J Cell Mol Med. 2010 Aug 16. [Epub ahead of print]

Red wine antioxidant resveratrol-modified cardiac stem cells regenerate infarcted myocardium.

Gurusamy N, Ray D, Lekli I, Das DK.

Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, Connecticut, USA.

Abstract

Abstract. To study the efficiency of maintaining the reduced tissue environment via pretreatment with natural antioxidant resveratrol in stem cell therapy, we pretreated male Sprague Dawley rats with resveratrol (2.5 mg/kg/day gavaged for 2 weeks). After occlusion of the left anterior descending coronary artery (LAD), adult cardiac stem cells stably expressing EGFP were injected into the border zone of the myocardium. One week after the LAD occlusion, the cardiac reduced environment was confirmed in resveratrol treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). In concert, cardiac functional parameters (left ventricular ejection fraction and fractional shortening) were significantly improved. The improvement of cardiac function was accompanied by the enhanced stem cell survival and proliferation as evidenced by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as evidenced by the expression of EGFP twenty-eight days after LAD occlusion in the resveratrol treated hearts. Our results demonstrate that resveratrol maintained a reduced tissue environment by overexpressing Nrf2 and Ref-1 in rats resulting in an enhancement of the cardiac regeneration of the adult cardiac stem cells as evidenced by increased cell survival and differentiation leading to cardiac function.

PMID: 20716127

That mechanism involves short stands of RNA, about 1/100^th the length of a typical gene, called microRNAs, which are only 22 nucleotides in length (nucleotides comprise the rungs on the DNA ladder), which may interfere with normal messenger RNA in the production of protein.

As a brief background to readers, when genes are activated, a process called gene expression, they produce protein. When genes are switched off, called gene silencing, no protein is made. MicroRNAs interfere with this protein-making process and thus switch genes off.

Here is how microRNA works. Normally a strand of RNA is copied from DNA and exits the cell nucleus (center of a living cell) into the watery compartment called cytoplasm. It is here that messenger RNA, which contains codes to assemble protein, puts gene-control into action. (See graphic above.) For a basic understanding of how DNA, RNA and how genes make protein, readers can click here

However, not all RNA copies made in the cell nucleus contain code to make protein. Some are short-strands of non-coding RNA and if these so-called microRNAs mesh with normal messenger RNA, they can interfere with protein making and silence a gene, or maybe we should say the entire genome. After all, biologists now realize microRNAs have a more pervasive effect over the entire library of 25,000 human genes (the genome) than first imagined.

Conventional understanding

Before this report proceeds further with an explanation of how microRNAs switch genes, readers will need a short explanation of the conventional understanding of how genes are switched on and off.

Only in recent years has the public learned that the 25,000 human genes housed in each cell of the body are not fixed, they do not predetermine biological destiny and they can be switched on or off by processes called epigenetics.

The two prominent mechanisms of switching genes are (a) the donation of methyl groups, for example vitamin B12 and vitamin B9 (folic acid), and the (b) coiling or uncoiling of a strand of histone around a bundle of DNA called chromatin. Resveratrol (rez-vair-ah-trawl), a red wine molecule, is an example of an agent that switches genes by this latter method. (See diagram below)

The high interest in resveratrol in recent years is that it is known to target the Sirtuin1 gene (also called SIRT1), known as a survival and longevity gene. Resveratrol inhibits an enzyme called histone deacetylase, which then influences the coiling of histone around chromatin bundles of DNA.

Discovered in 1993

The discovery in 1993 of a novel class of RNAs in roundworms, which control various genes and biological processes, changed the understanding of how genes are switched on and off. At first, the broad impact of microRNAs on gene expression went unrecognized. But a decade later, an article in Nature Magazine referred to small microRNAs “the genome’s guiding hand.”

Even as recently as 2004 it was believed that microRNAs represented just 1% of the human genome and influenced only 10% of genes

By 2008 it was estimated microRNAs influenced a third of the human genome

It is now predicted that microRNAs regulate up to 90% of the genes in the human body. MicroRNAs may control every cellular process in all cells and tissues of the human body! Normal microRNA function is required for maintenance of health and prevention of disease.

Today geneticists know that the role of microRNAs in gene expression could be as important as that of methylation or histone modification. microRNAs: definition & overview.

Biologists now recognize there are approximately 1000 molecules of microRNA per cell, with some cells exceeding 50,000 molecules.

Even as early as 2002 one forward-thinking biologist said:

“It is rapidly becoming apparent that another whole level of (gene) regulation lurks, unsuspected, in living cells, hidden from our notice in part by the conventional approaches that we usually use to study gene regulation, and in part because these regulators are very small targets and are not easily found from gene sequences alone. These stealth regulators, operating below our radar, if not that of the cell, are small regulatory RNAs, adding to control the genome.”

