly damaged by heating (boiling over 1 minute), or freezing (R).

Many Supplements do not provide active Myrosinase

Broccoli has long been known to provide many health benefits and as such, broccoli sprout supplements are not new to the market.  Unfortunately, many of these were developed before researchers realized the Myrosinase + Glucoraphanain = Sulforaphanin  equation required great care in processing to protect the Myrosinase.

As a result, most broccoli supplements do not provide ANY active myrosinase  (RR).

Sulforaphane was found to be 7 times greater in fresh broccoli sprouts vs supplements with inactive myrosinase  (R).

Glucoraphanin powder with inactive tyrosinase can be combined with a good source of tyrosinase such as broccoli sprouts or mustard seeds to greatly increase Sulforaphane absorption (R).

Mustard seed

The myrosinase found in broccoli is quite fragile and inactivated by freezing or heat. A much more robust form of myrosinase  is found in Mustard seeds. The addition of powdered mustard seed to heat processed broccoli dramatically increases sulforphane (R).

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Sulforaphane activates genes and enzymes that stimulate antioxidant production:

  • inhibits Phase I enzymes CYP1A1, CYP1A2, CYP1B1, CYP2B2 and CYP3A4 (RR).
  • activates (RR). SFN reacts with Keap1, thereby releasing Nrf2 from Keap1 binding (R).
  • increases other Phase II enzymes: NQO1, GSTA1, and HO-1 (RRRR).
  • blocks SXR (R).

Sulforaphane inhibits inflammation:

  • inhibits NfkB (RRR).
  • inhibits TNF-α (RR), NLRP3, IL-1β, IL-18 (R), IFN-gamma and IL6 (RRR).
  • inhibits IL-17 (RR).
  • inhibits TGF-β/Smad (R).
  • increases IL-10 (RRR), IL-4, Arg1, and YM-1 (R).
  • inhibits NO, iNOS and COX-2 (RRR).
  • silences Th17/Th1  (RR).
  • inhibits IL-23 and IL-12 (R).
  • inhibits MMP-9 (RR).
  • inhibits LDH and PGE2 (R).

Sulforaphane changes gene expression:

  • Sulforaphane inhibits DNMT1 and DNMT3A (R)
  • SFN is one of the most potent (histone deacetylase) HDAC inhibitors found to date (R).
  • SFN inhibits HDAC1, HDAC2, HDAC3, and HDAC4 (RR).
  • SFN decreases miR-21 and TERT (R).

Sulforaphane induces cell death (apoptosis) in cancer:

  • SFN activates caspase-3, caspase-7, caspase-8, caspase-9 (RR).
  • SFN decreases anti-apoptotic Bcl-2 (R) and Bcl-XL (R).
  • SFN increases pro-apoptotic Bax (R).
  • SFN induces p21 (CDKN1A) (R) and p53 (R).
  • SFN inactivates PARP (R).
  • SFN decreases HIF1A (R).
  • SFN decreases β-catenin (CTNNB1) (R).

More on Sulforaphane and Cancer

The mechanisms of SFN effects on cancer cells have been well studied. It suppresses the proliferation of cancer cells via diverse mechanisms including cell-cycle arrest, apoptosis induction, ROS production, and manipulation of some signaling pathways (166). SFN inhibits proliferation of PC-3 cells in culture in concentrationand time-dependent manner. Singh et al. (167) showed that oral administration of SFN led to >50% reduction in PC-3 xenograft tumor volume in SFN-treated mice in 10 days and more than 70% reduction in 20 days after starting treatment with no effect on body weight.

