No, Butter is NOT bad for your heart health

butter-and-knife-on-wooden-chopping-boardFor the past decades, butter has been implicated as a significant cause of heart disease.

However, studies provide mixed results and whether butter truly increases the risk of chronic disease is hotly debated.

A recent meta-analysis examined how eating butter affects heart disease, type 2 diabetes and mortality risk. Here is a detailed summary of the findings.



Butter is a dairy product made from cream. It is almost pure milk fat, which mainly consists of saturated fatty acids.

The role of butter in health and disease is uncertain and hotly debated. Several studies show that a high intake of saturated fat is linked with a poor blood lipid profile, which is a risk factor for heart disease.

Additionally, one controlled trial showed that a high intake of saturated palm oil, rich in palmitic acid, caused greater gains in belly fat and liver fat, compared to polyunsaturated fat (1).

However, the largest and most recent meta-analyses of observational studies suggest that reducing saturated fat itself has neutral effects on health, whereas replacing it with certain unsaturated fats may have benefits (2).

Additionally, growing evidence suggests that not all saturated fats are the same and demonizing saturated fats as a whole is an oversimplification.

Nevertheless, official dietary guidelines continue to recommend lower intakes of all saturated fat and higher intakes of non-hydrogenated unsaturated fat (3).

Studies suggest that butter is different from other sources of dairy fat. Specifically, the fat in butter is not enclosed in a milk fat globule membrane (MFGM).

Several randomized controlled trials show that eating butter fat has worse effects on the blood lipid profile than other sources of dairy fat with an intact MFGM, such as cream or cheese (456).

Whether these effects translate into an elevated risk of hard endpoints, such as heart attacks, remains unclear.

Article Reviewed

This meta-analysis examined the association between butter intake and heart disease, diabetes and all-cause mortality or death.

Is Butter Back? A Systematic Review and Meta-Analysis of Butter Consumption and Risk of Cardiovascular Disease, Diabetes, and Total Mortality.

Study Design

This was a systematic review and meta-analysis of prospective observational studies and randomized controlled trials examining the association of butter consumption with heart disease, diabetes and mortality.

The researchers searched scientific databases for all relevant studies that fulfilled the exclusion criteria. When conducting the analysis, they followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines.

A total of 9 publications were selected, including a total of 636,151 participants. No randomized controlled trials with hard endpoints were found.

Bottom Line: This was a systematic review and meta-analysis of prospective observational studies investigating the association of butter with the risk of heart disease, diabetes and death.

Finding 1: Butter Was Weakly Linked With All-Cause Death

Two large observational studies assessing the links between butter consumption and all-cause mortality (death) were included in the meta-analysis. These studies included a total of 379,763 participants.

The analysis showed that the risk of death increased by 1% for each tablespoon (14 grams) of butter consumed daily.

Previous, large meta-analyses examining the effects of total saturated fat intake found no significant effects on overall mortality (27).

Bottom Line: The researchers discovered that for each tablespoon of butter eaten, the risk of death from any cause increased by 1%.

Finding 2: Butter Reduced the Risk of Diabetes

The analysis included four studies examining the links between butter consumption and type 2 diabetes. They included a total of 201,628 participants.

Pooling the findings from these studies, the researchers discovered that a higher intake of butter was linked with a modest decrease in the risk of developing type 2 diabetes.

Specifically, the risk of type 2 diabetes decreased by 4% for each tablespoon (14 grams) eaten daily.

Other observational studies have found no association between dairy fat and type 2 diabetes, but a few support the present results, reporting a reduced risk with higher intakes (891011).

Bottom Line: The study showed that each tablespoon of butter eaten daily reduced the risk of type 2 diabetes by 4%.

Finding 3: Butter Was Not Linked With Heart Disease

Five of the included studies investigated the association of butter with heart disease.

When their results were combined, butter intake was not significantly linked with heart disease, including stroke and coronary heart disease.

These findings are supported by previous meta-analyses (1213).

Bottom Line: The meta-analysis showed that butter was not significantly associated with the risk of developing heart disease.


The main limitation of this meta-analysis was the observational design of the included studies. Observational studies cannot demonstrate causality.

Since high butter consumption is generally associated with unhealthy dietary patterns and lifestyle habits, the study might have overestimated the association of butter with mortality, and/or underestimated its links with type 2 diabetes.

Summary and Real-Life Application

This analysis suggests that butter is neutral when it comes to the risk of developing heart disease.

Additionally, it was associated with a lower risk of type 2 diabetes, but a slightly elevated risk of overall mortality. Since these findings were based on observational studies, they should be taken with a grain of salt.

It should be noted that the elevated mortality risk associated with butter is relatively small compared to many other foods, such as refined grains and sugar.

In conclusion, it seems there is no compelling reason to avoid butter. Moderate amounts should be fine. However, if you eat lots of it, it may be wise to replace some of it with oils that have proven health benefits, such as olive oil.

Does Frequent Snacking affect Weight Loss

pretzels-and-snacks-in-bowlsMany snack foods are unhealthy and easy to overeat.

In many cases, snack foods may contribute to the development of obesity. But does snacking itself affect body weight?

A recent review shows that cutting down on snacks may be as effective as popular weight loss supplements like Garcinia Cambodia. Below is an overview of its main points.


Article Reviewed

This review discussed how snacking affects satiety and body weight.

Snack Food, Satiety, and Weight.

What Are Snacks?

Snacks are any foods that are eaten between meals.

They often are ready-to-eat and do not require much preparation. These commonly include candy, chips, crackers, cookies, granola bars, nuts and popcorn.

Although beverages are usually not considered snack foods, they are consumed along with snack foods.

In this review, we place beverages under the broad definition of snack foods.

Bottom Line: Snacking is the act of consuming foods or beverages between meals.

Does Snacking Increase Weight Gain?

Snacking contributes to approximately 27% of the daily calorie intake among children. The percentage has been on the rise in recent decades in both children and adults (12).

The increased popularity of snacking goes hand in hand with increased screen time (watching TV and playing computer games) and less physical activity.

Additionally, highly processed, “unhealthy” snacks are the most popular, and there seems to be limited interest in healthy snacks in the general population (345).

For these reasons, unhealthy snacking often contributes to weight gain.

Bottom Line: Snacking may be largely responsible for weight gain among many people.

Is Snacking Healthy?

In general, nutrient-poor and calorie-rich snacks are considered unhealthy (67).

Unhealthy snacks include candy, chips, cookies and sugary beverages. Healthy snacks may include fruits, vegetables, nuts, seeds and whole grains.

That said, any snacks are unhealthy when snacking goes out of hand and starts contributing to excessive weight gain.

Studies also suggest that irregular eating habits, often associated with snacking, may adversely affect health.

Bottom Line: Healthy snacks can be very nutritious. However, snacking is unhealthy when it gets out of hand or involves unhealthy food.

What Factors Affect Snacking?

Snacking is affected by a variety of factors.

The following factors have been associated with snacking behavior:

  • Poor education: One study showed that snacking was more common among those from poorly educated families (8).
  • Knowledge: Being able to distinguish between healthy and unhealthy snacks affects food choice (9).
  • Emotional eating: Some people respond to negative emotions by eating, especially high-sugar, high-fat snacks (10).
  • Stress: Anxiety causes some people to snack, especially emotional eaters (10).
  • Family norms: One study showed that having strict parents reduced snacking among children (11).
  • Social norms: When young adults were led to believe that limiting junk foods was the social norm, their intake of high-calorie snacks decreased (12).
  • Availability: Limited access to healthy snacks affects intake (13).
  • Packaging: Bigger package sizes encourage overeating, regardless of people’s appetite or the taste of the snack (14).
  • Distractions: Mindful eating reduces snack intake, whereas distractions (like watching TV or playing a video game) increases it (15).
  • Variety: Having a wide variety of snacks to choose from can promote overeating (1617).

Bottom Line: Numerous factors influence snacking behavior, including how often people snack, when they do it and how much they eat.

How Do Snacks Affect Appetite?

Some researchers have speculated that eating snacks might promote fullness or satiety, thus reducing the risk of overeating at the next meal (18).

One study found that snacks are generally not very filling. However, of all the snacks evaluated, those with the highest protein content were the most satiating (19).

Another study found that a high-protein afternoon snack delayed the need for dinner, compared to high-fat or high-carb snacks. In fact, the high-carb snack did not delay dinner at all (20).

High-protein snacks that may suppress appetite include:

  • Peanuts and tree nuts (2122).
  • Greek yogurt (2324).

Snacks that are high in fiber may also promote fullness, compared to low-fiber snacks. Popcorn is a good example of a common high-fiber snack food.

Additionally, low-fat (air popped) popcorn is significantly more satiating compared to an equal amount of potato chips (25).

Bottom Line: Snack foods that are high in protein and/or fiber may reduce appetite, compared to other types of snack foods.

Can Snacking Reduce Body Weight?

A few randomized controlled trials have examined how different snack foods affect body weight.

One trial in women showed that snacking on dark chocolate daily reduced body weight and fat mass, compared to fruit-flavored licorice (26).

Other studies comparing a variety of different snack foods found no differences in daily food intake or changes in body weight (272829).

It appears that people may compensate for snacking by eating less food at meals. However, the calorie compensation is often only partial, and regular snacking may lead to weight gain over time (3031).

Additionally, not all snacks are equal. One study showed that eating nut-based snack bars for 3 months reduced body fat and abdominal fat in overweight people, compared to a cereal snack bar (32).

Observational studies have also provided mixed results. Some studies showed no significant links between snack foods and weight or body weight (7).

These findings are partially supported by other observational studies. One important exception is sugary soda, which was significantly linked to obesity (3334).

In general, it seems that snacking on calorie-dense, low-nutrient foods may promote weight gain. This especially applies to sugary soda.

That said, it is still unclear whether traditional snack foods reduce body weight, compared to not snacking. At this point, it seems unlikely.

Bottom Line: There is limited evidence that snack foods reduce body weight. In contrast, high-calorie snack foods may promote weight gain.

Summary and Real-Life Application

Taken together, studies investigating the effects of snack foods on body weight provide mixed results.

In general, high-calorie snack foods are believed to promote obesity, especially sugary soda. In contrast, people seem to partially compensate for consuming healthy snacks by eating less at the next meal.

Overall, there’s limited evidence that traditional snack foods lead to weight loss.


One of the more common questions is whether I recommend any supplements. I recommend very few of them. For longer fasts, I recommend a general multivitamin, although there is scant evidence that it is beneficial. In fact, almost all vitamin supplements have been proven to be useless. In some cases, like vitamin B, worse than useless. All vitamins go through periods of of popularity and unpopularity. It’s worse than high school. One minute, you’re the most popular kid in class, then next you’re the laughingstock.

In the 1960’s the king of vitamins was vitamin C. Linus Pauling is the only person to have won two unshared Nobel Prizes — once for chemistry and once for peace. He had the firm unshakeable belief that many of the problems of modern nutrition could be cured by mega doses of vitamin C. He suggested that high dose vitamin C could prevent or cure the common cold, the flu and even cancer. He even suggested that “75% of all cancer can be prevented and cured by vitamin C alone”. That, of course is wildly optimistic. Many studies were done over the next few decades that clearly proved that most of these vitamin C claims were simply false hopes. Turns out the only disease Vitamin C cures is scurvy. Since I don’t treat many 15th century pirates, it’s not too useful for me.

Once vitamin C supplementation was proven largely useless to prevent disease, the next great hope was vitamin E. Its main claim to glory was as an ‘antioxidant’. Supposedly, vitamin E would neutralize all the nasty free radicals that were causing untold damage to our vascular system. Taking vitamin E would prevent heart disease, we were told. Except, of course, it did nothing of the sort. The HOPE trial, best remembered now as one of the trials to establish the use of the ACEI class of medication in cardiovascular protection. However, this randomized controlled trial also tested whether vitamin E could prevent disease. Unfortunately, the answer was no. Vitamin E supplements did not prevent heart disease or stroke. Indeed, more patients in the vitamin group died, had heart attacks and strokes although this was not statistically significant. Vitamin C was a bust, and so was vitamin E. But the list of shame would not stop there.

The next great hope was vitamin B. In the early 2000s, there was a great flurry of interest in a blood test called homocysteine. High homocysteine levels were correlated with increased risk of heart disease. Vitamin B could lower homocysteine levels, but whether this would translate into better health outcomes was unknown. Several large scale trials were launched with this hope. One of these was the NORVIT trial, published in 2006 in the prestigious New England Journal of Medicine.

The news was stunning. Stunningly bad, that is. Compared to taking placebo (sugar pills), supplementation with folate, vitamin B6 and B12 was giving people more heart attacks and strokes. Yes. The vitamin group was not doing better, it was doing worse. But worse news was still to come, if you can believe it. In 2009, researchers studied the two randomized controlled trials of vitamin B supplementation and found that in addition to raising the risk of cardiovascular disease, the risk of cancer was increased by 21%! Aw snap! The risk of dying from cancer increased by 38%. Taking useless vitamins is one thing, taking vitamins that are actively harmful is something else.

The use of vitamin B supplements for kidney disease was similarly dismal. The DIVINe study randomized two groups of patients with chronic kidney disease (CKD) to either placebo or vitamin B supplements with the hope of slowing down the progression of kidney disease. Homocysteine levels are high in CKD and the vitamins were able to lower these levels. But did they make any real difference? Sure did. The use of vitamin B made things worse. Much, much worse. It doubled the incidence of poor outcomes. Another nail in the coffin of the homocysteine story and vitamin B supplements. Another 10 years of research money wasted.