MicroRNAs are now being referred to as “a fundamental regulator of gene expression.”

It is stunning to realize that out of 25,000 human genes or so, only about 6% are functionally classified as transcription factors that is, genes which facilitate protein making. However, since some 93% of our genome is transcribed from DNA into a copy called RNA, by far the greatest part of the expressed genome are non-protein-coding RNAs!

MicroRNAs and disease

Tantalizing correlations between microRNA and human diseases have recently been demonstrated. Genomics of microRNA, Kim, Nam, Trends in Genetics 22, 3, 2006

Since microRNAs exceed the action of most drugs, which are largely targeted at single genes, while microRNAs regulate complex networks of genes, there is feverish work being done to create drugs that influence microRNA.

For example, it is known that microRNA-21 is only weakly up-regulated in normal healthy hearts, but is markedly upregulated in most models of heart failure and enlargement. This suggests microRNA-21 plays a key role in this condition. Inhibition of microRNA-21 reduces fibrosis (scarring), heart enlargement and improves cardiac function.

But readers at ResveratrolNews.com are likely more interested in the influence microRNAs may have over aging rather than disease, and where resveratrol fits into the microRNA picture.

MicroRNAs and aging

Some 800 longevity-associated genes have been identified thus far. This suggests broad rather than narrow control of the human genome is required to achieve longevity.

This is why biologists are so excited by microRNAs and their broad effects over networks of genes.

In 2005 it was reported that microRNA determined the lifespan of a roundworm.

Boehm A developmental timing microRNA and its target regulate life span in C elegans.

At least one longevity pathway known as IGF-1 (insulin growth factor-1) is controlled by microRNAs. In particular, microRNA known as lin-4 is linked with this IGF-1 longevity. Animals which cannot produce lin-4 exhibit a shortened lifespan.

IGF-1 is linked to the SIRTUIN1 gene pathway, a gene that is widely known as a mimic of calorie restricted diets. microRNA miR-217 increases Sirtuin1 gene activity and therefore might act in a similar manner to resveratrol.

In the Ames dwarf mouse, known for its remarkable propensity to delay the onset of aging, it was found that microRNA-27a influences the metabolic pathways involved in its longevity.

Of interest to the field of aging, microRNA expression is more often up-regulated than down-regulated with advancing age. For example, in laboratory mice microRNAs 93 and 214 exhibit significant up-regulation beginning at 33 months of age, which is close to the end of this animal’s normal lifespan (~36 months). Very few microRNAs are down-regulated with age.

MicroRNA-34a has been shown to regulate the Sirtuin1 gene, known as a survival gene that is activated during calorie restriction, which is a known longevity pathway.

Controlled studies involving microRNA and resveratrol are new and recent. One such study shows how resveratrol influences genes involved in cancer.

Watch for further breakthroughs involving resveratrol and microRNA here at ResveratrolNews.com. – © 2010 Bill Sardi, ResveratrolNews.com

The regulatory status of resveratrol as both an investigational drug (SRT501, Sirtris Pharmaceuticals, Cambridge, Mass.) and widely sold dietary supplement has presented a number of conjectural problems.

What would happen if the drug version of this molecule were nothing better than what is available as a dietary supplement?  Furthermore, to further blur the difference between dietary supplement and pharmaceutical drug, the dietary supplement industry today is now working under FDA-prescribed Good Manufacturing Practices which, if complied with, may no longer give pharmaceuticals such a big advantage over dietary supplements in quality.

In fact, Sirtris Pharmaceuticals, in a Securities Exchange Commission filing a couple of years back, said one of the risks involved in marketing their SRT501 resveratrol pill is that it is a stabilized, emulsified, micronized resveratrol, kind of like saying it is resveratrol with chrome bumpers, a souped-up gear box and spinning hubcaps, but it was still just resveratrol.

Now today’s news story (August 12, 2010) issued by xconomy.com was a stunner — the highly-touted SRT-501 drug, heralded as the first credible anti-aging pill and Sirturin1 survival gene activator, which sold to Glaxo-Smith-Kline for $720 million along with other designer Sirtuin1 activators, just became a dietary supplement.

Former executives of Sirtris have started a non-profit venture to offer look-alike version of Sirtris’ SRT501 as a nutriceutical rather than a pharmaceutical.

But was the world holding its breath and waiting for the drug version of this molecule as claimed in today’s news story?  After all, resveratrol supplement users aren’t dropping dead like flies from poor quality pills and waiting for a better quality product to arrive.

Former Sirtris exec Michelle Dipp, who is marketing an SRT501 clone at the Healthy Lifestyle Institute website, says “that their group is the only one she knows of that manufactures resveratrol in a completely synthetic process with the same high standards for sterilization and purity that govern pharmaceutical production.” (Xconomy quote)

Uh, what are we missing here?  Sterilization of resveratrol would subject this light and heat-sensitive molecule to radiation and/or high temperature.  That doesn’t sound wise.