They also reported that SFN changes the Bax: Bcl-2 ratio, activates caspases 3, 8, and 9, and cleaves and inactivates PARP protein. The authors proposed that SFN induces apoptosis in PC-3 xenograft tumors in a p53-independent manner through cytoplasmic and mitochondrial pathways. Liquid chromatography–mass spectrometry (LC-MS) analyses performed by Rose et al. (17) showed the presence of 7-methylsulphinylheptyl isothiocyanates in watercress (Rorippa nasturtium aquaticum) extract and 4-methylsulfinylbutyl nitrile and 4-methylsulfinylheptyl isothiocyanates in the broccoli extract. Their investigations showed that these compounds contribute to the inhibitory effects of broccoli and watercress extracts on the invasion of MDA-MB-231 cancer cells through suppression of MMP-9 activity.

Treatment of HEK293 cells with different concentrations of SFN with and without TSA, as a HDAC1 inhibitor, leads to the increase in TOPflash reporter activity without affecting b-catenin protein levels. Further studies showed that this increase is due to the decrease in HDAC activity and consequently the increase in histone acetylation following SFN treatment (168).

It has been demonstrated that mamosphere formation in breast cancer cells is dependent on E-cadherin expression (168). It is showed that SFN could target breast cancer stem cells. The mammosphere formation test on two cancer cell lines, MCF7 and SUM195, indicated that SFN could reduce the proportion of cell with stem cell properties, and this was further supported by ALDEFLUOR assay. In vivo examination results of SFN effects on xenograft SUM159 cells in NOD/SCID mice were consistent with the in vitro results. More importantly, cells derived from SFN-treated primary tumors could not produce secondary tumors, while cells derived from the nontreated primary tumors rapidly produced the secondary tumors in the contralateral mammary fat pad of the same mice (168).

Aldehyde dehydrogenase activity is a stem cell marker for enriching tumorigenic stem/progenitor cells (169,170). Five mmol/L of SFN led to >80% reduction of ALDH-positive SUM159 cells in vitro, and daily treatment of xenograft of SUM159 tumors with 50 mg/kg of SFN for 2 weeks led to 50% reduction in tumor size through the reduction in ALDH-positive SUM159 cells by 50%, with no effect on body weight (171). ApcMin/C mice consumed SFN in their diet have fewer tumors with lower sizes in comparison with a control group, albeit, immunohistochemical (IHC) staining revealed that the b-catenin expression was not affected by SFN consumption (172).

Furthermore, the effect of SFN treatment on selfrenewal contributing to signaling pathway, Wnt pathway, was examined by analysis of b-catenin and some other downstream genes at mRNA and/or protein levels (171).
Treatment of T24 bladder cancer cells with SFN results in induction of miR-200c expression (173).

Previous studies demonstrated that miR-200c targets the E-cadherin repressors ZEB1 and ZEB2. Ectopic expression of miR-200c resulted in upregulation of E-cadherin in cancer cells (174). Therefore, treatment of T24 cells with SNF led to E-cadherin induction and EMT suppression (173). However, it seems that these results depend upon cell type and treatment conditions. Although clinical trials seem necessary, there is a large body of investigations about anticancer effects of SFN, and the explicit point is that SFN inclusion into the diet promises a safe and confident strategy.

Another active ingredient of broccoli and other cruciferous vegetables is Indole-3-carbinol (I3C) that has anticancer effects too. Meng et al. (175,176) reported despite a somehow prohibiting effect of I3C on cell attachment in vitro, and I3C could also suppress the invasion and motility of cells. The effect of I3C on cellular metastasis was also evaluated by injecting treated cells into the tail vein of mice and tracing surface metastasis in the lung of the sacrificed animal. Their results indicated that I3C treatment reduced the metastatic capability of the cells.