The ironic part of this flawed knowledge is that we are still paying the price. Enriched wheat flour, for example is wheat with all the goodness extracted and then certain vitamins replaced. So almost all the vitamins were removed, and replaced with huge doses of iron and vitamin B. So what we got was a huge surplus of vitamin B. Not that I believe this was malicious. People were mostly concerned about nutrient deficiencies like beri beri, iron deficiency anemia and not so much with anything else. The problem, of course, is that we now have data that show that giving large doses of vitamin B may increase rates of cancer and hear attacks.

But why should vitamin B supplements be bad? After all, folate supplements have reduced the incidence of neural tube defects in pregnancy significantly. Like everything else in medicine, it’s a question of context. Vitamin B is needed for growth of cells. During growth periods, like pregnancy and childhood, this is a good thing.

The problem is completely different during adulthood. Excessive growth is NOT good. The fastest growing cells are cancer cells, so they love, love, love the extra vitamin B. Not so good for us people. Even for regular cells, the excessive growth is not good, because it leads to scarring and fibrosis. This explains how you get more heart attacks, strokes and kidney disease. Cardiovascular disease is caused by atherosclerosis, the hardening of the arteries and excessive fibrosis likely makes it worse.

Calcium supplements, of course have been recommended by doctors for decades as a preventative strategy against osteoporosis. I explained everything in this lecture from a few years ago “The Calcium Story”. Almost every doctor has recommended calcium supplements to prevent osteoporosis.

Why? The rationale is that bones have lots of calcium so eating calcium must make bones stronger. This is, of course, the reasoning that a third grader might use, but that’s besides the point. Eating brains makes us smarter. Eating kidneys improves kidney function. Right…. But any who, this puerile reasoning lasted for about 50 years.

We pretend that we live in a world of evidence based medicine. Just as we discussed with calories, it seems that evidence is not needed for the status quo, but only for ‘alternative viewpoints’. They finally did a proper randomized controlled trial on calcium supplementation and published it in 2006. The Women’s Health Initiative randomized over 36,000 women to calcium and vitamin D or placebo. Then they followed them for over 7 years and monitored them for hip fractures. Did taking calcium every day for 7 years give women super-strong bones that never crack?

Hardly. There was no difference in total fractures, hip, vertebral or wrist fractures. In other words, calcium supplements were completely useless. Actually, that’s not true. There was a significant difference. Those people taking calcium had significantly more kidney stones. So, they were actually harmed by taking these pills. Nice. Are these women glad they faithfully took their pills every day for the last 7 years?

What is the reason why these supplements are not beneficial and mostly harmful? It’s really quite simple. You must understand the root cause (the aetiology) of disease in order to prescribe rational treatment. The diseases that we face today — obesity, type 2 diabetes, osteoporosis, cancer, heart disease etc. ARE NOT VITAMIN DEFICIENCY DISEASES. If these are not disease caused by a lack of vitamins, why would we expect supplementation to make a difference?

Let’s take an analogy. Suppose our car does not run because the engine has exploded. Somebody then says “Oh, hey, I had a time where our car did not run because it was out of gas. Therefore you should put more gas into the car”. But it doesn’t work. Because you must treat the root cause. The problem was that the engine exploded. I don’t really care how much gas is in the car in this situation.

So, if we are treating vitamin deficiency disease (scurvy, beri beri, osteomalacia) then replacing vitamins is very logical and effective. If we are treating obesity, then replacing vitamins is completely and utterly useless. I don’t worry about nutrient density of foods, because I am not treating a nutrient deficiency disease. However, people love trying to sell you the latest greatest weight loss supplement (green coffee, raspberry ketones, PGX, fibre, Sensa etc).

If you are asking the question “What can I eat/ take/ supplement to help me lose weight?” then you are completely going in the wrong direction. The question you need to ask is “What can i NOT eat/take/ supplement to help me lose weight?” The money to be made answering the latter question is orders of magnitude smaller than trying to answer the first.



Why Antioxidants, Vitamins, and Hormones failed to reverse agingThey treat the symptoms and not the causes

“Although many drivers of human aging can be slowed or delayed, many of these factors are thought to be non-reversible reversible. (Ex: DNA gene mutations are not reversible).

Most “anti-aging” supplements like anti-oxidants cannot reverse such aspects of aging.  Instead, they merely “control damage,” and many have a mixed track record of efficacy in clinical trials.

Likewise, hormone replacement therapy (HRT) does not reverse aging, despite the claims of some “anti-aging” clinics. In fact, emerging scientific studies have shown that exogenous anti-oxidant supplements and HRTs have a paradoxical effect.

For instance, exercise has been shown to increase the expression of anti-oxidant genes and increase the expression of endogenous hormones (hGH, etc.). However, when exogenous anti-oxidants are used, this reduces the expression of anti-oxidant genes induced by the exercise producing a negative effect.

Likewise, exogenous HRT use suppresses endogenous hormone production and thereby accelerates the decline in hormone gene expression that occurs with aging (via an epigenetic feedback-this is so good inhibition mechanism).

This is why many testosterone users have testicular atrophy. Thus antioxidants and HRTs treat the symptoms (i.e. “downstream effects) of aging, rather than the cause (“upstream events”) of aging.

Current initiatives to restore NAD+ levels in individuals are attempts to affect what we believe are “upstream events” in aging to produce positive downstream events, such as already has been shown to be possible in various studies – like reversal of muscle aging in rodents.

Because nuclear NAD+ deficiency has so many impacts that mimic those of caloric restriction (CR), we look next at how it affects upstream events in molecular aging.  We are also looking at CR for ideas for the best biomarkers for objectively evaluating the “upstream effects” of restoring NAD+ levels in the nucleus.”

Combating ROS with supplements derived from “healthy” fruits and vegetables that were found to possess Anti-oxidant properties in vitro was all the rage over the last 15 years or so, but has been falling out of favor as research shows that many of these supplements fail to reach individual cells in sufficient concentration to make a positive impact in vivo.

The attempts to attenuate aging processes including the increase in organismal longevity by antioxidants were largely unsuccessful (r)

“In the absence of malnutrition, there is little or no reason to take antioxidant supplements because the ability of these compounds to function as antioxidants is largely determined by the [NADP+]/[NADPH]”

That is why more and more researchers say Anti-oxidants don’t work, as Dr Sinclair explains in this video.

Vegetables are better with Mediterranean cooking

Mediterranean Cooking Makes Vegetables Better Dallas Clouatre

Sometimes lost in the public service messages regarding what to eat is another important component in nutrition—how food is cooked. This is the take home message from a recent article published in Food Chemistry.1 Moreover, it seems that cooking techniques and materials can cut both ways, either depleting nutrients or enhancing them.

Studies have shown, for instance, that using poor quality fats to deep fry fish containing high levels of omega-3 fatty acids can reduce the content of the beneficial omega-3 nutrients.

The Food Chemistry study shows that the opposite also can happen, that deep-frying vegetables in extra virgin olive oil can improve nutritional quality.

There are other reasons cooking can be both friend and foe of nutrition. Over-cooking can destroy nutrients because of heat and oxidation, cause them to be tossed out with cooking water, and so forth.

In contrast, cooking can make available compounds that otherwise digestion normally cannot extract from raw food.

In this particular case, it was found that frying in extra virgin olive oil transferred to vegetables polyphenols from the oil and thereby improved the antioxidant capacity of the vegetables in comparison with either raw or boiled alternatives.

The particular new healthful polyphenols were shown to be from the oil and not found originally in the raw vegetables.

The further implication is that frying in oils of lesser quality will lead to vegetables that are not as nutritious as those fried in extra virgin olive oil.

Frying with Extra Virgin Olive Oil for Extra Nutrition
Nutrients in raw vegetables often are less bioavailable than is true when the same vegetables are cooked. Likewise, cooking sometimes leads to beneficial transformations of nutrients.

The degree to which these claims are true varies from vegetable-to- vegetable and with the cooking method employed. In the present study in Food Chemistry, potato, tomato, eggplant and pumpkin (120 grams each) were deep fried, sautéed and boiled.

Extra virgin olive oil was used for the frying and also added to cooking water to create a water/oil mixture; the third cooked arm employed water only.

The methods used were typical of standard cooking techniques. Deep fat frying used five parts oil to one part vegetable, whereas sautéing used one half as much oil as vegetable; temperatures were approximately 360 °F and 175–212 °F, respectively.

Boiling used five parts water or five parts water/oil to one part vegetable. Cooking lasted 10 minutes; vegetables were drained for an additional five minutes and then refrigerated before being homogenized for testing.

Testing before and after cooking determined fat, moisture, total phenols (or phenolics, referring to the chemical structure of these nutrients), eighteen phenolic compounds and antioxidant capacity.

Not surprisingly, deep-frying led to the greatest moisture loss and the greatest gain in fat; sautéing increased fat content less without appreciably changing the moisture content versus the raw state.

Depending on the vegetable, boiling either increased moisture or exercised no significant effect; boiling in the water/oil mixture increased vegetable oil content.

Changes in phenolic nutrients were more complex. Deep-frying increased these significantly for all four vegetables by reducing moisture.

Sautéing led to nutrient increases in potato and pumpkin, but only non-significant increases in eggplant and tomato.

For eggplant, sautéing reduced total endogenous phenolics, primarily chlorgenic acid, apparently because of oxidation from exposure to air due to not being totally covered by the oil.

Total phenolics fell in both potato and pumpkin from either method of boiling. Both frying techniques increased phenolic nutrients typical of extra virgin oil indicating a transfer to the vegetables of oleuropein, pinoresinol, hydroxytyrosol, tyrosol, p-coumaric acid and hydroxybenzoic acid.

Phenolic nutrients already found in these vegetables, such as chlorogenic acid and rutin, increased except in the eggplant.

Interestingly, all the cooking methods conserved or increased antioxidant capacity in the order of deep-frying, sautéing, and then boiling. The best results with either form of boiling required that the cooking water be consumed along with the vegetable.

About that Olive Oil…
Extra virgin olive oil is oil that has undergone the least processing and that retains the highest levels of naturally occurring polyphenols.

Extra virgin and virgin olive oils are good sources of antioxidants and other healthful compounds.

The study in Food Chemistry shows that not all of these healthful compounds are lost in cooking, not even in relatively high-temperature deep-frying. Instead, significant amounts can be transferred to the food being cooked.

This is an important finding, in part because it extends the range of uses of olive oil beyond, for example, dressings for salads, and also because it indicates that olive oil is important for more than simply being a source of monounsaturated fatty acids.

A quick look at research publications limited even to just 2015 yields papers showing that olive oil polyphenols.

  • Inhibit oxidative damage to lipoproteins, including LDLcholesterol, and at the same time improve the functionality of HDL-cholesterol, including the cholesterol efflux capacity to pick up excess cholesterol from peripheral tissues and return it to the liver for disposal
  • Helps to lower blood pressure in both men and women
  • Improves the physiology of the endothelium, a type of cell that lines the interior surface of blood and lymph vessels
  • As part of the Mediterranean diet, exercise favorable effects on a large range of cardiovascular risk factors

Foods: Raw or Cooked?
As with many such debates, evidence for the raw versus cooked food dispute does not neatly come down to either/ or choices.

Are some nutrients damaged or otherwise lost during cooking? Yes, some are. However, as the study above shows, depending on the food, nutrients can become more concentrated and cooking itself can add nutrients. Digestive enzymes are not particularly good at breaking the cell walls found in many vegetables. In these cases, cooking makes much more bioavailable many nutrients.

The carotenoids from carrots are good examples of this phenomenon. Again, the lycopene in tomatoes is three or more times more bioavailable from cooked tomatoes, especially when cooked with oil, than from raw tomatoes.

Small changes in cooking technique, such as not cooking vegetables in large volumes of water and then tossing the cooking water, can save many vitamins, minerals and other nutrients.

A judicious balance of raw and properly cooked foods is more likely to yield a full range of healthful nutrients than is either approach alone.


  1. Ramirez-Anaya Jdel P, Samaniego-Sanchez C, Castaneda-Saucedo MC, Villalon-Mir M, et al. Phenols and the antioxidant capacity of Mediterranean vegetables prepared with extra virgin olive oil using different domestic cooking techniques. Food Chem. 2015 Dec 1;188:430—8.

Why AGED Garlic Extract is better

Garlic and Kyolic Aged Garlic Extract Immunity Boosters Carmia Borek

Garlic’s potential as a remedy to prevent and combat a wide range of diseases has been lauded and practiced for thousands of years. By the 21st century medicine has confirmed many of the medicinal benefits of garlic. Among the wide range of remedial and preventive properties, garlic has shown the ability to boost immunity. Other effects that are linked in some ways to immunological mechanisms including cardiovascular disease, neurodegenerative disease, cancer, and overcoming fatigue.

The pungent taste of garlic is not to everyone’s liking, as its odor may remain on the breath and skin for days; moreover, large quantities of ingested garlic, potentially needed to boost immunity may cause gastrointestinal disturbances. The supplement Aged Garlic ExtractTM (Kyolic® AGETM) that is odorless, has become a most popular preparation that provides the benefits of garlic, including boosting immunity, without the unpleasant side effects. Moreover, its efficacy and reliable standing as the preferred garlic formulation for research on the health benefits of garlic, has yielded over 700 peer reviewed research publications in scientific and medical journals.