Furthermore, Dipp alludes to the synthetically-produced pure form of resveratrol, produced by fermentation, rather than a botanical extract, usually derived from Giant Knotweed (botanical name Polygonum cuspidatum) or red grapes.  But the pure form of resveratrol is also widely available in dietary supplements and has not been shown to be advantageous over the botanically-extracted molecule.  In fact, at some times in the past, Sirtris said it was using the botanical version of resveratrol in its SRT501 drug.

So if souped-up resveratrol is worth $720 million, then what is a well-tested nutriceutical version of resveratrol worth, like Longevinex®, which filed patents ahead of Sirtris and was shown to exert a far greater genomic effect than plain resveratrol in an Affymetrix gene array study?

Inexplicably, Michelle Dipp says neither she nor any other Glaxo executive can endorse the nutriceutical version of resveratrol, but Healthy Lifestyle co-founder Christolph Westphal along with David Sinclair, who is still working with Sirtris, both claim to take the drug now morphed into supplement version of the product.  If that isn’t an endorsement, what is?

Certainly it is hoped that the Healthy Lifestyle Institute will bring a higher level of credibility to the resveratrol marketplace which is riddled with outlaw marketers making unfounded claims for their products.  But it is off to a specious start.

And this development also brings back Sinclair, the pied piper for so many resveratrol pill users, as a non-endorsing promoter of the product (whatever that is?).

Another point of contention is David Sinclair’s memorable recommendation that consumers wait for Sirtris’ more powerful Sirtuin1-gene activating drugs since the biological effects demonstrated in the laboratory with resveratrol would require the consumption of 1000-bottles of red wine per day.  Nobody knows how many 250-mg resveratrol pills the good associate professor at Harvard is taking per day, but at $1.50 per pill, 1000-mgs would cost $6.00 a day.

Subsequent dosing studies conducted in animals at the University of Connecticut show optimal benefit for the heart at 175 mg of resveratrol, with some loss of benefit starting at 350 mg and doses exceeding 1750 mg per day clearly increase the amount of damaged heart-muscle tissue in the event of heart attack and 3500-mg “kills” the rodent heart every time.

What does this say about resveratrol supplement companies like Rev Genetics and Biotivia which got into business offering mega-dose resveratrol pills (500 and 1000 mg per pill) based upon Sinclair’s recommendations, and suggested up to 7000 mg per day without adequate safety studies being performed?  Many resveratrol supplement companies today have no medical advisors and their executives can’t even decipher the lingo in a published journal article.

The cost for 250 milligrams of this miraculous SRT501 look-alike pill, which has now been abandoned as an investigational drug for diabetes, is $45 for 1-month supply, or $540 a year.  This is one and the same “drug” that was employed at a 5000-mg dose in a human cancer trial that had to be halted earlier this year due to severe side effects (kidney failure).

The Healthy Lifestyle Institute website states that resveratrol has been found to prolong the lifespan of yeast cells, fruit flies, roundworms and mice, though the latter longevity claim was only produced among rodents who were given a fat-engorged diet.  A later study showed that the human equivalent of 365 mg or 1565 mg of resveratrol slightly shortened the lifespan of animals fed a normal-fat calorie diet.

The best available evidence today shows modest doses of plain resveratrol best mimic the effect of a calorie restricted diet.

If all this doesn’t cast a dark cloud over the prospect of resveratrol as an anti-aging pill, the fact that resveratrol failed to activate the widely flaunted Sirtuin1 survival gene as originally reported in 2003, further muddies the prospect of a longer life for users of these pills.

Moreover, resveratrol has been publicized as a molecular mimic of a calorie-restricted diet.  But Leonard Guarente of MIT has clearly shown that the Sirtuin1 gene is not consistently up-regulated in all tissues and organs in an animal given a limited-calorie diet.  So Sirtuin1 may not be a universal gene target for longevity.

Imagine a dietary supplement that exhibited the same failed product claims and misdirection as SIRT501?  It would have been placed in the regulatory pillory box and demolished by the FDA and FTC.  But this pill carries the imprimatur of Harvard Medical School and the prestige of an ethical drug company such as GlaxoSmithKline.  Maybe the pill is meant to be a status symbol rather than confer any real longevity?  Who knows in today’s crazy world of drugs and circuses.

Still, there would be no impetus to develop an anti-aging pill had Sinclair not published that infamous report in Nature Magazine in late 2003 showing yeast cells lived longer when given a red wine molecule.

Sinclair and his laboratory brought everybody to the party.  His jaw-dropping presentations at scientific meetings and television interviews captured the imagination of many.  God knows, the world needs such a pill.  Putting the idea of super-longevity aside for a moment, if a confirmed anti-aging pill were available today and it would delay the onset of aging by just 7 years, it would spare Medicare from insolvency and avoid many nursing home admissions among retirees.