Bioavailability and new biomarkers of cruciferous sprouts consumption

There are epidemiological evidences of the benefit of consuming cruciferous foods on the reduction of cancer risk (Royston & Tollefsbol, 2015), degenerative diseases (Tarozzi, et al., 2013) and the modulation of obesity-related metabolic disorders (Zhang, et al., 2016), after cruciferous intake. Cruciferous sprouts are especially rich in bioactive compounds compared to the adult plants, due to their young physiological state, being an excellent choice for consuming healthy fresh vegetables (Pérez-Balibrea, Moreno, & García-Viguera, 2011; Vale, Santos, Brito, Fernandes, Rosa, & Oliveira, 2015). The highest benefit of cruciferous foods occurs when are consumed fresh, as young sprouts, avoiding degradation of the enzyme myrosinase by cooking, which is necessary to hydrolyse their characteristic sulphur and nitrogen compounds, the glucosinolates (GLS), to the bioactive isothiocyanates (ITC) and indoles. In case of sprouts, the degradation of GLS after consumption occurs during chewing, in presence of the plant’s enzyme myrosinase, and also is mediated by β-thioglucosidases in the gut microbiota (Angelino & Jeffery, 2014).

There is growing evidences that ITC, such as sulforaphane (SFN) and sulforaphene (SFE), as well as the indole-3-carbinol (I3C), play antioxidant, anti-inflammatory and multi-faceted anticarcinogenic activities in cells (Stefanson & Bakovic, 2014), through the in vivo inhibition of the activation of the central factor of inflammation NF-κB (Egner, et al., 2011), and the induction of the Keap1/Nrf2/ARE pathway related with antioxidant genes and detoxifying enzymes, such as glutathione S-transferases (GST) (Baenas, Silván, Medina, de Pascual-Teresa, García-Viguera, & Moreno, 2015; Myzak, Tong, Dashwood, Dashwood, & Ho, 2007), and also blocking carcinogenic stages in vitro and in vivo by induction of apoptosis, cell cycle arrest and inhibition of histone deacetylases, among others finally, metabolised in the liver with N-acetyl-L-cysteine (-NAC). During the last years, some conjugated ITC, such as SFN-NAC, and other secondary compounds, such as 3,3’diindolylmethane (DIM), which is released by I3C in acid medium (i.e. the stomach), have been used as biomarkers of cruciferous intake (Angelino & Jeffery, 2014; Fujioka, et al., 2016a). However, the bioavailability of the GLS glucoraphenin (GRE) and its isothiocyanate SFE, from radish sprouts, which only differ from SFN in a double bond between the third and fourth carbon (see Figure 2 in section 3), have not been yet investigated.

To our concern, there are no publications studying the bioavailability of radish sprouts compounds, and it is unknown if SFE is metabolised also by the mercapturic acid pathway. Furthermore, there are no commercially available conjugated metabolites of SFE, which would be needed for study its bioavailability by a rapid and sensitive UHPLC-QqQ-MS/MS method, stablishing their appropriate ionization conditions and MRM transitions.

On the other hand, the presence of SFN has been described in radish (Pocasap, Weerapreeyakul, & Barusrux, 2013), maybe by the hydrolysis of GRE, or through a modification of SFE once formed to SFN, suggesting its possible transformation by the mercapturic acid pathway. Therefore, the aim of this study was to evaluate and compare the bioavailability and metabolism of GRA and GRE, from broccoli and radish sprouts, respectively, and the study of possible different biomarker profiles of broccoli and radish consumption for the first time. Also the urine profile evolution of ITC, indoles and conjugated metabolites, after consumption of both broccoli and radish sprouts, were evaluated in a 7 days-cross-over trial with 14 healthy adult women.

2. Material and methods
2.1. Plant material
Broccoli (Brassica oleracea var. italica) and radish (Raphanus sativus cv. Rambo) 8-day-old sprouts were supplied by Aquaporins & Ingredients, S.L. (Murcia, Spain). These sprouts were bioestimulated during production (4 days previous to delivery) with the natural compound methyl jasmonate 250 μM (Baenas, Villaño, García-Viguera, & Moreno, 2016), in order to obtain cruciferous sprouts up to 2-fold richer in bioactive compounds. Three trays of sprouts

(n=3) were collected once a week during the study, then, samples were flash frozen and lyophilised prior analysis of GLS and ITC (see 2.3. section), through an hydro-methanolic (Baenas, Garcia-Viguera, & Moreno, 2014) and aqueous extraction (Cramer & Jeffery, 2011), respectively, as previously described.

