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.

Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions

This study is published here

INTRODUCTION
The world is in health transition, and obesity is greatest threat to human health (Jose Hernandez–Morante et al., 2011). Obesity associates with various lifestyle-related diseases, such as diabetes, cardiovascular disease, hypertension and fatty liver disease (Jebb and Moore, 1999; Nakamura et al., 1994), causing a major health burden in terms of morbidity and mortality (Sturm, 2007). Although there has a few drugs in market to ameliorate or prevent obesity, costs and side effects limited their application (Chuah et al., 2013). It is well known that anti-obesity food ingredients could control and reduce body weight (Kim et al., 2008b).

Garcinia Cambogia extracts are found in northeastern India and Andaman Islands, and it has been extensively used for centuries throughout Southeast Asia as a food preservative, flavoring agent, and carminative (Jena et al., 2002).

Garcinia cambogia extracts is now popularly used as an ingredient of dietary supplements for weight loss (Saito et al., 2005), anti-obesity (Kim et al., 2008a; Kim et al., 2004), hypolipidaemic (Altiner et al., 2012) and anticancer activity (Mazzio and Soliman, 2009).

(-)-Hydroxycitric acid (HCA) is the major active ingredient present in the fruit rind of Garcinia cambogia (Jena et al., 2002; Marquez et al., 2012). The Garcinia cambogia extracts contains approximately 10~30% (-)-HCA, which can be isolated in the free form (Lewis and Neelakantan, 1965). Previous studies had identified (-)-HCA as a potent competitive inhibitor of adenosine triphosphate-citrate lyase (Watson et al., 1969), which is an extra-mitochondrial enzyme catalyzing the cleavage of citrate to oxaloacetate and acetyl-CoA (Watson and Lowenstein, 1970). (-)

Hydroxycitric acid reduced the availability of acetyl coenzyme A for the fatty acids and cholesterol synthesis (Watson et al., 1969). Many studies had reported that supplemental with (-)-HCA can promote weight reduction through suppressing de novo fatty acid synthesis, increasing lipid oxidation and reducing food intake (Chuah et al., 2013; Downs et al., 2005).

Recently, our laboratory also certified that Garcinia cambogia extracts could attenuate fat accumulation through regulating lipolysis gene expression by affecting adiponectin-AMPK signaling pathway in rat obesity model induced with high-fat diet (Liu et al., 2015).

(-)-Hydroxycitric acid is structurally similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002). Thus, (-)-HCA supplementation is expected to alter metabolic pathways. Amino acids are the fundamental building blocks of proteins (Jiang et al., 2014). In body, amino acids can be transformed to α-ketoglutarate by transamination reactions associated with multiple metabolic pathways such as glycolysis, gluconeogenesis, and the citric acid cycle (TCA) (Fauth et al., 1990).

Previous study showed that α-ketoglutarate is not only a key intermediate in the TCA cycle but also can replenish the cycle in anaplerotic reactions (Fink, 2008). Thus, amino acids are involved in protein synthesis, the energy production, gluconeogenesis, and lipogenesis (Wang et al., 2013). In addition, some amino acids are important for body development because they are the precursors of hormones.

It had been shown that phenylalanine and tyrosine are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Also, it had reports that insulin and growth hormone enhance amino acid uptake and protein synthesis, meanwhile the increase of amino acid contents stimulates glucagon secretion that promotes rapid conversion of amino acids to glucose (Rhoades and Rhoades, 2012).

Although dietary supplements of Garcinia cambogia extracts may be a practical way to reduce excessive fat accumulation in human or animal production, the precise physiological mechanism of HCA has not yet been fully clarified. Therefore, present study was conducted to investigate the effect of a long-term Garcinia cambogia extracts supplement on body weight gain, energy metabolism and the changes of amino acids content in serum, liver, and muscle of rats.

To our knowledge, this is the first report to investigate the impact of Garcinia cambogia extracts on the amino acid profile in different tissue. Our results not only provided information about how Garcinia cambogia extracts exerts its action but also certified the use of Garcinia cambogia extracts to control body weight.

MATERIALS AND METHODS
Garcinia cambogia extracts. Garcinia cambogia extracts was purchased from An Ynn Co. Ltd (Zhengzhou, China). The Garcinia cambogia extracts contain 56.0% ~ 58.0% (-)-Hydroxycitric acid including its free and lactone form, and it also contains 12.0%~14.0% cellulose, 5.5%~6.0% α-Dmelibiose, 2.5% ~ 3.0% β-D-lactin, 1.5% ~ 2.0% D-mannopyranose, 11%~12% oxophenic acid, 2.0% ~3.0% octadecyl alcohol, 3.5%~4.0% Coenzyme A, and 1.5% ~ 2.0% inorganic elements.