Based upon best available evidence to date, the closest thing to a valid anti-aging pill, aside from French red wine, which is producing unprecedented numbers of centenarians in France, is Longevinex®.

This resveratrol-based nutriceutical offers an array of small molecules, just as red wine does, and in a dose (100 mg resveratrol, 250 mg total polyphenolic molecules) that is in the same range as the amount of red wine molecules provided in 3 to 5 glasses of dark red wine (60 mg per 5-oz glass, or 180-300 mg), which is considered to be optimal for human health.  And it also has some good science behind it.

When Longevinex® was put to the comparison test in a gene array study, it activated 9-times more longevity genes than a calorie-restricted diet or plain resveratrol.  Furthermore, it appears most consumers taking resveratrol pills may be wasting their money.

It has been demonstrated that short-term compliance to a limited-calorie diet activates 198 genes, while the full genomic effect of calorie restriction, activation of 832 genes, requires life-long adherence to a deprivation diet.

Resveratrol in the short-term (2 months) only activated 225 genes in laboratory mice.  It would presumably take many decades of use of a plain resveratrol pill to activate the same number of genes activated by a life-long calorie-restricted diet.

However, Longevinex® exerted a profound genomic effect in the short-term, 1711 genes, with 633 of these genes being switched in the same direction as calorie restriction.  Longevinex® seemed to deliver a similar gene-switching profile to a limited-calorie diet at an early point in time.

Furthermore, Longevinex® is poised to release a bevy of scientific studies showing its nutriceutical matrix is far superior to plain resveratrol.

The first of these studies, which has been submitted for publication in a scientific journal, shows Longevinex® is the first resveratrol pill that has been demonstrated to limit damage to the heart in the event of a heart attack, theoretically working better than a baby-aspirin at preventing mortal outcomes in animal studies.  About half of the people who die of a sudden mortal heart attack were taking a baby aspirin tablet on the day of their demise.  Similar human studies cannot be performed for ethical reasons.  It appears many thousands of adults are dying needlessly from sudden heart attacks – deaths that could be prevented with resveratrol pills.

A second scientific study, also submitted for publication, shows Longevinex® exhibits an unprecedented level of safety and non-toxicity in animals, even up to 7000 mg human- equivalent dose.   Given that a 3500 mg dose of plain resveratrol “kills” the rodent heart every time, this is an eyebrow-raising finding.

The third scientific study, now in peer review, shows Longevinex® exerted powerful beneficial effects in laboratory mice subjected to an induced heart attack.  Blood flow in the aorta (first blood vessel outside the heart) and coronary arteries was significantly better following a heart attack than what was produced with plain resveratrol, and the heart pumping action in the left side of the heart was superior to that of plain resveratrol as well.  Longevinex® limited damage to heart muscle slightly better than plain resveratrol.

All these heart-protective properties correlated with an unprecedented effect Longevinex® has on microRNA, short RNA strands that are known to control whole networks of genes.  Longevinex’® microRNA-controlling properties were superior to that of plain resveratrol.

There are an estimated 290 brands of resveratrol pills being sold today sharing a market that was estimated in 2008 at $30 million (~$100,000 of sales per brand).  Most of these brands use borrowed science, pointing to scientific studies that used research-grade resveratrol that is frozen in storage at -80°C and sealed in an airtight opaque vial.

Resveratrol is a light-sensitive molecule that is subject to degradation unless efforts are made to protect it from light, heat and oxygen.  Longevinex® was the first company to stabilize resveratrol and protect it from light and heat and today offers a microencapsulated resveratrol pill that is enfolded in plant starches and dextrins to protect it from environmental degradation.

Many outlaw companies sell resveratrol pills online today, using bloggers as front-men to generate inquiries and offering kickbacks to bloggers in the form of affiliate fees.  The public has been bilked by free-bottle offers, often made by off-shore companies that are beyond the reach of the law.  Online consumers have found their credit cards being billed for product they never ordered.  So far, the FTC has taken no action against these outlaw companies.

LATE BULLETIN – As this report was going to press, GlaxoSmithKline in England issued a press release stating its former executives at Sirtris are being sanctioned by the company for selling a resveratrol dietary supplement behind the company’s back.  Christolph Westphal and Michelle Dipp, former Sirtris execs, will resign from their positions with the Healthy Lifestyle Institute and the product sold online will be withdrawn from sale.  Glaxo says the version of resveratrol pill being sold as a dietary supplement by Healthy Lifestyle Institute is not as potent as the drug version.  Yet both pills offer the same exact molecule at the same dose per capsule.  Stay tuned for further developments.  © 2010 Bill Sardi, ResveratrolNews.com

Since the prospect of an anti-aging pill was first announced in late 2003, when a Harvard scientist first reported a red-wine molecule caused yeast cells to live far longer in a lab dish, the adoption of such red-wine pills has been puzzlingly slow.