2.2. Human subjects and study design. 

A total of 14 women, aged 27-36 years, non-smokers with stable food habits and not receiving medication, during the experimental procedure, were selected to participate in the study. Due to the sex-related disparities in pharmacokinetics and bioavailability results reported by different authors (Soldin, Chung, & Mattison, 2011; Soldin & Mattison, 2009), the choice of a single genus was chosen to avoid a high dispersion in the data, and the availability of healthy young adult female volunteers for the study. Written informed consent was obtained from all subjects. The present study was conducted according to guidelines and procedures approved by the CSIC Committee of Bioethics for the AGL-2013-46247-P project. Subjects were randomly assigned to a seven-by-seven cross-over design (Figure 1), one group receiving broccoli sprouts and the second receiving radish sprouts. Nobody dropped out of the study. A list of foods containing glucosinolates was given to all the participants in order to avoid consumption during the study. Experimental doses (7 trays of broccoli or radish sprouts of 20 grams each) were given at once, on Friday. Subjects were instructed to ingest 1 tray per day, at 10 a.m., according to the cross- over design and to keep trays refrigerated (4 °C) at home. The first day of the study, the urine samples were collected from 0 to 12 h, and from 12 to 24 h after ingestion. From day 2 to 7, the urine samples were collected in 24 h periods. All urine samples were kept refrigerated during collection and were frozen upon reception in the laboratory.

2.3. Metabolites analysis 

The quantitative analysis of GLS in sprouts was carried out by HPLC-DAD 1260 Infinity Series (Agilent Technologies, Waldbronn, Germany), according to UV spectra, and order of elution already described for similar acquisition conditions (Baenas, et al., 2014). For samples preparation, briefly, freeze-dried sprouts (50 mg) were extracted with 1mL of methanol 70% V/V in a US bath for 10 min, then heated at 70°C for 30 min in a heating bath and centrifuged (17500 xg, 15min, 4°C). Supernatants were collected, and methanol was completely removed using a rotary evaporator. The dry material obtained was dissolved in 1 mL of ultrapure water and filtered through a 0.22 μm Millex-HV13 filter (Millipore, Billerica, MA, USA). Measurement of metabolites in sprouts and urine (GRA, SFN, SFN-GSH, SFN-CYS, SFN- NAC) was performed following their MRM transition by a rapid, sensitive and high throughput UHPLC-QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) method, with modifications of the protocol of Dominguez-Perles et al. (2014), for the optimization of new compounds: GRE, SFE, glucobrassicin (GB), I3C, DIM; assigning their retention times, MS fragmentation energy parameters and preferential transitions (Supplemental File 1). The urine samples were centrifuged (11,000g, 5 min) and the supernatants (400 μL) were extracted using SPE Strata-X cartridges (33u Polymeric Strong Cation), following the manufacturer’s instructions (Phenomenex, Inc., Madrid, Spain), and the slight modifications of Dominguez- Perles et al., (2014). Briefly, the cartridges were conditioned and then aspirated until dryness. The target analytes were eluted with 1 mL of MeOH/formic acid (98:2, v/v) and dried completely using a SpeedVac concentrator (Savant SPD121P; Thermo Scientific, Waltham, MA). The extracts were reconstituted with 200 μL of mobile phase solvent A/B (90:10, v/v) previously to their UHPLC/MS/MS analyses. GRA and GRE were obtained from Phytoplan (Diehm & Neuberger GmbH, Heidelberg, Germany) and ITC and indoles were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). No commercially available conjugated metabolites of SFE from radish were found.