Aged Garlic Extract
The manufacturing of AGE, by Wakunaga of America, consists of harvesting organically grown garlic, and carrying out a procedure of extraction and aging, at room temperature, for 20 months The process converts harsh volatile compounds such as allicin to stable substances, thereby increasing antioxidant levels in AGE above levels found in fresh garlic.

Among the many components in AGE the major ones are organosulfur antioxidants, largely water soluble, and highly bioavailable including S-Allyl cysteine and S-Allyl mercaptocysteine. Also present are lipid soluble organosulfur compounds, carbohydrates, including fructans, which are immuo-boosters, micronutrients such as selenium and other antioxidants such as fructosyl arginine and alixin. The high antioxidant level in AGE prevent the damage induced by free radicals that are generated in metabolism and enhanced by environmental factors, such as radiation of different types including UV light from the sun and UV machines. Free radical damage plays a role in inflammation, and various pathological conditions including heart disease, dementia and cancer, so that the inhibition of free radicals by AGE is part of its action in helping prevent these pathological conditions, in part by boosting immunity.

Our Immune System
A healthy immune system is the secret to good health, protecting against infectious bacteria, viruses, fungi and helping block the development of cancer. A weak immune system exposes us to the damaging effects of infectious bacteria and viruses around us, from direct contact, exposure to a cough or a sneeze, from contaminated food and a wide range of sources that trigger serious illnesses, that may lead to death.

The immune system is complex and multilayered. Inflammation is one of the first responses of the immune system to infection and involves the release of substances called prostaglandins and leukotrienes that attract white blood cells (leukocytes) and interferons, that have anti-viral effects.

Among the white cells, the leukocytes there exist B and T lymphocytes. There are subtypes of T cells: killer T cell that kill cells infected with pathogens and helper T cell that regulate the immune response. Killer T cells kill cells that are infected with viruses (and other pathogens), or are otherwise damaged or dysfunctional. Helper T cells regulate the immune responses directing other cells to perform various tasks. Natural killer cell (NK) have in their power to kill tumor cells. Another group of lymphocytes are ãä-T cells that share the characteristics of helper T cells, killer T cells and NK cells.

AGE Enhances Immunity
Aged Garlic Extract has been shown in preclinical and clinical studies to enhance the immune response, mitigate infectious diseases, and kill cancer cells. AGE intake has been shown to increase the phagocytic (cell-killing) activity of macrophages, the activity of the T lymphocytes and increase the number and action of natural killer cells (NK) and antitumor action; AGE also was found to inhibit the allergy-causing histamine release and have anti-inflammatory effects, suppressing prostaglandins and enhancing interferon.

AGE Increases NK Activity
In a random double-blind clinical trial, Ishikawa and colleagues found that AGE given to patients with colorectal, liver or pancreatic cancer resulted in a significant increase in the number and activity of the NK cells, killing cancer cells.

Advanced-cancer patients with a decline in immune functions and quality of life, with inoperable colorectal, liver, or pancreatic cancer were recruited for the study. In a randomized six month double-blind trial, AGE was given to one group and a placebo to another. The patients consisted of 42 with liver cancer (84 percent), seven patients with pancreatic cancer (14 percent), and one patient with colon cancer (two percent). The study showed that both the number of NK cells and the NK cell activity increased significantly in the AGE group; showing that the administration of AGE to patients with advanced cancer of the digestive system has the potential to improve the anti-tumor NK cell activity.

AIDS patients show lower levels of NK cells. Abdullah and colleagues showed that a six week intake of AGE at 1800 mg/day increased the levels of NK cells to that of healthy individuals; Helper T cells were also increased and patients showed an improvement of several pathological conditions, including herpes virus infection, yeast infections and diarrhea; Comparing the efficacy of AGE to that of fresh garlic, investigators found that AGE was more effective as an immune-stimulator than fresh garlic; NK activity was increased by 160 percent with the intake of AGE capsules compared with an increase of 140 percent in patients taking the fresh garlic preparation.

AGE Helps Reduce Colds And Flu
A randomized, double-blind, placebo-controlled study recruited 120 healthy subjects (60 per group between ages of 21 and 50) and evaluated the effect of AGE supplementation (2.56 g/d) on the proliferation of immune cells and cold and flu symptoms.

After 45 days of intake of an encapsulated Aged Garlic Extract, NK and £^£_-T cells rose in number, compared to placebo. Following 90 days of supplementation, diary entries of illness showed that though the incidence of colds and flu, were not statistically different, the group consuming Aged Garlic Extract showed a reduced severity of both colds and flu noted by a reduction in the number of reported symptoms (21 percent fewer) and by a reduction in the number of days (61 percent fewer), and incidences (58 percent fewer) where the subjects¡¦ function was sub optimal.

The investigators concluded that supplementation of the diet with AGE may enhance immune cell function and potentially reduce inflammation, resulting in a reduced severity of colds and flu.

AGE Blocks Ultraviolet-Induced Immunosuppression.
Studies on human volunteers in Australia found that exposure to ultraviolet radiation (UV) causes immunosuppression resulting in an increase skin cancer frequency. There was a gender difference. UV doses that caused immunosuppression in men were three times lower than those causing immunosuppression in women. The investigators concluded that this phenomenon might underlie the higher incidence of skin cancer and mortality observed in the male population.

In a preclinical study, where the immune response as measured by contact hypersensitivity, Reeve and colleagues found that immunosuppression of 58 percent induced by a moderate exposure to UVB radiation was reduced to 19 percent by a diet containing AGE, at 4 percent of the diet. The preclinical studies suggest that AGE may help protect humans against immunosuppression induced by exposure to UV, and therefore have a potential to reduce the risk of UV induced skin cancer, for example by lengthy exposures to the sun.

AGE Reduces Inflammatory Prostaglandins
Oxidative damage by free radicals and immune-inflammatory activation are considered important factors in the development of cancer, neurodegenerative and cardiovascular diseases. Prostaglandins are substances associated with inflammation in that the release of local pro-inflammatory prostaglandins takes place, accompanied by the destruction of tissue. Rahman and colleagues have shown that dietary supplementation with AGE for 14 days reduced the plasma and urine levels of prostaglandin 8-iso-PGF (2 alpha) by 29 and 37 percent, respectively in nonsmokers and by 35 and 48 percent, respectively in smokers.

By stopping the intake of AGE, they found that in both groups, smokers and non smokers, the plasma and urine concentrations of prostaglandins reversed to values that were no different from those before ingestion of AGE, within fourteen days after cessation of the dietary supplementation. The study shows that a continuous intake of AGE is required to maintain the reduced levels of inflammatory prostaglandins.

The Bottom Line
Aged Garlic Extract is a powerful wide ranging health supplement that plays a role in helping enhance immunity and thus helping protect against diseases and conditions that involve inflammation and weakening of the immune system; such conditions have been reported to be associated with cancer development, neurodegenerative and cardiovascular disease as well as aging. In over 700 medical and scientific studies in both preclinical and clinical studies, AGE has shown its capacity to help reduce disease and maintain health.


  1. Nantz MP, Rowe CA, Muller CE et al Supplementation with aged garlic extract improves both NK and Υδ-T cell function and reduces the severity of cold and flu symptoms: a randomized, double-blind, placebo-controlled nutrition intervention. J Nutr. 2012; 31:337—44.
  2. Borek C. Dietary antioxidants and human cancer. Integr Cancer Ther. 2004;3:333—41.
  3. Reeve VE1, Bosnic M, Rozinova E, Boehm-Wilcox C. A garlic extract protects from ultraviolet B (280¡V320 nm) radiation-induced suppression of contact hypersensitivity. Photochem Photobiol. 1993;58:813—7.
  4. Dillon SA, Lowe GM, Billington D, Rahman K. Dietary supplementation with aged garlic extract reduces plasma and urine concentrations of 8-iso-prostaglandin F(2 alpha) in smoking and nonsmoking men and women. J Nutr. 2002 ;132:168—71.
  5. Ishikawa H, Saeki T, Otani T, Suzuki T, Shimozuma K, Nishino H, Fukuda S, Morimoto K. Aged garlic extract prevents a decline of NK cell number and activity in patients with advanced cancer. J Nutr. 2006;136:816S—820S
  6. Abdullah TH, Kirkpatrick DV, Carter J. Enhancement of natural killer activity in AIDS with garlic. J Oncology. 1989;21:52—3.

Asthma relief yet another reason to take Vitamin D

Vitamin D Pills Forming Letter D
Asthma is a chronic inflammatory disease that causes breathing difficulties. The underlying cause is incompletely understood.

A recent meta-analysis of randomized controlled trials examined the effects of vitamin D supplements on asthma symptoms. Here is a summary its conclusions.



Asthma is a chronic inflammatory condition of the lung airways. It is characterized by attacks of breathlessness, chest tightness and wheezing.

Several observational studies have associated low circulating levels of vitamin D with an increased risk of asthma attacks (123).

These findings are partly supported by some meta-analyses of randomized controlled trials (456).

Article Reviewed

This was a meta-analysis of randomized controlled trials examining the evidence linking asthma with vitamin D.

Vitamin D for the management of asthma.

Study Design

This was a systematic review and meta-analysis of randomized controlled trials investigating the effects of vitamin D supplements on asthma symptoms.

The researchers searched for all relevant studies using several databases.

Only studies that met the inclusion criteria were selected:

  • The studies had to be double-blind, randomized controlled trials.
  • The participants had to be asthmatic adults or children.
  • The main outcomes were asthma symptoms and/or the risk of a sudden worsening of symptoms.
  • The studies had to last at least 3 months.

A total of nine trials met the inclusion criteria and were included in the primary analysis — seven in children and two in adults.

The studies included a total of 1,093 participants — 435 children and 658 adults — and their length ranged from 4–12 months. Most of the participants had mild to moderate asthma.

Bottom Line: This was a meta-analysis of nine randomized controlled trials examining the effects of vitamin D supplements on asthma symptoms in children and adults.

Finding 1: Vitamin D Reduced the Risk of Severe Asthma Attacks

The analysis showed that supplementing with vitamin D reduced the risk of a sudden worsening of symptoms (asthma attacks) requiring asthma medication (corticosteroids) via injection.

Specifically, the risk decreased by 37%, on average. It also caused a 61% reduction in the risk of having at least one asthma attack requiring a visit to an emergency department or hospital.

The authors concluded that this reduction in severe asthma attacks had significant clinical relevance.

Bottom Line: Taking vitamin D supplements reduced the risk of severe asthma attacks requiring corticosteroid injections, hospitalization or a visit to an emergency department.

Finding 2: Vitamin D Did Not Affect Day-To-Day Asthma Symptoms

In contrast to the protective effects of vitamin D against severe asthma attacks, the researchers concluded that it did not significantly affect Asthma Control Test (ACT) scores.

The ACT score is used to determine if asthma symptoms are well controlled and what level of treatment may be required.

In addition, supplementing with vitamin D did not affect the percent predicted forced expiratory volume in one second, which is a measure of lung function.

Bottom Line: Taking vitamin D supplements did not affect day-to-day asthma symptoms or lung function.

How Does Vitamin D Reduce the Risk of Asthma Attacks?

The analysis shows that taking vitamin D supplements may reduce the risk of asthma attacks.

The way vitamin D may improve asthma is not completely understood but may be explained in the following way:

  1. Supplementing with vitamin D leads to increased levels of calcifediol (hydroxyvitamin D), which is the main form of vitamin D in the blood.
  2. When needed, an enzyme known as CYP27B1 changes hydroxyvitamin D into calcitriol, the bioactive form of vitamin D.
  3. Calcitriol is produced by various body tissues, mainly the kidneys. However, inflammation (as in asthma) leads to increased levels of CYP27B1 in the tissues surrounding the airways, promoting the formation of calcitriol in the lungs.
  4. Calcitriol binds with the vitamin D receptor, which acts to reduce inflammation and stimulates the production of antimicrobial compounds (7891011).
  5. In this way, vitamin D could reduce the inflammation associated with asthma, potentially reducing the risk of severe asthma attacks.
  6. Asthma attacks are also frequently brought about by lung infections. The way vitamin D strengthens the antimicrobial defenses of the lungs may also play a role.

Bottom Line: Vitamin D may reduce the risk of asthma by reducing airway inflammation and strengthening the antimicrobial defenses of the lungs.


This meta-analysis was conducted according to accepted standards and didn’t appear to have any flaws. However, caution should be taken when generalizing the results to certain groups.

First, the conclusion that vitamin D protects against severe asthma attacks was mostly based on studies in adults. As a result, it cannot be generalized to children.

Second, the researchers were unable to look into the effects of vitamin D supplements in subgroups, such as those with severe asthma or vitamin D deficiency.

Bottom Line: The findings cannot be generalized to children, and the effects of vitamin D supplements in those with severe asthma symptoms or good vitamin D status are unclear.

Summary and Real-Life Application

In short, this meta-analysis concludes that supplementing with vitamin D is likely to protect against severe asthma attacks. It also reduces the risk of having to visit an emergency department or stay in a hospital because of asthma.