Six years after that announcement, red-wine resveratrol (rez-vair-ah-trawl) pills have been adopted by fewer than 1 million Americans, generating sales of around $30 million (2008 estimate).  “Compare that to over $1 billion of sales of Viagra pills in their first year of availability,” says Sardi.

“This wasn’t another nostrum being promoted on late-night TV infomercials or by some self-proclaimed guru, it was being touted on the front pages of Scientific American and Science Magazine.  While the public was curious, they weren’t ready to make guinea pigs out of themselves just yet,” says Sardi.

In his newly released e-book, Sardi says, in the 1960s and 1970s, midway on the timeline of a tremendous upsurge in life expectancy, American adults were still mentally ready to adopt such a development should science provide it.

Americans naively bought millions of bottles of Geritol, a liquid elixir that was widely advertised on TV game shows like Twenty-One and The-Sixty-Four-Thousand-Dollar Question, thinking it was good for their health, when it provided an array of B vitamins combined with an over-dosage of iron and a swig of 12% alcohol (six times more than a glass of wine!).  It actually led to their early demise, says Sardi.  Geritol was essentially an anti-anti-aging pill, he adds.

Inexplicably, Americans bought far more bottles of Geritol in the 1960s and 1970s than they bought red wine resveratrol pills in the first decade of the 21^st century, he says.

Americans fear longevity over death

The primary hang-up Americans have about super-longevity is that they are now more fearful of longevity than death.  Americans have witnessed a generation of their own relatives who have lived the last 10-15 years of their lives in chronic disease and debilitation.  They want nothing of this.

Sardi says if Americans are shown a photograph of a man blowing out 100 candles on his 100^th-birthday cake, the vast majority will respond by saying “I don’t want to ever live that long.” “One would think our grandparents and parents are saying they are ready to line up for processing at the Soylent Green factory,” says Sardi.  (He alludes to a 1973 movie that took viewing audiences to a time in the future when food shortages caused humans, upon their death, to be processed into a food called Soylent Green.)

Asked if they would take such an anti-aging pill if it ever became a reality, Sardi cites an online poll taken a few years ago which showed about 9 of 10 retirees would pass it up for three primary reasons: (1) they wouldn’t accept more quantity of life without more quality of life; (2) they are fearful they would run out of retirement money; (3) and they are concerned about contributing to the overpopulation of the planet.

Genetic discoveries concealed

The next longevity side-tracker Bill Sardi reveals in his book was unexpected, he says.  His investigation uncovered clear evidence that genetic engineering technology, developed and published in the scientific literature in the early 1970s, was hidden from public view till recently because it would demolish industries such as life and health insurance and medical and pharmaceutical research, as well as topple most retirement funds.

Out of their own selfish interests, elitists made a decision that humanity was not ready for the introduction of technology that could make a 250-year life expectancy common, says Sardi. The news media was also complicit in concealing these discoveries, which could have resulted in the availability of an anti-aging pill 35 years sooner, he adds.

That decision produced the current drugs and circuses that threaten to topple modern society today, says Sardi.  Had an anti-aging pill been introduced in the 1970s, and served to delay the onset of age-related disease by just 7 years, it would have averted the impending bankruptcy of Medicare, now facing a $60 trillion shortfall.

Not mentally ready for such a pill

And finally, Sardi says, while there may be, for the first time, a bona fide anti-aging pill in the works, most American adults would not be ready to adopt it because of mental hang-ups and faulty thinking.

For example, Sardi says most Americans errantly believe the set of genes they inherited from their parents dictate the length of their lifespan.  “There is no such thing as a longevity gene,” Sardi emphasizes.  Everybody has the same set of ~25,000 genes, with a small number of structurally-defective (mutated) genes responsible for just 2% of all disease.  The remaining genes are environmentally controlled.  Aging involves the switching of many genes.

Etched in the public’s mind is the proud idea that longevity genes have been passed down to them when in fact a large body of scientific evidence points to the unequivocal fact that people with long-lived relatives who move to another country – such as the Japanese who have moved to Hawaii – will live a shortened life due to a change in their diet and environment.

Your DNA is not your destiny

The discovery that genes are controlled by environmental factors such as exposure to solar radiation, hot and cold temperature and provision or shortages of food, points to small natural molecules that can get into the genetic machinery within living cells and mimic these environmental factors.  This is called molecular medicine.  These mimetic molecules can be taken in the form of a pill – an anti-aging pill, notes Sardi.