2.4. Statistical analysis 

All assays were conducted in triplicate. Data were processed using SPSS 15.0 software package (LEAD Technologies Inc., Chicago, IL, USA). First of all, data were tested by Shapiro-wilk normality test, as these values do not follow a normal distribution (non-parametric data), statistical differences were determined by the Wilcoxon signed-rank test when comparing two samples and by the Friedman test when comparing multiple samples. Values of P<0.05 were considered significant.

3. Results and discussion
3.1. Bioactive compounds present in cruciferous sprouts
Broccoli and radish sprouts from each week of the study were characterised in GLS and ITC (Table 1). Results are presented as commercial serving dose (20 grams of fresh weight (F.W.)). The amount of cruciferous sprouts consumed daily by the participants was considered a normal serving according to the EFSA (2009), which consider that there is no a perfect way of measuring habitual intake, however, the portion size should be convenient for the use in the context of regular dietary habits.
In broccoli sprouts, glucoraphanin (GRA), in the hydromethanolic extract, and its hydrolysis compound sulforaphane (SFN), in the aqueous extract were the predominant compounds, according to previous findings (Angelino & Jeffery, 2014; Cramer & Jeffery, 2011).
Results showed that radish sprouts presented glucoraphenin (GRE) and glucoraphasatin (GPH) as predominant GLS (Table 1), which were hydrolysed to sulforaphene (SFE) and raphasatin (RPS), respectively. Only SFE was detected in the aqueous extract, as RPS is very unstable and rapidly degraded to less bioactive compounds in aqueous media, such as raphanusanins, E- and Z-3-methylsulfanylmethylene-2-pyrrolidinethiones and E-4-methylsulfanyl-3- butenyldithiocarbamate (Kim, Kim, & Lim, 2015; Montaut, Barillari, Iori, & Rollin, 2010). GRA was not detected in radish sprouts, but the hydrolysis product SFN was present in the aqueous extract (Pocasap, et al., 2013). This could be due to the formation of SFN derived from SFE, losing its double bond, or directly hydrolysed from GRE (Figure 2). Forthcoming evaluations of GRE hydrolysis under different conditions would provide more information about its possible transformations.

3.2. Bioavailability and metabolism of GLS/ITC 

After ingestion of a serving portion of broccoli sprouts (20 g F.W.), GRA (64 μmol) was hydrolysed, absorbed and metabolised, through the mercapturic acid pathway, by a 12 % on average. SFN and its conjugated metabolites, with glutathione (-GSH), cysteine (-CYS) and N- acetyl-L-cysteine (-NAC) (Angelino & Jeffery, 2014), were found in urine (~7.6 μmol/ 24 h as the sum of SFN, SFN-GSH, SFN-CYS and SFN-NAC) (Figure 3A), considered markers of bioavailability.

In the case of radish sprouts, the metabolism of SFE, the predominant ITC, has not been described to the present date, and there are not any conjugated SFE metabolites commercially available to evaluate its bioavailability, by optimization of MRM-transitions in UHPLC-QqQ- MS/MS system. Therefore, it was hypothesised that conjugated SFN metabolites could be found also in urine after radish sprouts consumption, being also possible biomarkers of intake.

Results showed that GRE (61 μmol in 20 g F.W.) was metabolised in SFE, SFN and SFN metabolites (SFN-NAC, SFN-CYS, SFN-GSH), corresponding to 8% on average (~4.9 μmol/24 h) of the GRE consumed (Figure 3B). Therefore, SFN metabolites (SFN-NAC, SFN- CYS, SFN-GSH) could act also as biomarkers of radish consumption. In this sense, this analysis method allowed us to evaluate radish compounds bioavailability, in addition to differentiate it from the bioactives in broccoli, as well as other cruciferous foods, finding intact SFE metabolite as characteristic biomarker of radish consumption.