Although vitamin D itself won’t cure asthma, supplementing with it seems to reduce the risk of severe asthma attacks in people with mild or moderate asthma.

However, studies in severely asthmatic people are lacking. Additionally, it is unclear if these benefits are confined to people with poor vitamin D status or a deficiency.

Alternate day fasting is effective weight loss plan

Following a standard weight loss diet is difficult for most people who too often depend on the fad weight loss pills like Garcinia Cambogia extract.

Recently different methods such as intermittent fasting are gaining popularity.

A randomized controlled trial compared the safety and effectiveness of alternate-day fasting to a traditional, calorie-reduced diet. Here is a detailed summary of its findings.


When people diet, they eat less than they normally would.

Typically, an effective weight loss diet involves a 20–30% calorie deficit, relative to the amount of calories needed to maintain weight. It generally leads to a moderate 5–10% weight loss over a 6-month period (12).

However, sticking to a calorie-reduced diet for a long period is extremely difficult for most people (3).

For this reason, alternative strategies are growing in popularity. One such strategy is intermittent fasting, which involves eating little or nothing for specified periods and normally the rest of the time.

One common intermittent fasting approach is alternate-day fasting (ADF), which involves eating little or nothing every other day.

Like most other weight loss methods, ADF reduces the risk of heart disease and diabetes. It may also cause beneficial changes in appetite hormones (456).

Studies in overweight or obese adults indicate that ADF may cause 3–8% weight loss over a period of 2–12 weeks (78).

Yet, it’s still unclear whether ADF is an effective weight loss strategy. Until now, no studies have compared ADF to a traditional weight loss diet (9).

Article Reviewed

screen-shot-2016-10-26-at-10-41-04-pmThis was a randomized controlled trial comparing the effectiveness of alternate-day fasting to a standard weight loss diet.

A randomized pilot study comparing zero-calorie alternate-day fasting to daily caloric restriction in adults with obesity.

Study Design

This was a small, 2-month randomized controlled trial examining the safety and effectiveness of alternate-day fasting, compared to a traditional weight loss diet.

A total of 26 obese adults participated in the study. They were randomly assigned to one of two groups:

  • Alternate-day fasting (ADF): Participants fasted every other day. On non-fasting days, they could eat as much as they wanted. On fasting days, they were only allowed to consume water, calorie-free beverages and stocks or broths.
  • Traditional weight loss diet (TWD): Participants followed a calorie-restricted diet (a deficit of 400 calories per day) for two months.

In both groups, all food was provided by the study kitchen, and food intake was closely monitored. Additionally, the participants’ macronutrient intakes were standardized with 55% of calories from carbs, 15% from protein and 30% from fat.

At the start and end of the study, the researchers measured the following:

  • Body weight.
  • Body composition: Measured using dual-energy X-ray absorptiometry.
  • Blood lipids: Total cholesterol, triglycerides and HDL were measured in fasting blood samples.
  • Blood sugar control: Evaluated with a glucose tolerance test.
  • Resting metabolic rate: Assessed in the morning using standard indirect calorimetry.
  • Appetite hormones: Leptin and ghrelin were measured in fasting blood samples.
  • Brain-derived neurotropic factor (BDNF).

When the study was over, the participants received standard weight maintenance advice. The above measurements were repeated after a 6-month unsupervised follow-up.

Summary: This was a randomized controlled trial comparing the safety and effectiveness of alternate-day fasting to a traditional weight loss diet.

Finding 1: Alternate-Day Fasting and Standard Dieting Caused Similar Weight Loss

Alternate-day fasting (ADF) and the traditional weight loss diet (TWD) caused similar weight loss.

Specifically, those who fasted every other day lost 18.1 pounds (8.2 kg), on average, whereas those who dieted every day lost 15.7 pounds (7.1 kg), as shown in the chart below.

Weight Loss ADF TWD

Although the weight loss was slightly higher among those who fasted every other day, the difference was not statistically significant. However, the relative weight loss (percentage of body weight) was nearly significant.

Further studies with a greater number of participants and more statistical power are needed to determine whether this difference is real or just a chance occurrence.

Summary: Alternate-day fasting led to weight loss similar to that of a standard weight loss diet with a moderate calorie deficit.

Finding 2: Alternate-Day Fasting Led to a Greater Calorie Deficit

Participants who fasted every other day achieved a greater calorie deficit.

They consumed 376 fewer calories per day, on average, compared to those who were on the traditional weight loss diet.

The chart below shows the differences in calorie deficit between groups.

Calorie Deficit ADF TWD

This is a large reduction in calories that should lead to considerable weight loss over two months.

However, this extra calorie deficit didn’t seem to significantly affect weight loss, as shown in the previous section.

Possible explanations include the underreporting of food intake in the ADF group or a reduction in the number of calories burned.

Summary: Alternate-day fasting seemed to cause a greater calorie deficit, on average, compared to a traditional weight loss diet.

Finding 3: Alternative-Fasting Had Favorable Effects on Body Composition

After the intervention part of the study had ended, the researchers followed the participants for an additional six months.

During these six months, there were no significant changes in weight regain between groups.

However, when the researchers compared values from the start of the intervention, changes in percent fat mass (FM) and lean mass (LM) were significantly more favorable among those who fasted every other day.

These findings are presented in the chart below.

Change FM Trunk FM LM

These findings should be interpreted with caution since there were some between-group differences in body weight at the start of the study.

Summary: Alternate-day fasting appeared to beneficially affect body composition, compared to a traditional weight loss diet.

Finding 4: Resting Metabolic Rate Decreased in Both Groups

Both alternate-day fasting and traditional dieting caused a drop in the number of calories burned at rest (resting metabolic rate).

This effect is known as metabolic adaptation or starvation mode — the body’s response to a calorie deficit.

When the decrease in resting metabolic rate (RMR) was adjusted for fat mass and lean mass, the difference between groups became marginally significant. The findings are presented in the chart below.

Change In Resting Metabolic Rate

Summary: Both alternate-day fasting and a calorie-reduced diet caused a decrease in resting metabolic rate.

Finding 5: Alternate-Day Fasting Caused an Increase in BDNF

Previous studies suggest that fasting may improve mental performance, possibly due to changes in brain-derived neurotrophic factor (BDNF).

BDNF may also be involved in the regulation of energy balance (10111213).

In the current study, there were no differences in BDNF levels between groups.

However, at the end of the follow-up period, the researchers discovered that levels of BDNF had increased significantly among those in the ADF group, compared to the TWD group, as shown in the chart below.


These findings suggest that ADF may lead to long-term changes in the formation of BDNF, which might promote weight loss maintenance. This needs to be studied further.

Summary: Alternate-day fasting led to an increase in brain-derived neurotropic factor. The health relevance of this is unclear.

Finding 6: Effects of Alternate-Day Fasting on Appetite Hormones

Previous studies indicate that alternate-day fasting increases fullness after meals, as well as levels of the satiety hormone peptide YY (14).

In the present study, the researchers measured leptin (a satiety hormone) and ghrelin (the hunger hormone) at the start and end of the study. The findings are presented in the chart below.

Change Leptin Ghrelin ADF TWD

There were no significant differences in hormone changes between groups.

ADF also led to improvements in blood lipids. Once again, there were no significant between-group differences.

Summary: Alternate-day fasting and a traditional weight loss diet similarly affected the appetite hormones ghrelin and leptin.


The main limitation of the study was its small size. The low statistical power may explain the lack of significant differences in some of the outcomes.

Second, physical activity levels weren’t monitored. This might have affected the results.

Third, the researchers didn’t know how many of the participants continued following the ADF or TWD during the follow-up period.

Finally, food intake was strictly controlled, and the findings may not be generalized to a free-living population.

Summary and Real-Life Application

In short, this study suggests that alternate-day fasting is safe and at least as effective as a moderate, calorie-reduced diet.

It did not raise the risk of weight regain during the first six months after the weight loss program finished.

Although weight loss wasn’t significantly different between groups, there were some signs that alternate-day fasting may be more beneficial than continuous dieting. These findings need to be confirmed by larger studies.

Health risks of excessive Red meat consumption

Numerous observational studies have associated a high intake of red meat with an increased risk of heart disease and cancer.

In contrast, a high intake of fruits and vegetables is linked with a reduced risk of heart disease and cancer.

A recent observational study investigated whether high intakes of fruits and vegetables would counterbalance the link between red meat intake and poor health. Here is a summary of its findings.


Observational studies suggest that eating a lot of red meat may increase the risk of diabetes, heart disease, certain types of cancer and premature death (12345).

These associations are quite consistent across studies and populations. However, observational studies have a major weakness — they cannot prove causality.

As a result, scientists are still not entirely sure if high red meat intake itself is harmful or if other factors related to high red meat intake are responsible (16).

The unhealthy lifestyle habits sometimes associated with high red meat intake include:

  • Lower-quality diet (78).
  • Low consumption of fruits and vegetables (9).

Nevertheless, some scientists believe that certain components of red meat, especially in processed red meat or overcooked meat, may at least partially explain these associations.

Accordingly, some observational studies have linked processed meat with adverse health effects but not unprocessed, fresh red meat (51011).

Researchers have hypothesized that eating a lot of fruits and vegetables might partially offset the harmful effects of high red meat intake (9).

This hypothesis is supported by evidence indicating that high meat intake does not increase the risk of death when intake of fruits and vegetable is comparable to that of vegetarians (1213).

Article Reviewed

This observational study investigated the association of red meat intake with the risk of death from chronic disease at different levels of fruit and vegetable intake.

High red meat intake and all-cause cardiovascular and cancer mortality: is the risk modified by fruit and vegetable intake?

Study Design

This observational study examined whether high intakes of fruits and vegetables could offset the adverse health effects of red meat intake.

The researchers evaluated data from two large prospective studies, including a total of 74,645 Swedish men and women.

Food intake was evaluated using self-administered questionnaires asking how often people consumed fruits, vegetables, fresh meat or processed meat.

Fresh meats included fresh and minced pork, beef and veal, whereas processed meat included sausages, hot dogs, salami, ham, processed meat cuts, liver pate and blood sausage.

Bottom Line: This was an observational study investigating whether high intakes of fruits and vegetables can counterbalance the negative health effects of high red meat intake.

Finding 1: Red Meat Was Linked With an Increased Risk of Death

The study showed that eating a lot of red meat increased the risk of death from heart disease by 29% and the overall risk of death by 21%. However, it was not significantly associated with an increased risk of death from cancer.

The chart below shows the percent changes in the risk of death, compared to the lowest quintile of red meat consumption (less than 46 grams per day).

Interestingly, these associations were largely reduced when limiting the analyses to non-processed (fresh) red meat, suggesting that processed meat may be to blame.

Bottom Line: The study showed that a high intake of red meat, especially processed red meat, was significantly linked with an increased risk of death from heart disease or other causes.

Finding 2: Eating Fruits and Vegetables Didn’t Reduce the Health Risks of Red Meat Intake

The researchers discovered that red meat intake was associated with an increased risk of death, irrespective of how many fruits and vegetables people were eating.

The harmful effects of red meat were clearly dose dependent. The higher the intake, the more likely the participants were to die during the follow-up period.

Additionally, this association of red meat with death was independent of education status or unhealthy lifestyle habits, such as smoking or alcohol consumption.

The researchers also found that fruit and vegetable intake was not associated with total red meat intake or the intake of processed meat.

In other words, those who ate a lot of red meat didn’t necessarily eat less fruit and vegetables (FV), as shown in the chart below.

However, a high fruit and vegetable intake wasn’t associated with reduced risk of death from heart disease or other causes.

Bottom Line: High red meat intake was consistently associated with an increased risk of death at all levels of fruit and vegetable intake.


The study’s main limitation was its observational design – it couldn’t demonstrate causality.

Second, food intake was self-assessed using food frequency questionnaires (FFQs), which are often inaccurate.

In addition, the questionnaires didn’t ask about lamb and game meat intake, which might have skewed the results.

Third, the study revealed no protective effects from fruit and vegetable consumption. This might explain why they didn’t counterbalance the increased risk associated with red meat intake.

Summary and Real-Life Application

In short, this observational study indicates that eating a lot of fruits and vegetables does not counterbalance the harmful effects of high red meat intake.

However, since the study had a few limitations, its findings should be taken with a grain of salt. The results need to be confirmed in future studies.

Quercetin slows NAD+ consumption to combat Aging

Raising NAD+ levels back to youthful levels to combat aging is an exciting new field of research.

Supplementing with NAD+ precursors such as Nicotinamide Riboside  and NMN are getting a lot of the attention, and recent research is proving it is effective.

Rather than boosting NAD+ levels, an alternative and/or complementary approach seeks to remedy the cause of WHY NAD+ levels drop as we age.

Recent studies have found the enzyme CD38 RISES at the same time NAD+ levels decline, and seems to destroy NAD+. They found that inhibiting CD38 results in much higher NAD+ levels.

Other studies have found Flavonoids like Quercetin and Apigenin are effective at inhibiting CD38, resulting in higher NAD+ levels.


Quercetin  is a  flavonoid and known anti-inflammatory agent (1) that has been shown to have beneficial effects against cancer (2),  and atherosclerosis (3).

It is found in many vegetable, tea, coffee and red wine.

Quercetin supplements are  highly bioavable with a half-life of 11-28 hours which means that supplementation results in greatly increased blood plasma levels.