The objective of an anti-aging pill is for small molecules like resveratrol to act as a short-cut or mimic of a limited-calorie diet, switching the same genes.  A limited-calorie diet has been shown to nearly double the lifespan of all living organisms tested, including yeast cells, fruit flies, roundworms, rodents, and recently, primates.

Putting an anti-aging pill to the test

Sardi says longevity seekers only need to switch about 800 or so genes in a favorable manner to achieve longevity.  That is what a life-long, calorie-restricted diet achieves in rodent heart tissue.  Mice have about the same number of genes as humans and the position and length of their genes are similar to humans, so they can serve as a laboratory model of human aging.

Sardi points to a laboratory study where mice were given a calorie-restricted diet, plain resveratrol, or resveratrol combined with other small molecules like those found in red wine. A short-term, limited-calorie diet controlled 198 genes, plain resveratrol a similar number of genes (225), while a specially formulated resveratrol pill combined with other molecules, similar to what is provided in red wine, controlled an unprecedented 1711 genes. [Experimental Gerontology 2008]

This means the beneficial effects of a limited-calorie diet can be mimicked in the short-term with a resveratrol-based nutriceutical (Longevinex®) that only many decades of adherence to a limited-calorie diet a daily use of a plain resveratrol pill can accomplish.

“If this laboratory test with mice is reflective of what happens in humans, those consumers who take plain resveratrol pills will have to take them for decades before they achieve a genomic effect similar to a calorie-restricted diet,” notes Sardi. “Many longevity seekers simply don’t have that many remaining years of life to live,” he says.

“This study was an exciting moment in the history of biology, and the research community has ignored it,” says Sardi.  “The nutriceutical combination switched 633 genes in the same direction as a calorie-restricted diet,” he noted.  “That is as close as modern biology has come to an anti-aging pill so far,” he says.

More faulty thinking

There is a lot of other faulty thinking about longevity, says Sardi.  Among them are: (a) the mistaken belief they are too old to benefit from an anti-aging pill; (b) not wanting to interfere with God’s timing for the end of their life; and, (c) overwhelmed by this science, choosing to delegate their decision to take such a pill to their disease-oriented doctors.

Sardi says there is no category in the public’s mind for an anti-aging pill.  “They don’t believe it could ever become a reality, or they fear it actually will.  And they have fallen for so many nostrums against aging before.”

Proof it works

Sardi says there will never be an “FDA-approved anti-aging pill.” The problem in evaluating an anti-aging pill is conclusive proof of its effectiveness can only be shown in decades-long human studies, something that is prohibitive in cost and frankly impractical.

Sardi says he facetiously offers people who take anti-aging pills a guarantee: he will send them a cake and the candles if they reach their 100^th birthday; but if they don’t reach the age of 100, they can send in receipts of their anti-aging pill purchases and receive a full refund!

He says biological markers of aging can provide sufficient proof these pills can slow and even reverse human aging.  Such a proof-of-principle study was already conducted in a human by non-invasive measurement of cellular debris (called lipofuscin – li-poh-fusk-in) in the back of the eyes by digital imaging.  Accumulation of cellular debris (lipofuscin) is an established marker of biological aging.  After 5 months of taking an anti-aging pill, an 80-year-old man experienced a reduction of lipofuscin in his retinas, evidence for reversal of his biological age.

Sardi says technology is widely available today to assess a person’s biological age, apart from their chronological (calendar) age, by ocular measurement of lipofuscin.

Where it started

Sardi, who fashioned a patent-applied-for, anti-aging pill in 2004, says he was prompted to do this when he visited the laboratory of a Harvard professor in late 2003.  That professor showed him a drawer full of different brands of resveratrol pills that didn’t prolong the life of yeast cells.  They were biologically inactive.  His company, Resveratrol Partners LLC, was first to formulate a stabilized resveratrol-based pill (Longevinex®) that was equivalent to what researchers use in the laboratory.

As for the slow public adoption of such a pill, Sardi says: “One should have guessed that the pursuit of Ponce de Leon, that long sought-after fountain of youth, would be a poisoned spring before the world would find it.”

A free viewing copy of the newly-released e-book, entitled How The World Got Lost On The Road To An Anti-Aging Pill, is available at www.resveratrolnews.com © 2010 Bill Sardi

Niki Chondrogianni, , Suzanne Kapeta^a, Ioanna Chinou, Katerina Vassilatou, Issidora Papassideri and Efstathios S. Gonos

^a National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vassileos Constantinou Avenue, Athens 11635, Greece
^b University of Athens, School of Pharmacy, Division of Pharmacognosy and Chemistry of Natural Products, Zografou Campus, Athens 15771, Greece
^c Korres S.A. Natural Products, 57th Athens-Lamia National Road, 32011, Inofyta, Greece
^d Department of Cell Biology and Biophysics, University of Athens, Athens, Greece

Received 20 January 2010;
revised 31 May 2010;
accepted 1 July 2010.
Available online 7 July 2010.