The values of bioavailability ranged from 9 to 100 % according to different GLS/ITC profiles of the cruciferous vegetables administered, and the consumption as raw or cooked foods and the influence of the microbiota (Shapiro, Fahey, Wade, Stephenson, & Talalay, 1998; Vermeulen, Klopping-Ketelaars, van den Berg, & V aes, 2008). In our case, after broccoli sprouts consumption, the SFN-NAC was the predominant metabolite found in urine (~80 %), followed by SFN-CYS (~11 %), SFN (7.5 %), and SFN-GSH (~0.9 %) (Figure 3A), as previously found (Clarke, et al., 2011; Dominguez-Perles, et al., 2014). In the case of radish sprouts, the SFE was excreted in higher amounts (~65 %) (Figure 3B), followed by the conjugated metabolites: SFN- NAC (~19 %), SFN-CYS (~4%), SFN (~1.1%) and SFN-GSH (~0.7%). SFN and SFE present the isothiocyanate group (−N=C=S), which central carbon is highly electrophilic and actively interacts with cellular nucleophilic targets; such as, the GSH and/or cysteine residues (Kim, Kim, & Lim, 2010). Little information is available about SFE bioactivity and, only recently, Byun, et al., (2016) showed that SFE reduced the cellular GSH levels in vitro, which could indicate its conjugation with GSH. Thus, the synthesis of SFE conjugates with GSH, CYS or NAC could help to generate knowledge on the metabolism of this compound.

On the other hand, the higher excretion of pure SFE after radish consumption compared to pure SFN after broccoli ingestion, may suggest that this compound could follow a different transformation pathway. Contrary to SFN, SFE contains a double bond between the third and fourth carbon, which could result in a decrease in the electrophilicity of the –N=C=S group (Kim, et al., 2010), and different excretion kinetics and transformation.

According to Holst & Williamson (2004), human studies about Phase I metabolism may contribute considerably to understand the biotransformation of ITC and, consequently, the limit of their bioavailability and health-promoting effects. Therefore, further studies about SFE metabolism and bioactivity are needed to support the health-promoting activities of SFE, now insufficiently studied. For instance, Myzak, et al, (2006) showed differences in the bioactivity of SFN conjugated metabolites, as being SFN-CYS and SFN-NAC, significantly active as HDAC inhibitors, with cancer therapeutic potential in vitro and in vivo, but not for SFN and SFN-GSH (Myzak, Ho, & Dashwood, 2006). On the other hand, pure SFN and SFE have shown bioactivities, inhibiting growth of several colon cancer cells (Byun, et al., 2016). Therefore, whether SFE is metabolised by the mercapturic acid pathway or acting in the cell without transformation, this ITC may provide health-promoting effects through induction of detoxification enzymes and antioxidant activity, which did not appear to be affected by their hydrophilicity or other structural factors (La Marca, et al., 2012).

3.3. Urine profile evolution of SFN, SFE and their metabolites. 

The values of individual metabolites excreted, analysed in urine samples, were represented according to two different criteria: 1) the levels of excretion during 24 h were normalised, first from 0 to 12 h after consumption and then from 12 to 24 h, using creatinine as an index to which refers the results, since the creatinine excretion is a relatively constant value between subjects; 2) when comparing the daily excretion during 7 days, the data were represented in volumes collected every 24 h.

The excretion of SFN and its conjugated metabolites, as well as SFE after radish sprouts ingestion was higher during the first 12 h after consumption of sprouts (Figure 4). It has been described that urinary excretion of SFN metabolites after consumption of fresh broccoli reaches peak concentration 3-6 h after consumption, but it could be delayed until 6-12 h (Vermeulen, et al., 2008). After broccoli sprouts ingestion, non-significant differences were found (Figure 4A) between the two periods. In contrast, significant differences were shown in the values of excretion after radish sprouts consumption in all metabolites except for SFN-GSH and SFN- CYS (Figure 4B). The delayed excretion of metabolites, after broccoli sprouts consumption, could be related to a saturation of the membrane transporters (such as P-glycoprotein) in the cells, as SFN is conjugated with GSH and cysteinylglycine in the cells and exported after protein binding, being available for metabolism and excretion (Hanlon, Coldham, Gielbert, Sauer, & Ioannides, 2009). SFE is mostly excreted during the first 12 h after ingestion of radish sprouts (Figure 4B), suggesting that it might be not subjected to the mercapturic acid metabolism. Nevertheless, the biological activity of the pure ITC has shown to be similar than the N -acetylcysteine conjugates (Tang, Li, Song, & Zhang, 2006), being of great interest either if are metabolised or not.