Possible health benefits: anti-carcinogenic, anti-inflammatory, antiviral, antioxidant, psychostimulant, capillary permeability, mitochondrial biogenesis

Quercetin is a natural product reputed and used traditionally for its beneficial effects on health. It is categorized as a flavonol, one of the six subclasses of flavonoid compounds. The name Quercetin is derived from quercetum (oak forest), after Quercus.

Quercetin has unique biological properties that offer potential benefits to overall health and disease resistance, including anti-carcinogenic, anti-inflammatory, antiviral, antioxidant, psychostimulant activities, capillary permeability and stimulation of mitochondrial biogenesis. Quercetin is present in various vegetables as well as in tea and red wine.

In a typical Western diet the daily intake of quercetin is estimated to be in the range of 0 and 30 mg.

Quercetin is also one of the most complex flavonol compounds to understand due to its metabolism. It involves intestinal uptake and/or deglycosylation, glucuronidation, sulfation, methylation, possible deglucuronidation and so on.

From the various quercetin metabolites generated in the body it has been recently demonstrated that quercetin “3-O-β-D-glucuronide (Q3GA)” and quercetin “3′-sulfate” are the dominant quercetin conjugates in human plasma. Typically, the human quercetin plasma concentration is in the order of nanomolar, but upon quercetin supplementation it may increase upto the low micromolar range.

This together with a half-life of the atom and its metabolites in the range of 11-28h it can be assumed that continuous supplementation leads to an  increased plasma concentration.

Quercetin2In human studies, quercetin has been mostly well tolerated.

Doses up to 1,000 mg/day for several months did not produce adverse effects on blood parameters, liver and kidney function,hematology, or serum electrolytes. In general, there is plentiful available evidence that support the safety of quercetin for addition to food. A few items should be noted however.

At high dosing, quercetin has been shown to inhibit topoisomerase II which is an essential enzyme in DNA replication and inhibition leads to diplochromosomes.

These results have led to some  questioning the usefulness of quercetin as this introduces for example a possible risk of liver cancer while other studies find that this same property has anti-cancer treatment qualities.

The second note is with regards to other drug interactions. Due to is complexity it may interfere with these prescription medications.

Therefore, it is great to see the continuing research into characterizing the promising benefits and also potential side effects of this novel dietary supplement.



Quercetin the key behind Rutin’s many benefits

rutin-quercetinThe more calories you store over time — the more you’ll be stuck feeling:

  • Bloated and sleepy
  • Unusually weak
  • Mentally drained
  • Hungry even after you just ate

But there’s good news. Doctors and scientists are now rethinking EVERYTHING they know about weight loss, thanks to a new discovery. No, we’re not talking about Garcinia Cambogia – that is so 2016…

An ingredient that burns BIG amounts of fat… even if you’re just sitting down!

I know… when I looked at the research I almost couldn’t believe it myself. But this incredible ingredient is really catching the attention of the scientific community…

… and could start making a HUGE difference in people’s lives sooner than you think.

It’s called Rutin, an all-natural compound that’s theorized to kickstart your Brown Adipose Tissue fat — or “Brown fat.”

You may have heard of this before. Brown fat is the incredible “permanent fat” that keeps you warm by using your regular calorie-packed fat as fuel.

So in a study published just last month by The FASEB Journal,scientists tested Rutin’s fat-burning effects on obese mice…

And incredibly, Rutin didn’t just work…

It significantly boosted metabolism and weight loss in 100% of the obese mice… without any exercise! 1

Brown Fat Is GOOD

brownfatgraphicSo can activating Brown fat work on YOU?

Well, unfortunately, the older humans get — the less Brown fat they have…

In fact, the humans with the most Brown fat are actually babies

Luckily, adults still keep a tiny fraction of their original Brown fat…

And cutting-edge research now shows activating this tiny amount of “helpful” fat could work on HUMANS at any age.

So to test this, researchers from the elite University of Sherbrooke “activated” the Brown fat of healthy human subjects.

And over the course of a three-hour “sitting” period, the researchers couldn’t believe the numbers they were seeing…

The human subjects with “activated Brown fat” burned an incredible 1.8 times more calories compared to the others! 2

That’s right — by activating their own Brown fat, the human subjects nearly DOUBLED their fat-burn… by just sitting there.

So how can you start feeling the powerful effects of Rutin?

Well, one of the BIGGEST sources of Rutin is actually in mulberries — a flavorful fruit jam packed with lots of other vitamins and minerals, too…

And conveniently, this potent berry is available everywhere — so you can start feeling the powerful effects of Rutin right now.

I’d say the best thing to do is go stock up on mulberries at your local grocery store.

Not all mulberries are the same. In fact, the mulberries with the highest levels of healthy vitaminsantioxidant contentand Rutin are the mature, dark berries. So when you’re at the grocery store DO NOT get the hard, colorless mulberries. Because they aren’t RIPE and won’t give you nearly as much good stuff as the dark, mature mulberries!



This June 2016 study shows that CD38 increases dramatically with age and plays a key role in destroying Nicotinamide MonoNucleotide (NMN), a NAD+ precursor.

The authors show that protein levels of CD38 increased in multiple tissues during aging.

They then compared the relationship of CD38 levels to NAD+ of normal mice  vs CD38 Knockout mice (Mice bred without the gene to product CD38).

PowerPoint Presentation

The NAD+ levels of normal mice aged 32 months are about 1/2 that of young mice, whereas the CD38 knockout mice showed no decrease in NAD+ levels at the same age.

This study did not suggest a cause for the rapid increase in CD38 activity but other studies have shown a link to CD38 increase with inflammation from disease and injury as we age.

The exponential rise suggests that CD38 may deplete NAD+ needed for other processes and directly relate to the aging process.

Finally, the authors addressed how CD38 may affect therapies designed to raise NAD+ levels. Currently, the favored approach in mouse and humans is to treat with NAD+ precursors, such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN).

Interestingly, CD38 not only degrades NAD+ in vivo, but also NMN. When CD38 knockout mice were given injections of NAD+, NMN, or NR (which is converted to NMN), circulating levels of NAD metabolites remained stable after 150 min, long after they began to fall in the wild-type animals.

Furthermore, when compared to the wild-type, CD38 knockout mice on a high-fat diet exhibited a much larger improvement in glucose tolerance when given NR.

Conclusion:CD38 degrades NAD+ and its precursors. Inhibiting CD38 can lead to increased NAD+ levels


Prior research shows that declining NAD+ levels is linked to many age related diseases and metabolic disorders such as diabetes, and a possible contributing factor to aging (4).

David_Sinclair_solo_mid_0_0_4_1A  landmark 2013 study by Dr David Sinclair demonstrated that increased levels of NAD+  have been  shown to reverse age related degeneration in mice, giving older mice the muscle capacity, endurance and metabolism of much younger mice (5).

Other studies have shown that supplementing old mice with  NAD+ precursors can greatly improve metabolic health such as  increased insulin sensitivity, improved mitochondrial function, reduced stem cell senescence, and increased lifespan (6,7,8)

This suggests that many of the the normal age related conditions are at least partly driven by decreased mitochondrial functioning, and that increasing NAD+ levels can restore mitochondrial functioning and reverse many age related problems (9).

Conclusion: Top researchers have been investigating raising NAD+ levels for treatment of disease and degenerative aging.



While many of the health benefits of Quercetin are well documented, the mechanisms of action  are not completely understood.

In this study  published April 2013, obese mice that received Apigenen or Quercetin showed improved glucose homeostasis, glucose tolerance, and lipid metabolism.

The authors theorized the mechanism may be the anti-inflammatory properties inhibit  CD38 which results in  increased NAD+ levels in tissues.

First, they measured the effect of quercetin on endogenous cellular CD38 activity and found that quercetin promotes an increase in intracellular NAD+ in a dose-dependent manner (Fig. 4B).

They also compared NAD+  levels of normal mice with CD38 knockout mice after supplementation with Quercetin and found it  promotes an increase in NAD+ in normal mice but does not further increase NAD+ levels in CD38 knockout mice (Fig. 4D), indicating that the effect of quercetin on NAD+ levels is CD38 dependent.

They were able to demonstrate that Quercetin  inhibits CD38 and promotes an increase in NAD+ levels.

Conclusion: The researchers concluded that apigenin and quercetin as well as other CD38 inhibitors may be used to raise NAD+ levels and treat metabolic syndrome and obesity-related diseases.


Supplementation with NAD+ precursors such as Nicotinamide Riboside is generating a lot of excitement as recent research is proving it is effective at raising NAD+ levels in humans, which has tremendous  therapeutic potential to treat metabolic and age-related disease.

Dr Sinclair is at the forefront of research on NAD+ levels and their effects on aging. In this video (18 minute mark), he demonstrates recent research supplementing with NAD+ precursors to return old mice to youthful states.

He recently published this article that reviews the studies on CD38, with the following findings:

    • Results from  these and other studies with CD38 demonstrate that combating the rise of CD38 is also a promising approach to protect NAD+ levels.
    • These findings suggest that the efficacy of NAD+ precursors may be enhanced by co-supplementation with CD38 inhibitors
Conclusion: Inhibiting CD38 to prevent NAD+ destruction AND supplementing with NAD+ precursors so the body can create more NAD+ is a promising new avenue in the anti-aging battle

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Results from this study need to be folded into the article above

Several epidemiologic studies have indicated that coffee consumption has a neuroprotective effect against Parkinson disease (PD) and Alzheimer disease (AD). Liu et al. (2012) analyzed 304,980 participants in the National Institute of Health (NIH) study and found a mild protective effect of coffee against PD. Palacios et al. (2012) found a similar effect in a smaller cohort, again attributing it to caffeine intake. Qi and Li (2014) did a meta-analysis of 13 articles on coffee, tea, and caffeine consumption and concluded there was a linear dose-response effect reaching a maximum at approximately 3 cups of coffee per day. As far as AD is concerned, Eskelinen and Kivipelto (2010) reported that caffeine and other factors in coffee were possible protective factors. On the basis of the Cardiovascular Risk Factors, Aging and Dementia study they concluded that drinking 3e5 cups of coffee at midlife was associated with a decreased risk of dementia/AD of about 65% in late life, and this was due to caffeine and/or other mechanism such as antioxidant capacity by other components. Eskelinen et al. (2009) showed that drinking 3e5 cups of decaffeinated coffee at midlife decreases the risk of AD.


Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson’s disease and Alzheimer’s disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-g or interferon-g released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-a and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/ nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson’s disease and Alzheimer’s disease.

Attributing these effects to caffeine in coffee was recently challenged by the findings of Ding et al. (2015). They examined the effects of consuming total, caffeinated, and decaffeinated coffee in 3 very large cohorts of men and women. They found significant inverse associations with coffee consumption and deaths attributed to cardiovascular disease, neurological diseases, and suicide. There was no significant difference between consuming caffeinated or decaffeinated coffee. This latter report indicated that other constituents of coffee than caffeine must be responsible for the protective effect. For example, flavonoids are polyphenolic bioactive compounds that are found in foodstuffs of plant origin (Babu et al., 2013). Flavonoids are classified into subgroups based on their chemical structure: flavanones, fla- vones, flavonols, flavan-3-ols, anthocyanins, and isoflavones. Flavonoids have a backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one). Specifically, adding hydroxyl groups to the backbone generates flavonols including quercetin and flavones such as apigenin and luteolin. It is known that they functions generally to remove oxidant and to inhibit inflamma- tion (Marzocchella et al., 2011).

In this report, we examined the effects of caffeine and other well-known coffee components, such as quercetin, flavone, and chlorogenic acids (CGAs), on neuroinflammation and neurotoxicity mediated by toxic factors from activated microglia and astrocytes. We also investigated changes in oxidative stress markers caused by quercetin and caffeine because depletion of glutathione (GSH) in glial cells induces neuroinflammation resulting in neuronal death (Lee et al., 2010a).

We found that CGA, flavone, quercetin, and caffeine reduced the release of proinflammatory cytokines such as tumor necrosis factor- a (TNFa) and interleukin-6 (IL-6) from lipopolysaccharide/inter- feron-g (LPS/IFNg)-stimulated microglia and THP-1 cells, as well as from IFNg-stimulated astrocytes and U373 cells. The toxicity was attenuated in a concentration and incubation-time dependent manner. This was due to reduced activation of intracellular inflammatory pathways such as P38 mitogen-activated protein ki- nase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB) proteins and decreased release of proin- flammatory cytokines such as TNFa and IL-6. We found that quer- cetin was the most potent anti-inflammatory and neuroprotective coffee component. In addition, quercetin has antioxidative prop- erties, but caffeine does not.

The data indicate that quercetin, but not caffeine, is a major component reducing the risk of pathogenesis in degenerative neurological diseases such as PD and AD. It may prove to be a useful therapeutic agent.

2. Methods

2.1. Materials

All reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. The following substances were applied to the cell cultures: bacterial LPS (Escherichia coli 055:B5) and human recombinant IFNg (Bachem California, Torrance, CA, USA).

2.2. Cell culture and experimental protocols

The human monocyte THP-1 and astrocytoma U373 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The human neuroblastoma SH-SY5Y cell line was a gift from Dr R. Ross, Fordham University, NY, USA. These cells were grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 100 IU/mL penicillin and 100 mg/mL streptomycin (Invitrogen) under humidified 5% CO2 and 95% air.