Abstract

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.

Second, resveratrol pills have been marketed avidly in the U.S. now for about 6 years and we simply haven’t received reports from patients or families of Alzheimer’s patients that any resveratrol pill restored memory, though we do frequently receive reports from healthy individuals that they experience improved mental alertness and clarity of thinking and that their recall (memory) is a lot faster.

Third, yes, resveratrol does pass the protective blood-brain barrier as it is a small molecule (this will be elaborated upon below).  However, resveratrol traverses the blood circulation complexed with glucuronate or sulfate, two detoxification molecules that attach to resveratrol as it passes through the liver, temporarily rendering resveratrol as a non-bioavailable molecule (can’t pass through cell walls attached to these other molecules).  However, an unlocking enzyme, glucuronidase, which is 16 times more abundant at sites of inflammation, infection and malignancy, frees resveratrol as an unbound molecule, which is nature’s drug delivery system.  Metabolism of resveratrol in the liver also prolongs the half-life of resveratrol from ~14 minutes to 9 hours.

Third, what is reported in The New York Times by Nicholas Wade, whose articles about SIRTRIS pharmaceuticals over the past six years have been quite popular, appears to be a defense of Sirtuin1 gene activating drugs.  Sirtris’ SRT501 botanical-based stabilized, emulsified, micronized resveratrol pill has come under fire with the recent discovery a fluorescent compound in the gene activation test was responsible for the Sirtuin1 activation, not resveratrol.  This doesn’t diminish the promise of resveratrol, but it does cast a cloud over its intended gene target.

Nicholas Wade’s article (Potential Found In New Approach to Alzheimer’s) emanates from a newly published report in Cell (Volume 142, Issue 2, pages 320-32, July 23, 2010), which can be viewed online here (click the tabs to view all the parts of the report).  The title of the report is “SIRT1 Suppresses β-Amyloid Production by Activating the α-Secretase Gene ADAM10.” (Interpretation: A gene called Sirtuin1and its proteins inhibit the formation of beta-amyloid plaque via the activation of an enzyme, alpha secretase — plaque that is implicated in the onset of memory loss typically seen in Alzheimer’s disease.

That nasty brain plaque

You can visualize beta amyloid accumulation in the brain in the PET scan images presented below.   The PET scans revealed beta-amyloid plaque in the brains of three Alzheimer’s disease patients (left) and three normal controls (right). The yellow indicates high uptake of a label that targets beta-amyloid plaque, and the red indicates medium uptake.

A problem with this study is that overproduction of beta amyloid plaque is not the primary problem in the aged brain.  The problem is the inability to get rid of beta amyloid.  It hardens and becomes a toxic form of plaque. This is believed to be due to the accumulation of copper and other heavy metals in the brain over time.  Very small amounts of inorganic copper (not bound to a protein) in tap water, 10-times less than what the Environmental Protection Agency allows, inhibits the efflux (removal) of beta amyloid.

What researcher George Brewer at the Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, points out is that inorganic copper, iron and other heavy metals, largely acquired from drinking water and vitamin supplements, hardens the beta amyloid plaque and inhibits the efflux of beta amyloid from the brain.  Resveratrol, being a copper chelator, is certainly a potential antidote in this regard.  However, unbound iron is also a problem and resveratrol does not chelate (key-late) iron.  The addition of an iron chelator such as quercetin or IP6 would be desirable in this regard.

If you attempt to upregulate Sirtuin 1 but fail to stop the intake or inorganic copper, what have you accomplished? As an aside, it is interesting to note that Alzheimer’s is a disease largely exhibited in advanced civilizations that rely upon copper plumbing.  More primitive cultures do not exhibit Alzheimer’s among their aged.

Gene expression: cause or consequence?

One of the problems with gene expression studies like this is that it is not known if this is a cause or consequence of the disease.  In the New York Times today Leonard Guarente of MIT shows that increased expression of Sirtuin1 protein not only inhibits beta amyloid plaque formation but slows the typical decline in memory seen in this inbred species of laboratory animal.  (Note that it was not demonstrated to reduce existing memory impairment in this experiment, which would make it therapeutic rather than just preventive.  However, other resveratrol studies do point to therapeutic or restorative action.)

It is important to understand the workings of epigenetics here in contrast to structural genetics (gene sequencing, mutation, etc).  The science of epigenetics, in contrast to gene structure that was discovered by Charles Crick and Francis Watson in 1953, is that gene protein-making is governed by environmental factors such as food, lack of food, radiation exposure (sunlight), and temperature.  Small molecules known to activate the same genes as a calorie restricted diet, for example, would serve as an example of molecular medicine, a short-cut where these environmental factors are mimicked.  However, if the environmental factor (i.e. copper accumulation) continues, it may override any gene activator.