Animals and diets. Five-week old male Sprague– Dawley (SD) rats weighing 200 ± 20 g were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (China). Rats were housed individually under constant temperature of 25 ° C and humidity of 50% ~ 60% and maintained on a 12:12 h light/dark cycle. All animal handling procedures were performed in strict accordance with guidelines established by Institutional Animal Care and Use Committee of Nanjing Agricultural University.

Before initiation of experiment, rats were acclimatized to the environmental conditions for 1 week. A total of 60 rats were randomly assigned to one of four groups: control group, low dose of (-)-HCA-treated group, medium dose of (-)-HCA-treated group, and high dose of (-)HCA-treated group.

Rats were supplemented with Garcinia cambogia extracts at 0, 25, 50, and 75 g/kg diet, and the contents of Garcinia cambogia extracts were equivalent to 0, 1000, 2000, and 3000 mg/kg diet of (-)HCA level. Rats were fed ad libitum with free access to water for 8 weeks, diet was removed for the last 12 h of the experimental term, and then the rats were anesthetized with ether and scarified by decapitation. Rats were weighed at the beginning and the end of experiment to determine average daily gain.

Daily feed consumption per day was recorded; the average daily feed intake and feed conversion ratio were then determined. At the end of the experiment, blood samples were allowed to clot at 4 °C and centrifuged at 1520 × g for 20min before harvesting the serum. Then, the serum, liver, and muscle samples were collected and kept at 70 °C until further analysis. Liver was weighted to determine the liver index, which indicated by the liver weight (mg)/body weight (g).

Measurement of serum glucose and glycogen content.
Serum glucose (catalog#: F006), hepatic glycogen (catalog#: A034), and muscle glycogen contents (catalog#: A034) were measured using commercial kits according to the manufacturers’ protocol (Nanjing Jiancheng Biotechnology Institution, Nanjing, China).
Measurement of protein content in liver and muscle.
Protein content in liver and muscle was measured using commercial kits (catalog#: P0012), which were purchased from the Beyotime Biotechnology Institution Shanghai, China.

Measurement of serum hormone content.
Serum triiodothyronine (T3, catalog#: A01PZC), thyroxine (T4, catalog#: A02PZC), insulin (catalog#: F01PJB), glucagon (catalog#: F03PJB) and Leptin (catalog#: C16DJB) contents were measured using Radioimmunoassay Kit according to the manufacturers’ protocol (Beijing North Institute of Biotechnology, Beijing, China). The intracoefficients of variation for all hormones detection kit were less than 10% and inter-coefficients of variation were less than 15%.

Analyses of amino acids profile by high pressure liquid chromatography
Instrumentation and reagents. High pressure liquid chromatography (HPLC) analyses were carried out on a benchtop Agilent1100 series LC chromatographic system (Agilent Technologies, Waldbronn, Germany) equipped with a vacuum degasser, autosampler, thermostated column compartment, quaternary pump and a diodearray detector. The chromatographic column (XTerra®MS C18, 5 μm, 4.6 × 250 mm) was purchased from Waters (Waters Co., Milford, MA, USA).

The standards of alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine(Gly), glutamine (Gln), leucine (Leu), valine(Val), tryptophan (Trp), phenylalanine (Phe), arginine (Arg), asparagine (Asn), threonine (Thr), tyrosine (Tyr), isoleucine (Ile), serine (Ser), methionine (Met), proline (Pro), cysteine (Cys), histidine (His), and lysine (Lys) were purchased from Sigma. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Tedia Company Inc. (Fairfield, OH, USA). Tetrahydrofuran (HPLC grade) and Ophthaldialdehyde (OPA) were purchased from Merck KGaA (Darmstadt, Germany).

High-purity water was prepared from a Milli-Q gradient water purification system (Millipore, MA, USA) and was used for all protocols in this study.