Human astroglial and microglial cells were isolated from sur- gically resected temporal lobe tissue as described earlier (Lee et al., 2010b). Briefly, tissues were rinsed with a phosphate-buffered saline (PBS) solution and chopped into small (<2 mm3) pieces with a sterile scalpel. They were treated with 10 mL of a 0.25% trypsin solution at 37 C for 20 minutes. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d’Urfé, PQ, Canada) was added to reach a final concentration of 50 mg/mL. Tissues were incubated for an additional 10 minutes at 37 C. After centrifugation at 275g for 10 minutes, the cell pellet was re-suspended in the serum-containing medium and passed through a 100-mm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA). The cell sus- pension was centrifuged again (275g for 10 minutes) and re- suspended in 10 mL of DMEM/F12 with 10% FBS containing gentamicin (50 mg/mL), and plated onto tissue culture plates (Becton Dickinson) in a humidified 5% CO2, 95% air atmosphere at 37 C for 2 hours. This achieved adherence of microglial cells. The nonadherent astrocytes along with myelin debris were transferred into new culture plates. Astrocytes adhered slowly and were allowed to grow by replacing the medium once a week. New pas- sages of cells were generated by harvesting confluent astrocyte cultures using a trypsineEDTA solution (0.25% trypsin with EDTA,

Invitrogen). Human astrocytes from up to the fifth passage from 4 surgical cases were used in the study.

For estimating the purity of astrocytic and microglial cell cul- tures, aliquots of the cultures were placed on glass slides at 37 C for 48 hours. The attached cells were then fixed with 4% para- formaldehyde for 1 hour at 4 C and made permeable with 0.1% Triton X-100 for 1 hour at room temperature. After washing twice with PBS, the astrocytic culture slides were treated with a mono- clonal anti-glial fibrillary acidic protein antibody (1/4,000, DAKO) and the microglial slides with the polyclonal anti-ionized calcium- binding adapter molecule 1 (Iba-1) antibody (1/500, Wako Chem- icals, Richmond, VA, USA) for 3 hours at room temperature. The slides were then incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG antibody (Invitrogen, 1:500) and Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody (Invitrogen, 1:500) in the dark for 3 hours at room temperature to yield a positive red fluorescence. To visualize all cells, the slides were washed twice with PBS and counterstained with the nuclear dye 40,6-diamidino- 2-phenylindole, dihydrochloride (DAPI) (100 mg/mL, Sigma) to give a blue fluorescent color. Images were acquired using an Olympus BX51 microscope and a digital camera (Olympus DP71). Fluorescent images were colocalized with ImagePro software (Improvision Inc, Waltham, MA, USA). The purity of microglia and astrocytes were more than 99% (2.54 ` 0.54 astrocytes in 500 total cells in micro- glial culture and 3.17 ` 0.62 microglia in 500 total cells in astrocytic culture, n 1⁄4 30).

To achieve SH-SY5Y differentiation, the undifferentiated cells were treated for 4 days with 5-mM retinoic acid (RA) in DMEM/F12 medium containing 5% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Singh et al., 2003). The RA-containing medium was changed every 2 days. Differentiated SH-SY5Y cells demonstrated neurite extension, indicative of their differentiation (Lee et al., 2013).

2.3. Experimental protocols

2.3.1. Protocol 1

Human astrocytes, U373 astrocytoma cells and THP-1 cells (5 105 cells), and human microglial cells (5 104 cells) were seeded into 24-well plates in 1 mL of DMEM/F12 medium con- taining 5% FBS. Caffeine, chlorogenic acid, quercetin, and flavones (Sigma, St. Louis, MO, USA) were then introduced at concentra- tions of 100 ng/mL to 1 mg/mL. The stock solutions were prepared with organic solvents; dimethyl sulfoxide (DMSO) for quercetin, caffeine, and CGA and acetone for flavone. Incubation of the mixtures was carried out for 2, 4, 8, or 12 hours. One set of cells was then incubated at 37 C for 2 days in the presence of in- flammatory stimulants. For microglia and THP-1 cells, the stimu- lants were LPS at 1 mg/mL and IFNg at 333 U/mL. For astrocytes and U373 cells, the stimulant was IFNg alone at 150 U/mL. A compa- rable set of cells was incubated in media without inflammatory stimulants. After incubation, the supernatants (400 mL) were transferred to differentiated human neuroblastoma SH-SY5Y cells (2 105 cells per well). The SH-SY5Y cells were incubated for further 72 hours, and MTT assays were performed as described in the following section.

2.3.2. Protocol 2

Because it is perceived that caffeine, chlorogenic acid, quercetin, and flavones could be useful as pharmaceutical agents, they must be shown to be nontoxic to humans. To determine whether they were directly affecting SH-SY5Y cell viability in the presence of LPS/ IFNg-stimulated THP-1 conditioned medium (CM) or IFNg-stimu- lated U373 CM, each compound was added to a glial cell superna- tant (400 mL) just before the supernatants were added to the SH-SY5Y cells. The glial cell supernatants were from THP-1 cells or

U373 cells that had been activated for 2 days with the inflammatory stimulants previously described. The subsequent procedures were the same as in protocol 1.

2.4. SH-SY5Y cell viability assays

The viability of SH-SY5Y cells after incubation with glial cell supernatants was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays as previously described (Lee et al., 2011). Briefly, the viability was determined by adding MTT to the SH-SY5Y cell cultures to reach a final concentration of 1 mg/mL. After a 1 hour incubation at 37 C, the dark crystals formed were dissolved by adding an SDS/DMF extraction buffer (300 mL, 20% sodium dodecyl sulfate, 50% N, N-dimethylformamide, pH 4.7). Subsequently plates were incubated overnight at 37 C, and optical densities at 570 nm were measured by transferring 100 mL aliquots to 96-well plates and using a plate reader with a corre- sponding filter. Data are presented as a percentage of the values obtained from cells incubated in fresh medium only.

2.5. Measurement of TNFa and IL-6 release

Cytokine levels were measured in cell-free supernatants after 48-hour incubation of THP-1 cells, U373 cells, microglial cells, and astrocytes. The cell stimulation protocols in these experiments were the same as that used in protocol 1. Quantitation was per- formed with enzyme-linked immunosorbent assay (ELISA) detec- tion kits (Peprotech, NJ, USA) following protocols described by the manufacturer.

2.6. Activation of P38 MAPK and NFkB protein by Western blotting

Western blotting on cell lysates was performed as described previously (Lee et al., 2011). Briefly, microglia and astrocytes were exposed to quercetin and caffeine at 10 mg/mL each for 8 hours and were subsequently exposed to stimulants for 2 hours. Human microglia and astrocytes were treated with a lysis buffer (150 mm NaCl, 12 mm deoxycholic acid, 0.1% Nonidet P-40, 0.1% Triton X-100, and 5 mm Tris-EDTA, pH 7.4). The protein concentration of the cell lysates was then determined using a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Proteins in each sample were loaded onto gels and separated by 10% sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) (150 V, 1.5 hours). The loading quantities of lysate proteins were 100 mg. Following SDS- PAGE, proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, CA, USA) at 30 mA for 2 hours. The mem- branes were blocked with 5% milk in PBS-T (80 mm Na2HPO4, 20 mm NaH2PO4, 100 mm NaCl, 0.1% Tween 20, pH 7.4) for 1 hour and incubated overnight at 4 C with a polyclonal anti-phospho- P38 MAP kinase antibody (9211, Cell Signaling, Beverly, MA, USA; 1/2000) or anti-phospho-P65 NFkB antibody (3031, Cell Signaling; 1/1000). The membranes were then treated with a horseradish peroxidase-conjugated anti-IgG (P0448, DAKO, Mississauga, Ontario, CA, USA; 1:2000) or the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3000) for 3 hours at room temperature, and the bands were visualized with an enhanced chemiluminescence system and exposure to photographic film (Hyperfilm ECL, Amer- sham Biosciences). Equalization of protein loading was assessed independently using a-tubulin as the housekeeping protein. The primary antibody was anti-a-tubulin (T6074, Sigma, 1/2000) and the secondary antibody was anti-mouse IgG (A3682, Sigma, 1/ 3000). Primary antibody incubation was overnight at 4 C, and the secondary antibody incubation was for 3 hours at room temperature.

2.7. Glutathione level

The GSH level was assessed by the method of Hissin and Hilf (1976) and Lee et al. (2010a). This assay detects reduced GSH by its reaction with o-phthalaldehyde at pH 8.0. Cells (106) in 1.5-mL tubes were washed twice with PBS, and 200 mL of 6.5% trichloro- acetic acid (TCA) was added. The mixture was incubated on ice for 10 minutes and centrifuged (13,000 rpm, 1 minute). The superna- tant was discarded, and the pellets were re-suspended in 200 mL of ice-cold 6.5% TCA and centrifuged again (13,000 rpm, 2 minutes). Supernatants (7.5 mL) were transferred to 96-well plates containing 277.5-mL phosphate-EDTA buffer (pH 8.0) in 1 M NaOH solution. Then 15 mL o-phthalaldehyde (1 mg/mL in methanol) was added. The reaction mixture was incubated in the dark at room tempera- ture for 25 minutes. The fluorescence at 350 nm excitation/420 nm emission was measured in a multiwell plate reader. The concen- tration was calculated from a standard curve using serial dilutions of reduced GSH. The concentration was expressed as mmol/g protein.

2.8. Protein carbonyls

Protein carbonyl content was determined by a modification of the procedure of Lyras et al. (1996). Cells (3 106) were lysed with a buffer containing 0.2% Triton X-100, 60 mL of protease inhibitor cocktail, and 1 mL of phenylmethylsulfonyl fluoride in 100 mM KH2PO4/K2HPO4 (pH 7.4). The lysate was incubated at 37 C for 2 hours, centrifuged at 8000 g, and the supernatant collected. A 10% (w/v) streptomycin sulfate solution was added to the supernatant at a final concentration of 1% to remove remaining nucleic acid. The solution was mixed at room temperature for 10 minutes and centrifuged for 10 minutes at 8000g. The supernatant was removed and 800 mL divided equally into 2 12 mL plastic centrifuge tubes. For each 1 mL of supernatant, 400 mL of 10-mM 3, 4-dinitrophenylhydrazine in 2 M-HCl was added to one tube and 400 mL of 2-M HCl to the other tube. The tubes were then incubated for 1 hour, and the protein was precipitated by adding an equal volume of 20% (w/v) TCA. The tubes were centrifuged at 8000g, the supernatants discarded, and the pellets washed several times with 1.5 mL of an ethyl acetate ethanol mixture (1:1) to remove excess 3, 4-dinitrophenylhydrazine. The final protein pellets were dissolved in 1 mL 6-M guanidium hydrochloride and the absorbance of both solutions measured at 280 nm and 370 nm as per Reznick and Packer (1994).

2.9. Measurements of 8-OHdG, lipid peroxide and 3-nitrotyrosine by ELISA assays

Levels of 8-hydroxy-2’-deoxyguanosine (OHdG; JaICA, Shi- zuoka, Japan), lipid peroxide (Cayman Chemical, Ann Arbor, MI, USA) and 3-nitrotyrosine (Nitrotyrosine assay kit, Millipore, Temecula, CA, USA) were measured in cell extracts after 2e3 days of incubation. The protocols were the same as previously described measuring cell viability. Quantitation was performed with ELISA detection kits following protocols described by the manufacturer.

2.10. Data analysis

The significance of differences between data sets was analyzed by 1-way or 2-way analysis of variance tests. Multiple group com- parisons were followed by a post hoc Bonferroni test. p values are given in the figure legends.

3. Results

We purchased small amounts (10 oz 1⁄4 approximately 280 mL) of coffee from 3 different companies and dried it completely at room temperature. Dried weights were found to average 140 mg. To assess the neuroprotective effect of various coffee constituents, we dissolved the dried coffee in sterilized water. We then added supernatants from stimulated THP-1 cells and U373 cells and incubated the mixture for 2 hours (final concentrations: 140 mg/ 280 mL 1⁄4 0.5 mg/mL). The stimulants for THP-1 cells were LPS/IFNg and for U373 cells, IFNg was applied for 2 days. The resultant CM from both THP-1 cells and U373 cells was transferred to RA-differentiated SH-SY5Y cells (experimental protocol 1). MTT assays were performed after 3 days. Data are shown in Fig. 1. There were significant increases in SH-SY5Y cell survival after treatment with CMs from LPS/IFNg-stimulated THP-1 cells and IFNg-stimu- lated U373 cells that had been exposed to coffee and decaffeinated coffee.

In coffee beans, the constituents most likely to exert anti- inflammatory and antioxidative function are caffeine, quercetin, flavonoids, and CGA as well as their derivatives (Kreicbergs et al., 2011). The amounts of the 4 compounds included in 100 grams of coffee beans are 280 mg for CGA, 200 mg for quercetin, 60 mg for flavones (a representative of flavonoids), and 40 mg for caffeine. We calculated the amount of each compound in 140 mg dried weight of coffee (CGA: 392 mg, quercetin: 280 mg, flavones: 84 mg and caffeine: 56 mg). We found that mixtures of the 4 components protected SH-SY5Y cells from toxicity of LPS/IFNg-stimulated THP-1 CM and IFNg-stimulated U373 CM just as much as both types of coffee (Fig. 1). The data indicate that the protective function of coffee beans must come almost entirely from these 4 compounds.