The levels of excretion after 7 days of ingestion (Supplemental files 2 and 3) were also studied and no differences were found in the median daily excretion of SFE, SFN and its metabolites in both broccoli and radish studies. This suggests that repetitive dosing of sprouts should not produce accumulation of any metabolite in the body, as any factor that increases the metabolites amount in the body will cause a decrease in its excretion (Hanlon, et al., 2009). Also, there is a high interindividual variation in excretion values related to human bioavailability studies, which could be explained by different factors, such as the intensity of chewing, where myrosinase enzymes come into contact with intact GLS, gastric pH, intestinal transporters and the activity of the microbiota, where one subject could metabolize three times more GLS into ITC than another, and also the polymorphisms of GST enzymes may affect ITC metabolism (Clarke, et al., 2011; Egner, et al., 2011; Fujioka, Fritz, Upadhyaya, Kassie, & Hecht, 2016b). Low amounts of intact GRA and GRE, on average 0.011 and 0.04 μmol/24 h, respectively, were also recovered in the urine.

One of several challenges in the design of clinical trials is the selection of the appropriate dosage. In this work, commercial trays of sprouts were used, facilitated and quality certified by the company. The average amount of sprouts (~ 20 g per tray) was chosen as one serving, representing a realistic dietary supply. Different specific dietary intervention studies have estimated that the consumption of 3-5 servings per week of cruciferous foods (broccoli, red cabbage, Brussels sprouts, among others) may produce upregulation of detoxification enzymes, responsible for clearance of chemical carcinogens and ROS (Jeffery & Araya, 2009). Therefore, the daily consumption of cruciferous sprouts may result in potential effects decreasing the risk for cancer, even though further epidemiological trials and in vivo studies testing broccoli and radish sprouts are necessary to further understand these effects.

3.4. Bioavailability, metabolism and urine profile evolution of GLS/indoles 

Glucobrassicin (GB) present in broccoli and radish sprouts is an indole GLS derived from tryptophan and releases bioactive indole-3-carbinol (I3C) upon hydrolysis. This bioactive compound requires acid modification in the stomach to form 3,3’-diindolylmethane (DIM) and other condensates to optimize activity, increasing levels of Phase II enzymes, related to detoxification against lung, colon and prostate cancers (Egner, et al., 2011), and the antiproliferative effects on estrogenic-sensitive tumours (Fujioka, et al., 2016a). In particular, DIM has been associated with the suppression of epigenetic alterations related to carcinogenesis, by suppression of DNA methylation and aberrant histone modifications (Fujioka, et al., 2016b).

Additionally, the induction of Phase I enzymes, including the CYP 1 family, catalysing the oxidation of xenobiotics may also be responsible of the action (Ebert, Seidel, & Lampen, 2005; Watson, Beaver, Williams, Dashwood, & Emily, 2013).
Because of the rapid hydrolysis of I3C to DIM in vivo, high stability of DIM, and the strong correlation between GB intake and the amount of DIM excreted, this compound has been described as a biomarker of cruciferous vegetables consumption (Fujioka, et al., 2014). Other condensated compounds such as indol-[3,2-b]-carbazole and related oligomers, were in non- quantifiable concentrations in other studies (Reed, et al., 2006).

Little is known about the bioavailability of other indole GLS present in broccoli and radish sprouts, such as hydroxyglucobrassicin (HGB), methoxyglucobrassicin (METGB) and neoglucobrassicin (NEOGB), which might be also hydrolysed leading indolyl-3-methyl isothiocyanates, unstable and hydrolysed to their corresponding carbinols (Agerbirk, De Vos, Kim, & Jander, 2008; Hanley & Parsley, 1990).