Chromatographic conditions.
High pressure liquid chromatography was performed according the method described by Shen et al (Shen et al., 2010). Briefly, a ternary system was used as mobile phase: solvent A is methanol, solvent B is acetonitrile, and solvent C is 10 mmol · L 1 Na2HPO4–NaH2PO4 (pH = 7.2, containing 0.3% tetrahydrofuran). A gradient elution program is mobile phase A, B, and C is 9%, 6%, and 85% for 10min, and then change to 12%, 8%, and 80% for 25 min, finally 15%, 15%, and 70% of mobile phase A, B, and C was used for another 25min. Flow rate: 1.0 mL · min 1. Fluorescence: excitation wave length = 340 nm and emission wavelength of 450 nm. Oven temperature: 40 °C. Injection volume: 20 μL.

Samples prepared and analyzed. Approximately 100 mg of liver and muscle was homogenized on ice with 1 mL of saline, and then centrifuged at 3000 g for 15 min before harvesting the supernatant. One hundred microliter of serum or tissue supernatant samples were mixed with 200 μL acetonitrile for 30 min at room temperature and then centrifuged at 12000 g for 30 min to harvest the supernatant for HPLC analysis. High pressure liquid chromatography analysis was performed after automatic pre-column derivatization with O-phthaldialdehyde (OPA) according the method described by Zeng et al.

(Zeng et al., 2013). Briefly, 20μL samples were mixed with 40 μL OPA-solution for 2 min at room temperature, and then 20 μL of the mixture was loaded into column. For identification purposes, the amino acid standards were used by spiking the samples, as well as by comparing the relative retention time. For quantification purposes, calibration curves using external standard methodology were performed. For recovery calculations, peak areas obtained from each sample was compared with the peak areas of standard used for spiking (Di Pierro et al., 2000; Uhe et al., 1991).

Statistical analyses.
Data were analyzed using the Statistical Package for Social Science (SPSS Inc., Chicago, IL, USA) and expressed as mean values ± SE. Treatment differences were subjected to a Duncan’s multiple comparison tests, and the differences were considered significant at P < 0.05 RESULTS
Effect of (-)-HCA on body weight and feed intake in rats
Compared with the control group, the body weight gain was significantly decreased in 2000 mg/kg (-)-HCA treatment group (P < 0.05) (Fig. 1A). No significantly differences were observed on the feed intake (p > 0.05)
No statistical differences were observed on the serum glucose content in (-)-HCA treatment groups at different doses compared with the control group (p>0.05) (Fig. 2A). The hepatic glycogen (Fig. 2B) and muscle glycogen (Fig. 2C) contents were significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05). These results indicated that (-)-HCA supplement could promote the glycogen synthesis in rats. Effect of (-)-HCA on protein content in rats As shown in Fig. 3, the protein contents in liver (Fig. 3A) and muscle (Fig. 3B) were significantly increased in (-)-HCA treatment groups at three doses compared with the control group (p<0.05). These results indicated that (-)-HCA supplement could enhance protein synthesis in rats. Effect of (-)-HCA on serum metabolic hormone content in rats
A significantly increase of serum T4 content was observed in 1000 mg/kg and 2000 mg/kg (-)-HCA treatment groups when compared with the control group (p < 0.05) (Fig. 4A). The T3 content was significantly higher in 1000mg/kg (-)-HCA treatment group than that in control group (p < 0.05) (Fig. 4B). No noticeable changes were observed on glucagon content (p>0.05) (Fig. 4D), while the insulin content was significantly increased in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups than that in control group (p < 0.05) (Fig. 4C). In addition, serum Leptin content was significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05) (Fig. 4E). These results indicated that (-)-HCA supplement might regulate the glucose metabolism by regulating serum metabolic hormones content in rats. Effect of (-)-HCA on amino acid profile in rats
A chromatogram of synthetic mixture of amino acid standards was shown in Fig. 5. Each peak represents one of specific amino acid, and 15 amino acids were separated (T<50min) under the experimental conditions used. As shown in Table 1, in 7.8125 ~ 500 μmol/L concentration range, amino acid standard concentration was linear related to the peak area and the correlation coefficients is 0.9983 ~ 0.9999. The Intra-day RSD and Inter-day RSD is between 0.47% ~ 2.37% and 1.68% ~ 5.50%, respectively, which are within 6%. In addition, the recovery rate of 15 amino acid standards was between 93.88% ~ 105.20%. These parameters results indicated that this sensitive procedure could be used for the quantitative analysis of amino acid in tissues. Amino acid content in serum
As shown in Fig. 6, most of the amino acid contents were higher in (-)-HCA treatment groups than that in the control group. Among glucogenic amino acid, the threonine content in 2000 mg/kg (-)-HCA treatment group (p < 0.05) and the threonine, arginine, alanine, valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.05) were significantly increased than that in the control group (Fig. 6A). Among aromatic amino acid, the tyrosine and phenylalanine contents were significantly increased in 3000 mg/kg (-)-HCA treatment group (p < 0.05) than that in the control group (Fig. 6B). Among branched amino acid, the valine and leucine contents were significantly increased in 3000 mg/kg (-)HCA treatment group (p < 0.05) than that in the control group (Fig. 6C). These results indicated that (-)-HCA supplement could increase the glucogenic amino acid, aromatic amino acid, and branched amino acid in serum of rats. Amino acid content in liver
Similarity, most of the amino acid contents were also higher in (-)-HCA treatment groups than that in the control group (Fig. 7). Among glucogenic amino acid, the asparagine and threonine contents in 1000mg/kg (-)-HCA treatment group (p<0.05) and the aspartic acid, glutamic acid, threonine, and valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased than that in the control group (Fig. 7A). Among aromatic amino acid, the tryptophan and phenylalanine contents in 1000 mg/kg (p < 0.05) and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly increased (Fig. 7B). Among branched amino acid, the isoleucine content in (-)-HCA treatment group at various doses (p < 0.05) and the valine and leucine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased (Fig. 7C). These results indicated that (-)-HCA supplement could increase amino acid contents, especially aromatic amino acid and branched amino acid, in the liver of rats. Amino acid content in muscle
Contrary to serum and liver, most of the amino acid contents were lower in (-)-HCA treatment groups (Fig. 8). Among glucogenic amino acid, the glutamic acid content in (-)-HCA treatment group at all dose (p < 0.05) and the valine and glutamine contents in 1000 mg/kg (-)-HCA treatment group (p < 0.05) and aspartic acid content in 2000mg/kg (-)-HCA treatment group (p < 0.05) were significantly decreased (Fig. 8A). Among aromatic amino acid, 1000 mg/kg (-)-HCA treatment significantly decreased tyrosine and phenylalanine contents in muscle (p < 0.05) when compared with the control group (Fig. 8B). Among branched amino acid, the valine content in 1000mg/kg (-)-HCA treatment group (p < 0.05) and the leucine content in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly decreased (Fig. 8C). These results indicated that (-)-HCA supplement could decrease amino acid contents, especially aromatic amino acid and branched amino acid, in the muscle of rats. DISCUSSION