We then investigated the protective effects of caffeine, CGA, flavones, or quercetin (10 mg/mL each), alone, or in combination, against the toxicity of CMs toward RA-differentiated SH-SY5Y cells. The CMs were obtained by 2 days treatment of LPS/IFNg-activated

Fig. 2. Effects of caffeine (CAF in X-axis), chlorogenic acid (CGA in X-axis), flavone (FLA in X-axis), and quercetin (QUE in X-axis) alone (10 mg/mL each) or their combination on SH- SY5Y cell viability changes mediated by (A) LPS/IFNg-stimulated THP-1 and (B) IFNg-stimulated U373 CMs. THP-1 cells and U373 cells were pretreated with each compounds or their mixtures for 2 h and were exposed to stimulants for 2 d. Then the cell-free supernatants were transferred to RA-differentiated SH-SY5Y cells (Experimental protocol 1). MTT assays were carried out in 3 d. Values are mean ` standard error of the mean, n 1⁄4 4. Two-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. *p < 0.01 for LPS/IFNg-activated THP-1 (A) or IFNg-activated U373 groups (B) compared with control (CON) groups, **p < 0.01 for CGA and FLA groups compared with LPS/IFNg-activated THP-1 (A) or IFNg-activated U373 groups (B) or CAF groups, ***p < 0.01 for QUE groups compared with CGA and FLA groups, #p < 0.01 for CAF þ CGA and CAF þ FLA groups compared with CAF groups, ##p < 0.01 for CAF þ QUE groups compared with CAF þ CGA and CAF þ FLA groups, $p < 0.01 for CGA þ FLA groups compared with CAF þ CGA and CAF þ FLA groups, and $$p < 0.01 for CGA þ QUE, FLA þ QUE groups compared with FLA þ CGA groups. Note that quercetin was most potent anti-inflammatory and neuroprotective compounds in coffee components and caffeine was not effective. Abbreviations: CGA, chlorogenic acid; CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.

THP-1 cells or IFNg-activated U373 cells and were then transferred to differentiated SH-SY5Y cells. MTT assays were performed after 3 days (see experimental protocol 1). It was found that CGA, flavones, and quercetin, but not caffeine, attenuated SH-SY5Y cell viability loss by THP-1 CM (Fig. 2A) as well as U373 CM (Fig. 2B). Quercetin demonstrated the most protective effect. It was also the most protective when it was added to any of the 3 other compounds.

3.1. Neuroprotectiveeffectofquercetin,caffeine,chlorogenicacid,or flavones against microglial, astrocytic, THP-1, and U373 cell toxicity

The effects of pretreatment with quercetin, caffeine, CGA, or flavones (100 ng/mLd1 mg/mL each) for 2, 4, 8, or 12 hours before incubation on the toxicity of LPS/IFNg-stimulated THP-1 CM toward SH-SY5Y cells were investigated (experimental protocol 1). It was found that quercetin, caffeine, CGA, or flavones attenuated SH-SY5Y cell viability loss by THP-1 CM in a concentration and preincubation time-dependent manner [Fig. 3AeD; (A): 2 hours before incuba- tion, (B): 4 hours before incubation, (C): 8 hours before incubation, and (D): 12 hours before incubation]. Quercetin was the most protective [p < 0.01 compared with untreated group from 3 mg/mL and 2 hours before incubation (A), from 1 mg/mL and 4 hours before incubation (B), from 0.3 mg/mL and 8 hours before incubation (C), and from 0.1 mg/mL and 12 hours before incubation (D)]. Caffeine was the least effective [p < 0.01 compared with untreated group from 100 mg/mL and 2 hours before incubation (A), from 30 mg/mL and 4 hours before incubation (B), from 10 mg/mL and 8 hours before incubation (C), and from 3 mg/mL and 12 hours before in- cubation (D)]. CGA and flavone were also effective but less so than

quercetin [p < 0.01 for quercetin group compared with CGA or flavone groups from 10 mg/mL and 2 hours preincubation (A), from 3 mg/mL and 4 hours preincubation (B), from 1 mg/mL and 8 hours preincubation (C), and from 0.3 mg/mL and 12 hours preincubation (D)]. At the shortest time interval of 2 hours and at the lowest concentration of 3 mg/mL, the protective effect of quercetin was minimal. But the toxicity was reduced to about half at a concen- tration of 1 mg/mL (p < 0.01 from 1 mM). By 12 hours the protective effect had increased to the point where the lowest concentration reduced the toxicity by about 1/4 while the highest concentration reduced it by about 4 fold.

We also investigated the effect of supernatants from LPS/IFNg- stimulated microglia and IFNg-stimulated astrocytes on SH-SY5Y cell viability after treatment of both types of glia with the agents in coffee. Owing to the limited availability of microglia and astrocytes, we only examined the protective effects of quercetin against glial-mediated neurotoxicity. Caffeine was used for com- parison. Both microglia and astrocytes were pre-exposed to the compounds at only 1 time period (8 hours) before treatment with stimulatory agents (experimental protocol 1). For measuring SH-SY5Y cell viability, MTT assays were utilized. It was observed that both quercetin and caffeine reduced the toxicity of both LPS/ IFNg-stimulated microglia (Fig. 5A) and IFNg-stimulated astro- cytes (Fig. 5B) toward SH-SY5Y cells (quercetin: for microglia p < 0.01 compared with control at 0.1 mg/mL and for astrocytes p < 0.01 compared with control at 0.3 mg/mL; and caffeine: for microglia and astrocytes p < 0.01 compared with control at 10 mg/ mL). Again, quercetin had a greater neuroprotective effect than caffeine (for microglia: p < 0.01 compared with caffeine group at the same concentration [0.1 mg/mL] and for astrocytes: p < 0.01 compared with caffeine group at the same concentration [0.3 mg/ mL]). These data demonstrate that the effects observed with cultured microglia and astrocytes are comparable to those observed with the THP-1 and U373 cell lines.

However, although CGA, flavones, quercetin, and caffeine had a protective effect against neuronal SH-SY5Y cells, they had no pro- tective effect against glial cells. Treatment of THP-1 cells or U373 cells for 12 hours, and microglia and astrocytes for 8 hours, with stimulated CM did not change the viability of any glial cells (Supplemental Fig. 1). Fig. 6A and B demonstrates that quercetin and caffeine acted indirectly and were not directly protective of SH-SY5Y cells. When they were added to the CM after stimulation had taken place (experimental protocol 2), they had no effect (A: THP-1 cells and B: U373 cells). As the figure shows, there was no difference between the agents and there was no effect of concen- tration. This establishes that the agents were working by inhibiting

The table summarized the IC50 results of the studies in Figs. 3 and 4. Values are mean ` standard error of the mean, n 1⁄4 4. Two-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. Note that there was a significant reduction in IC50 values between any pre- incubation time groups and that there was a significant reduction in IC50 values of quercetin compared with those of CGA and flavone in the same incubation time groups and of CGA and flavones compared with those of caffeine.

the glial inflammatory response. In summary, quercetin, caffeine, CGA, and flavone were not toxic to the glial cells in the presence of inflammatory stimuli. The viability of SH-SY5Y cells treated with the CM from LPS/IFNg-stimulated THP-1 and IFNg-stimulated U373 cells was unchanged.

3.2. Release of inflammatory cytokines

Inflammatory stimulation of microglia or THP-1 cells causes them to release the inflammatory cytokines TNFa and IL-6 (Hashioka et al., 2007; Klegeris et al., 1999). Fig. 7 shows the effect on TNFa release of treatment of glial cells with quercetin and caffeine (100 ng/mL to 1 mg/mL for 8 hours before incubation). THP-1 release of TNFa (Fig. 7A) and IL-6 (Fig. 7B) and human microglial release of TNFa (Fig. 7C) and IL-6 (Fig. 7D) are illustrated. The release of TNFa (Fig. 7A) and IL-6 (Fig. 7B) was reduced by quercetin and caffeine in a concentration-dependent manner (for quercetin: p < 0.01 for 100 ng/mL or higher and for caffeine: p < 0.01 for 10 mg/mL or higher). The inhibitory effects of quercetin were more powerful than caffeine (p < 0.01 for 300 ng/mL or higher for TNFa and p < 0.01 for 3 mM or higher for IL-6). The pattern was similar in microglia (Fig. 7C and D). LPS/IFNg stimulation caused a 9.5-fold increase of TNFa and IL-6. Treatment with quercetin and caffeine reduced this release (for quercetin: p < 0.01 for 100 ng/mL or higher and for caffeine: p < 0.01 for 300 ng/mL or higher).

For astrocytes, IL-6 is the main inflammatory mediator that is generated (Van Wagoner et al., 1999). Fig. 8 shows comparable data for IL-6 release from U373 cells (Fig. 8A) and cultured astrocytes (Fig. 8B). When cells were activated with IFNg, U373 cells and hu- man primary-cultured astrocytes release IL-6 (7-fold increase in U373 cells and 15-fold increase in astrocytes). However, both quercetin and caffeine reduced the release of IL-6 from IFNg-acti- vated U373 cells or astrocytes in a concentration-dependent manner (for quercetin: p < 0.01 for 100 ng/mL or higher in both cells and for caffeine: p < 0.01 for 30 mg/mL or higher in U373 cells and for 10 mg/mL in astrocytes). Again, quercetin is stronger in reducing IL-6 release than caffeine in both cell types (p < 0.01 for 100 ng/mL or higher in both).

3.3. Activation of intracellular inflammatory pathway in microglia and astrocytes

We investigated the effects of pretreatment with quercetin and caffeine at 10 mg/mL for 8 hours on activation of intracellular inflammatory pathway such as phospho-P38 MAPK and phospho- NFkB production. The data are shown in Fig. 9. On exposure to the stimulants, both microglia and astrocytes showed an increase in these proteins. For microglia, P38 MAPK was increased 8-fold and NFkB 9-fold. For astrocytes, both proteins were increased 8- to 10-fold. Both quercetin and caffeine at 10 mg/mL for 8 hours before

Fig. 5. Effect of pretreatment with caffeine or quercetin for 8 h on SH-SY5Y cell viability changes induced by (A) LPS/IFNg-activated microglial CM and (B) IFNg-activated astrocytic CM as followed by MTT assays. See experimental protocol 1 in Section 2. Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated (A) or IFNg-activated (B) groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated (A) or IFNg-activated groups (B) #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.

Fig. 6. Effect of treatment with caffeine, CGA, flavones, and quercetin on SH-SY5Y cell viability changes induced by LPS/IFNg-activated THP-1 cell CM (A) or IFNg-activated U373 cell CM (B) as followed by MTT assays. (A) THP-1 cells and (B) U373 cells. After THP-1 cells and U373 cells were stimulated for 2 d with LPS/IFNg or IFNg, respectively their supernatants were transferred to SH-SY5Y cells. Then caffeine, CGA, flavones, and quercetin were added. MTT tests were performed after 3 d (See experimental protocol 2 in Section 2). Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests where necessary. Note that there are no viability changes when all the compounds were exposed to SH-SY5Y cells after LPS/IFNg-activated THP-1 cell CM (A) or IFNg-activated U373 cell CM (B) were transferred. Abbreviations: CGA, chlorogenic acid; CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.

Fig. 7. Effect of pretreatment with caffeine or quercetin on released levels of TNFa (A, C) or IL-6 (B, D) from LPS/IFNg-activated THP-1 cells (A and B) or LPS/IFNg-activated microglia (C and D). 8 h preincubation with caffeine or quercetin was performed before LPS/IFNg was exposed to the cells for 2 d. Values are mean ` standard error of the mean, n 1⁄4 4. One- way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated groups, and #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: IFNg, interferon-g; IL-6, interleukin-6; LPS, lipopolysaccharide; TNFa, tumor necrosis factor-a.

incubation attenuated these increases (quercetin: 65%e75% decrease in both phospho-P38 MAPK and phospho-NFkB proteins, and caffeine: 30%e40% decrease in both proteins). Quercetin was again more powerful than caffeine (approximately 50%e60% less than caffeine, p < 0.01).

3.4. Alteration in oxidative stress markers by quercetin and caffeine

In the final set of experiments, we measured the antioxidant properties of quercetin and caffeine on oxidative stress in SH- SY5Y cells caused by LPS/IFNg-stimulated microglial CM and

IFNg-stimulated astrocytic CM. For these experiments, microglia and astrocytes were pretreated with 10 mg/mL caffeine and quercetin for 8 hours and then treated with stimulants (LPS/IFNg for microglia and IFNg for astrocytes). After 2 days incubation, microglial and astro- cytic CMs were transferred to SH-SY5Y cells. Levels of GSH and oxidative damages to DNA, proteins, and lipids were assessed in SH-Sy5Y cells. Data are shown in Figs. 10 and 11. Treatment with quercetin increased intracellular GSH levels by 37% (p < 0.01) but caffeine did not (Fig. 10). Exposure of SH-SY5Y cells to microglial CM and astrocytic CM for 3 days decreased GSH levels by 85%e90%. Quercetin, but not caffeine, attenuated this reduction (p < 0.01).

Fig. 8. Effect of pretreatment with caffeine or quercetin on released levels of IL-6 from IFNg-activated U373 cells (A) or IFNg-activated astrocytes (B). 8 h preincubation with quercetin and caffeine was performed before IFNg was exposed to the cells for 2 d. Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg- activated groups, #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: IFNg, interferon-g; IL-6, interleukin-6; LPS, lipopolysaccharide.