Additional studies are required to confirm if these indole GLS could be hydrolysed also in I3C and DIM, as well as to evaluate the possible health-promoting effects of their hydrolysis and condensate metabolites.
Results demonstrated that broccoli and radish sprouts content in GB were ~11.4 and ~7.7 μmol/20 g F.W, respectively. After ingestion of broccoli sprouts, 49 % of GB was suitably metabolised and excreted as hydrolysis metabolites, calculated as the sum of I3C and DIM (~5.57 μmol /24 h). Following radish ingestion, the percentage of GB hydrolysed and absorbed was 38 % (~2.92 μmol /24 h). It is remarkable that the DIM excreted correspond to over 99 % of these total metabolites. These results of bioavailability contrast with the extremely low percentage (< 1 %) of GB excreted as DIM after consumption of Brussels sprouts and cabbage in a previous study (Fujioka, et al., 2014). Nevertheless, results show relevant bioavailability of GB and the successful use of DIM as biomarker of cruciferous intake. Further studies about conversion of other indole GLS to I3C and DIM are needed to know more about bioavailability of these compounds, as there is no information in literature.

When urine samples were collected in two periods after the ingestion of the sprouts, higher values of excretion of I3C from 12 to 24 h than from the first period were detected (Figure 5), although non-statistically different. Regarding excretion values of DIM, no differences among results were found from 0 to 12 h and from 12 to 24 h (Figure 5). Even if a previous study in humans has shown that the majority of DIM was excreted in the first 12 h (Fujioka, et al., 2016b), other authors have detected DIM in plasma at 12 and 24 h post ingestion (Reed, et al., 2006). Therefore, the excretion of this compound might be longer than for the ITC, which were almost totally excreted during the first 12 h. According to these results, in vivo evidences show that I3C condensation products were absorbed, preferentially targeting the liver, and were detected within the first hour in urine. However, the amount increased significantly between 12 and 72 h, implying effects on the xenobiotic metabolism (WHO, 2004).

No statistical differences were found in the 24 h urine excretion values of I3C and DIM after 7- days of consumption of sprouts (Supplemental File 3). The high variability between subjects has been described before within a low dose level of GB consumed (50 μmol), which was considerably higher than in this study (Fujioka, et al., 2016a).

Furthermore, the results showed no accumulation of metabolites after 7 days of intervention, an important result for safe consumption, also proven with hyper-doses of GB (400-500 μmol) in humans (Fujioka, et al., 2016a).
Low amounts of intact GB (~0.011 and ~0.04 μmol/24 h, after broccoli and radish ingestion, respectively) were also recovered in the urine, but the biological activities of I3C, administered orally to humans, cannot be attributed to the parental compound but rather to DIM and other oligomeric derivatives (Reed, et al., 2006). Therefore, the evaluation of DIM in urine after broccoli and radish consumption provided a susceptible tool to design future clinical trials.

4. Conclusions 

The measurement of ITC, indoles and conjugated metabolites are useful biomarkers of dietary exposure to cruciferous foods. The SFN-NAC is not the only metabolite present in urine and, as along with DIM, could be used as biomarker of the consumption of cruciferous vegetables.

After ingestion of radish sprouts, SFE, together with SFN-NAC and DIM, could be considered as biomarkers, however, metabolites of SFE are not commercially available yet and, consequently, understudied. Repeated dosing of sprouts does not lead to accumulation or higher urine levels of metabolites over time. Furthermore, human short-term pharmacokinetics (e.g. 48 – 72 h) as well as long-term intervention studies (e.g. 10 – 12- weeks) using different doses, and collecting urine and plasma (several time points), as well as faecal samples (microbial metabolites and potential beneficial changes in the gut/colonic microbiota), are strongly recommended for future research in this area.