Present results showed that diet supplement with (-)-HCA reduced the body weight gain in male rats, and 2000mg/kg (-)-HCA treatment significantly decreased the body weight gain. This observation was consistent with the study of Leonhardt et al. (2001), who reported that (-)-HCA could promote the body weight loss in male rats. Many studies demonstrate that (-)-HCA reduced the body weight gain in rats (Kim et al., 2008b), human (Marquez et al., 2012) and broilers (Liu et al., 2015), and suggesting feed intake inhibition maybe a major mechanism of how Garcinia cambogia extracts exerts its function in controlling body weight (Leonhardt et al., 2001).
Nevertheless, no differences was observed on feed intake in rats supplemented with (-)-HCA, and this results indicated that the inhibition effect of (-)-HCA on body weight gain is not mainly via regulating feed intake in male rats.

The results presented here were consistent with the previous study, which demonstrated that 2 weeks (-)-HCA supplement has no significant effects on appetite in human (Kovacs et al., 2001). In addition, it is reported that Garcinia cambogia leaf supplementation had no effect in male Ross 308 broiler chickens on feed intake in finisher stage (Sebola et al., 2011).

We presumed that the differences in the preparation of (-)-HCA and the different animal or rat strains used in those study could contribute to such discrepancy. As a weight loss agent, it had presume that an increased fatty acid oxidation and decreased fat accumulation in animal with (-)-HCA-treated contributed to decrease its weight (Sullivan et al., 1972).

In addition, our recently study demonstrated that Garcinia Cambogia extracts could attenuated fat accumulation and body weight gain through activating the Adiponectin-AMPK signaling pathway in rat obesity model induced by high-fat diet (Liu et al., 2015). Although we did not investigated the effect of (-)-HCA on fat accumulation in this study, taken our results and other reported, we think that suppression fat accumulation may be a major mechanism of weight loss by (-)HCA-induced.

It is well known that body weight gain depends on the balance between energy intake and energy expenditure. Previous study suggests that Garcinia cambogia extracts could enhance energy expenditure in rats (Vasselli et al., 1998), and the suppressive effect of Garcinia cambogia extracts on body weight gain might also depend on increased thermogenesis, except for the reduction in feed intake (Leonhardt et al., 2001).