Fig. 9. Effect of pretreatment with caffeine or quercetin (10 mg/mL each) for 8 h on levels of phospho-P38 MAPK and phospho-P65-NFkB in LPS/IFNg-activated human microglia (A, left panel) and IFNg-activated astrocytes (A, right panel). Cell extracts were prepared and the proteins separated by SDS-PAGE. Representative blots are shown in (A) and quantitative results in (B). To ensure equal loading, the densitometric value of each band was normalized to the corresponding band for a-tubulin. Values are mean ` standard error of the mean, n 1⁄4 3. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated microglial or IFNg-activated astrocytic groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated microglial or IFNg-activated astrocytic groups, #p < 0.01 for quercetin groups compared with caffeine groups. Abbreviations: IFNg, interferon-g; LPS, lipopolysaccharide.

Fig. 11AeD demonstrates production of the oxidative damage products, 8-OHdG (A), protein carbonyl (B), lipid peroxide (C), and 3-nitrotyrosine (D). These products were substantially generated under normal conditions and were significantly reduced by quercetin (p < 0.01). Again, caffeine was not effective. When SH- SY5Y cells were exposed to microglial and astrocytic CM for 3 days, these damage products were increased (8-OHdG: 2 fold increase, protein carbonyl: 2-fold increase, lipid peroxide: 4.2- to 4.5-fold increase and 3-nitrotyrosine: 7.5- to 9-fold increase, p < 0.01). Treatment with quercetin attenuated the increase (8-OHdG: 35%e40% decrease, protein carbonyl: 35% decrease, lipid peroxide: 40%e50% decrease, and 3-nitrotyrosine: 40% decrease, p < 0.01). Caffeine was without effect.

4. Discussion

Brain injury results in neuroinflammation by activating micro- glia and astrocytes to release proinflammatory factors such as cytokines, toxic free radicals, and proteases. Oxidative stress in neuronal cells plays important direct and indirect roles in their death (Cobb and Cole, 2015). This can be reduced by the simple expedient of drinking coffee. Several epidemiologic studies attest to

Fig. 10. Effect of pretreatment with caffeine or quercetin (10 mg/mL each) for 8 h on changes in levels of intracellular GSH concentration in SH-SY5Y cells induced by LPS/ IFNg-activated microglial CM and IFNg-activated astrocytic CM. Microglia and astro- cytes were pretreated with caffeine and quercetin and exposed to stimulants (LPS/IFNg for microglia and IFNg for astrocytes) 2 d. Cell-free supernatants were transferred to RA-differentiated SH-SY5Y cells. The cells were collected to measure the 4 damage parameters in 3 d. One-way analysis of variance was carried out to test significance. *p < 0.01 for quercetin groups compared with CON groups in normal condition, $p < 0.01 for LPS/IFNg-activated microglial or IFNg-activated astrocytic groups compared with CON groups in normal condition, and **p < 0.01 for quercetin groups in LPS/IFNg-activated microglial or IFNg-activated astrocytic CM treated condition compared with LPS/IFNg-activated microglial or IFNg-activated astrocytic groups. Abbreviations: CM, conditioned medium; GSH, glutathione; IFNg, interferon-g; LPS, lipopolysaccharide; RA, retinoic acid.

a reduction in the risk of developing PD and AD in late life by developing the habit of drinking coffee in early midlife. Caffeine was assumed to be the active agent (Arendash and Cao, 2010; Eskelinen and Kivipelto, 2010; Hernán et al., 2002). However, this assumption was not supported by results of Ding et al. (2015), who found there was no difference in protective effects between caffeinated and decaffeinated coffee. These studies all assessed the protective effects over the long term. Such protective effects may not be revealed by short term studies. For example, Laitala et al. (2009) studied coffee consumption in 2006 middle-aged Finnish twins between 1975 and 1981. They found that coffee consumption over this 6-year interval was not an independent predictor of cognitive performance in old age. Similarly van Boxtel et al. (2003) measured cognitive performance in 1376 individuals from the Maastricht Aging Study over the same 6-year interval. They found no significant difference in cognitive decline from ages 24 to 81.

In this study, we have shown that quercetin, not caffeine, is the major constituent in coffee which inhibits glial-mediated toxicity against neuronal SH-SY5Y cells. In a standard cup of coffee, caffeine occurs in smaller amount than quercetin, CGA, and flavonoids (40 mg/100 grams of dried coffee) (Barone and Roberts, 1996). However, it needs very high concentrations to show anti- inflammatory and neuroprotective properties (at least 100 mg/mL in both THP-1 and U373 cells, Figs. 3 and 4). We found there was no significant difference in a coffee mixture of compounds with and without caffeine in our experimental condition (Fig. 1). This finding is consistent with the epidemiologic study of Ding et al. (2015).

Quercetin protected against SH-SY5Y cell loss after exposure to LPS/IFNg-stimulated microglia and IFNg-stimulated astrocytes. The effect was concentration and incubation time-dependent (Figs. 3e5). Quercetin also inhibited activation of proin- flammatory pathways such as P38 MAP kinase and NFkB stimula- tion (Fig. 8). This led to a reduction in the release of proinflammatory factors such as TNFa and IL-6 from LPS/IFNg- stimulated microglia and their surrogate THP-1 cells and IFNg-stimulated astrocytes and their surrogate U373 astrocytoma cells (Figs. 6 and 7). It was demonstrated that caffeine has these prop- erties but only very weakly. The other major coffee components, CGA and flavones, are more effective than caffeine (Fig. 2).

Quercetin also has antioxidative properties, which significantly increased intracellular GSH levels ([GSH]i) in SH-SY5Y cells under normal conditions. It also attenuated the reduction in [GSH]i in SH-SY5Y cells caused by glial CMs (Fig. 9). This phenomenon reflected a decrease in 8-OHdG, a biomarker of oxidative damage to DNA (Kasai, 1997) (Fig. 10). Quercetin also attenuated the increase in protein carbonyls, a general marker of oxidative damage to amino acids in proteins (Stadtman and Burlett, 1998). It reduced lipid peroxide, a product of the attack of reactive oxygen species on unsaturated fatty acids in lipids (Adibhatla and Hatcher, 2010), as well as 3-nitrotyrosine, a product of the attack of reactive nitrogen species such as peroxinitrite on tyrosine in proteins (Ischiropolous, 1998). Caffeine did not show any of these antioxidant functions.

Previously, we reported that depletion of GSH in both microglia and astrocytes induces neuroinflammation and results in neuro- toxicity (Lee et al., 2010a). Therefore, it can be concluded that quercetin protected SH-SY5Y cells not only by reducing the release of proinflammatory factors from glial cells but also by inhibiting attack by reactive oxygen species/reactive nitrogen species in glial CMs.

In our studies, we found that CGA and flavones also have anti- inflammatory and neuroprotective properties although they are weaker than quercetin (Figs. 2e4). It is possible that other unidentified coffee constituents could also exert antioxidative, anti- inflamatory, and neuroprotective effects via other mechanisms such as by anti-amyloid, anti-caspase, or other mechanisms.

Chronic neuroinflammation is closely associated with the pathogenesis of several neurodegenerative diseases, including AD and PD (McGeer and McGeer, 2002; McGeer et al., 2003; Whitton, 2007). Quercetin is a readily available protective. It may have potential to be a useful therapeutic agent in AD, PD, and possibly other neurodegenerative disorders.

Try Magnesium to help lower blood sugar levels

Assortment Of Magnesium Rich FoodsSupplementing with magnesium has been popular in recent years and is claimed to improve health in many ways.

Not all of these claims are backed by science, but there is convincing evidence linking magnesium supplementation to a lower risk of type 2 diabetes.

A recent meta-analysis examined the effects of magnesium supplementation in diabetics or people at a high risk of developing type 2 diabetes. Here is a summary of its findings.



Observational studies suggest that magnesium insufficiency or deficiency is linked with heart disease and several metabolic disorders, including hypertension, metabolic syndrome and type 2 diabetes (T2D) (123).

One large meta-analysis of observational studies including more than half a million participants showed that higher magnesium intake was associated with a lower risk of T2D (4).

Other studies have also shown that diabetics tend to have lower levels of magnesium, compared to healthy people (56).

However, the direction of causality is unclear. Diabetes might promote magnesium depletion or, alternatively, magnesium deficiency might increase the risk of T2D.

Randomized controlled trials support the second option. They show that supplementing with magnesium improves the symptoms of T2D, indicating that poor dietary intake of magnesium may, at least partly, contribute to its development (7).

But there is also some evidence suggesting that T2D may increase magnesium depletion, creating a vicious cycle (8).

Article Reviewed

This was a meta-analysis of randomized controlled trials examining the effects of magnesium supplementation on blood sugar control in people with type 2 diabetes (T2D) or those at a high risk of developing it.

Effect of magnesium supplementation on glucose metabolism in people with or at risk of diabetes: a systematic review and meta-analysis of double-blind randomized controlled trials.

Study Design

This was a systematic review and meta-analysis of randomized controlled trials examining the effects of magnesium supplementation on markers of blood sugar control and insulin sensitivity.

The researchers searched for relevant articles using five of the largest scientific databases.

The inclusion criteria were the following:

  • A double-blind, randomized controlled trial.
  • Participants were diabetic or at a high risk of developing T2D.
  • The studies examined oral magnesium supplementation.
  • Outcomes included markers of glucose metabolism or insulin sensitivity.

A total of 18 randomized controlled trials fulfilled all of the inclusion criteria — 12 studies included people with T2D and 6 included people at a high risk of diabetes.

Bottom Line: This was a meta-analysis of randomized controlled trials examining the association of magnesium supplementation with blood sugar control in diabetics or people at a high risk of developing type 2 diabetes.

Finding 1: Magnesium Reduced Fasting Blood Sugar Levels

The analysis showed that supplementing with magnesium significantly reduced fasting blood sugar levels in people with T2D or at a high risk of it, compared to a placebo.

Specifically, the standard mean difference was -0.4, which means that magnesium caused a moderate improvement (decrease) in blood sugar levels.

However, magnesium did not affect fasting blood sugar levels in people who didn’t have type 2 diabetes.

Additionally, levels of fasting insulin, insulin sensitivity (HOMA-IR) or HbA1c didn’t change significantly.

Bottom Line: Supplementing with magnesium reduced fasting blood sugar levels in people with type 2 diabetes.

Finding 2: Magnesium Reduced the Rise in Blood Sugar After Eating Sugar

An oral glucose tolerance test (OGTT) measures the changes in blood sugar after eating a dose of pure sugar (glucose). It is currently the gold standard for diagnosing diabetes.

Three of the included studies investigated the effects of magnesium on blood sugar levels during an OGTT in people at a high risk of developing diabetes.

Based on these studies, the researchers concluded that supplementing with magnesium significantly reduces blood sugar levels after eating sugar.

Specifically, the standard mean difference was -0.35, which is a moderate effect.

These findings were supported by one study that measured OGT in diabetics (9).

Bottom Line: Supplementing with magnesium significantly reduced the rise in blood sugar during an oral glucose tolerance test.

Finding 3: High Magnesium Levels Associated With Lower HbA1c Levels in Diabetics

Glycated hemoglobin (HbA1c) is a marker of the previous three-month average in blood sugar levels.

Analyses revealed that high magnesium levels correlated with lower levels of glycated hemoglobin (HbA1c) in people with T2D, supporting the overall findings of the meta-analysis.

However, supplementing with magnesium did not affect HbA1c levels, probably because changes in HbA1c are slow and many of the studies were short in duration.

Magnesium status was also associated with improved insulin sensitivity in people at a high risk of T2D, but no significant links were detected in diabetics.

Bottom Line: Observational studies showed that high magnesium levels were linked with lower HbA1c levels in diabetics.

How Does Magnesium Deficiency Impair Blood Sugar Control?

The meta-analysis strongly suggests that supplementing with magnesium benefits diabetics and those at a high risk of it.

There are several mechanisms by which magnesium insufficiency could impair blood sugar control.

  • Sustained magnesium deficiency appears to reduce blood sugar uptake (1011).
  • Low magnesium levels may impair insulin release from the beta-cells in the pancreas (1213).
  • Poor magnesium status might increase oxidative stress, possibly increasing insulin resistance (10).

Bottom Line: Magnesium deficiency could impair blood sugar control by reducing sugar uptake (insulin sensitivity), impairing insulin release and/or increasing oxidative stress.


This meta-analysis seems to have been designed well, and it followed accepted guidelines.

Although the included trials were generally high-quality, they included few participants and were generally short in duration. Long-term trials are needed.

Finally, the included studies varied in their design. For example, the doses of magnesium, as well as its form, differed considerably across studies.

Bottom Line: The included studies were generally of short duration, and their designs varied.

Summary and Real-Life Application

The researchers concluded that supplementing with magnesium may improve blood sugar control in diabetics, as well as people at a high risk of developing diabetes.

If you are diabetic or at risk of developing diabetes, supplementing with magnesium may be a good idea. However, you can also get all the magnesium you need from whole foods.

Magnesium-rich foods include whole grains, nuts, seeds, beans, dark leafy greens and fish. It is also abundant in coffee, cocoa and dark chocolate.