Thyroid hormones, including triiodothyronine (T3) and thyroxine (T4), are recognized as the key metabolic hormones in body. Thyroid hormones essentially modulate all metabolic pathways through alterations in oxygen consumption and carbohydrate metabolism (Smith et al., 2002).

Serum thyroid hormones contents are associated with energy expenditure and other effects, such as lipid metabolism and protein synthesis (Hornick et al., 2000).

Present study showed that serum T4 content in 1000 mg/kg and 2000mg/kg (-)-HCA treatment groups and serum T3 content in 1000mg/kg (-)-HCA treatment group were significantly increased in rats. Leptin, the ‘satiety hormone’, is an important hormone that helps to regulate energy balance (Pan et al., 2014).

In present study, it was demonstrated that 1000 mg/kg (-)-HCA treatment significantly increased serum Leptin content in rats.

One of the major functions of Leptin is control energy balance by binding to receptors in hypothalamus, which results in the increase energy expenditure (Lerario et al., 2001; Watson et al., 2000). Thus, the above data suggested that one possible mechanism of (-)-HCA supplement reduced body weight gain via enhancing energy expenditure in rats, which might be associated with the increase of thyroid hormones and Leptin levels.

It had been reported that aromatic amino acids, including phenylalanine, tryptophan, and tyrosine, have important roles in body development because they are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Our results showed that (-)-HCA treatment significantly increased serum tyrosine and phenylalanine contents in rats, and the tryptophan and phenylalanine contents also significantly increased in liver. Tyrosine and phenylalanine are premise material for the synthesis of thyroid hormones, and up to 80% of the T4 is then converted to T3 by organs such as the liver, kidney, and spleen (Koehrle and Brabant, 2010).

The changes of aromatic amino acids content were consistent with the significant increase of serum T4 and T3 contents in rats after (-)HCA treatment. In addition, tryptophan is premise material for the synthesis of serotonin (Boopathi and Ramasamy, 2014); thus, an increase of serum tryptophan levels could affect food intake behavior (Lopez et al., 2015). Although the serotonin content was not detected in this study, the significant increase of tryptophan contents indirectly indicated that (-)-HCA treatment might increase the serotonin release and availability.

This results was also consistent with previous reports that shows the increase of serotonin content after (-)-HCA treatment might be the main reason for appetite suppression (Ohia et al., 2002; Preuss et al., 2004; Roy et al., 2004).
Taken together, these results further confirmed that (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormones levels.

(-)-Hydroxycitric acid inhibits ATP-citrate lyase and increases the cellular pool of citrate, which in turn inhibits glycolysis and thus redirects the carbon sources for glycogen production within the liver or muscle (Cheng et al., 2012; Shara et al., 2003). No changes were observed on the glucose content, while 1000 mg/kg (-)HCA treatment significantly increased the hepatic glycogen and muscle glycogen contents in rats. Our results is similar to previous study results that show glycogen levels in skeletal muscle are increased after (-)-HCA supplementation in animal models (Ishihara et al., 2000) or in human (Cheng et al., 2012). Our results showed that 2000 mg/kg and 3000 mg/kg (-)-HCA treatment significantly increased insulin content in rats. Insulin can promote the storage of glucose and inhibit lipolysis and gluconeogenesis (Bernard et al., 2013; Bernard et al., 2011).

It has been reported that treated with Garcinia cambogia extracts for 4weeks significantly increased plasma insulin content (Hayamizu et al., 2003). In addition, previous study certifies that (-)-HCA can inhibit phosphofructokinase, a key enzyme controlling glycolysis (McCune et al., 1989). Once glucose was absorbed, it is rapidly phosphorylated to glucose-6-phosphate and then converted into glycogen through the glycogen synthesis pathway or lactate through the glycolytic pathway. Although we did not measure phosphofructokinase activity in this study, the inhibitory action of (-)-HCA on phosphofructokinase that results in the inhibition of glycolysis is consistent with the higher glycogen content in liver and muscle reported in here.

Therefore, the mechanism that (-)-HCA could increase the glycogen content in liver and muscle might be due to its inhibitory effect on the glycolytic pathway, and this action might be related to its ability to increase insulin content in rats.

Our results showed that (-)-HCA treatment significantly increased the protein contents in liver and muscle, which indicated that administration of (-)-HCA could promote protein synthesis in rats. This was consistent with the feed conversion ratio, which was significantly increased in rats supplemented with (-)HCA. Amino acids are not only the fundamental building blocks for protein synthesis (Jiang et al., 2014); they are also used for energy dissipation or other metabolic purposes (Conceicao et al., 2003). (-)-HCA has a structure similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002).

Thus, the indirect inhibition of cytosolic pool of citrate by (-)-HCA and subsequent reduction in acetyl coenzyme A and oxaloacetate alter the citric acid cycle (TCA) that is expected to alter metabolic pathways. Importantly, oxaloacetate is not only an important intermediate of the TCA cycle but also the first designated substrate of the gluconeogenic pathways of all other cycle intermediates, glycerol, or amino acids (Homem de Bittencourt et al., 1993).

Thus, we presume that (-)-HCA supplement may alter metabolic directions of amino acids, which in turn promoted protein synthesis in rats. In the present study, most of amino acid contents in serum and liver were increased, while its content in muscle were decreased in rats supplemented with (-)-HCA. Amino acids are used in a variety of cellular metabolism pathways, such as provision of energy (Conceicao et al., 2003), protein, and nucleotide precursors (Jiang et al., 2014), signaling molecules (Wu et al., 2000) and protection against oxidative stress (Nasresfahani et al., 1992). As energy requirements of body are met, amino acids will be mainly used for protein synthesis rather than for provision energy.

Our results showed that no changes were observed on serum glucose content, while hepatic glycogen and muscle glycogen contents were significantly increased in rats supplemented with (-)-HCA, which indicated that there was sufficient energy to meet the requirement of body. Under this condition, the increased of amino acid contents in serum and liver might be used to promote protein synthesis. In addition, alanine in muscle can be used to transport the ammonia to liver, and then the liver delivers glucose to muscle through serum, which is called as alanine-glucose cycle, and it can provide the adequate glucose for muscle (Rijkers, 2015).

Present study showed that (-)-HCA treatment obviously increase alanine contents in serum and liver of rats, this indirectly indicated that sufficient glucose in muscle provided an essential prerequisite for the protein synthesis. As mentioned earlier, insulin can enhance amino acid uptake and protein synthesis (Bernard et al., 2013; Bernard et al., 2011). Our result showed that serum insulin content was significantly increased in rats after (-)HCA treatment, which was consistent with the significant increase of the protein content in liver and muscle of rats. Therefore, we conjecture that (-)-HCA treatment could promote protein synthesis via regulating metabolic directions of amino acids in rats.

The amino acids predominantly involved in energy metabolic processes are branched amino acids, which include leucine, isoleucine, and valine (Assenza et al., 2004). Under normal conditions, the branched amino acids are selectively excluded from hepatic uptake and are metabolized predominantly in the skeletal muscle (Adibi, 1980; Tsuchiya et al., 2005; Urata et al., 2007).

Our results showed that (-)-HCA treatment significantly increased the contents of valine and leucine in serum and liver, and decreased their contents in muscle of rats. Previous study shows that excessive oxidation of branched amino acids may inhibit citric acid cycle via depleting the glutamate and ketoglutarate pool (Trottier et al., 2002). The changes of serum glucose and glycogen contents in this study indicated that branched amino acids might be mainly used for protein synthesis in muscle rather than for the source of energy. Muscle is one of the main target organs of insulin action, and insulin can promote the protein synthesis (Rijkers, 2015).

Escobar et al. demonstrated that insulin can stimulate the influx of branched amino acids into the skeletal muscle (Escobar et al., 2006). In addition, previous study suggests that branched amino acids play important role in skeletal muscles, specifically in the regulation of protein synthesis metabolism (Desikan et al., 2010). Wang et al. reports that amino acids can enhance protein synthesis (Wang and Proud, 2008). Considering the increase of serum insulin level and muscle protein content, we suspected that decrease of branched amino acids content after (-)-HCA treatment might be due to its enhance on protein synthesis.

In conclusion, in spite that (-)-HCA could promote protein synthesis, it can also promote weight loss by suppressing de novo of fatty acid synthesis and promoting energy expenditure. This study demonstrated that supplement with (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormone levels. Meanwhile, (-)-HCA treatment could promote protein synthesis in male rats by altering the metabolic directions of amino acids. The elucidation of the precise mechanism involved in this action of (-)-HCA needs further investigation.

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].

Don’t believe the LIES about Garcinia Cambogia

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.

PHARMACEUTICAL COMPANIES HATE GARCINIA CAMBOGIA

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].

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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].

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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.

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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].

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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.