Resveratrol DOES clear plaque from the brain

It is encouraging to learn that resveratrol has been shown to promote the clearance of beta amyloid plaque from the brain.  The chart below compares resveratrol with two other small natural molecules in their ability to reduce beta amyloid plaque.  (Total sAβ = total beta amyloid plaque in nanograms per milliliter.  The numbers across the bottom indicate microMolar concentration of resveratrol.)

Another problem with this research is that while over-activation of Sirtuin1 gene protein (SIR2 in animals) may be beneficial in the brain, it may be deleterious in other organs, such as the heart.  Furthermore, Leonard Guarente, MIT professor, the lead researcher in this study presented in the New York Times, conducted research which shows that calorie restriction (CR), the unequivocal dietary practice that doubles lifespan of all living organisms tested, does not universally differentiate the Sirtuin1 gene in all tissues and organs.  Therefore, stimulation of this gene cannot be used as a gauge of whether a molecule like resveratrol is a mimic of a CR diet.

Alzheimer’s does not involve a single gene

Since Alzheimer’s disease and aging itself involve many genes, a gene array study may be revealing.  Resveratrol is a small molecule (228 Daltons molecular weight), so it passes the blood-brain barrier, and through cell walls, where it influences genetic machinery indirectly via the FOXO1 gene in the cytoplasm or directly in the nucleus.

This is in contrast to other Sirtuin-activating drugs (SRT1720, SRT 1460, SRT2183) which are larger molecules and unable to traverse the blood-brain barrier.

Transresveratrol

The glory of resveratrol and other small molecules like it is it regulates many genes, not just one.  There is also synergism noted when multiple small molecules like quercetin are employed with resveratrol.

For example, Longevinex® (resveratrol, quercetin, IP6) was put to the test in a laboratory mouse study.  In a gene array study, Longevinex® was shown to activate, in the rodent brain:

Total # genes analyzed: 20,341 in rodent brain tissue

(Note: half of these genes will be duplicate or dysfunctional genes)
# genes changed by CALORIE RESTRICTION: 124
# genes changed by RESVERATROL:  424
# genes changed by LONGEVINEX: 2,560
# genes changed by All three treatments: 5

A more profound genomic effect, 6-fold greater than plain resveratrol and 21 times greater than a calorie-restricted diet, was produced by a resveratrol-based nutriceutical matrix (Longevinex®).

Multiple etiologies

While it is true that very low nanomolar concentrations (parts per million) of resveratrol have been shown to reduce beta amyloid plaque in the brain of animals, Alzheimer’s disease involves many factors, not just a single gene, nor just beta amyloid plaque.  Some people have a lot of brain tangles and beta amyloid and experience no loss of memory in their retirement years.

It is clear that many factors (poor circulation, gene expression, heavy metals (particularly iron, copper, lead, mercury, aluminum, manganese), chronic viral infection, lack of thiamin (vitamin B1) which is often caused by over-consumption of sugar or alcohol, may be involved and any proposed therapy will have to address multiple etiologies.

Chronic inflammation and brain aging

Chronic inflammation is a hallmark of aging and what has been termed “inflammaging” is believed to be central to the onset of Alzheimer’s disease.  This is likely due to the unequivocal fact that chronic brain inflammation, as evidenced by an inflammator agent (cytokine) known as tumor necrosis factor (TNF), interrupts transmission of brain chemicals across the synapse (gap) between brain cells (neurons).

The injection of a TNF-inhibiting drug (etanercept) directly into the brain via a neck vein has been demonstrated to produce almost immediate memory repair in humans, but the effect only lasts about 6 days and costs ~$10,000 to $40,000 a year.  While unaffordable and impractical, this does tell us that neurons are intact and inflammation plays a strong role in brain deterioration.

Of interest to readers here, resveratrol, quercetin, and vitamin D3, ingredients in Longevinex®, are strong TNF-inhibitors.  However, it should be noted that over-inhibition of TNF is potentially deleterious as it stunts the immune system and drastically elevates the risk for leukemia (cancer of the blood) and lymphoma as well as tuberculosis (lung infection).  This suggests modest dosage, which is what Longevinex® provides (100 mg resveratrol).  Over-dosage may be why mice on high-dose resveratrol (365 and 1565 milligrams, human equivalent dose) experienced a shortened life span and mostly succumbed to lymphoma.

Why do humans age?

While experiments like these gain much attention in the news media, they don’t help to explain why humans age at different rates.  Aging is not a shortage of Sirtuin1 protein per se, as researchers may mislead us into believing.   We may or may not have been producing a lot of Sirtuin1 protein in brain tissues when we were young and our brains were working well.

I maintain the rate of aging is controlled by the accumulation of minerals.  There are three speeds of human aging: (a) no significant biological aging in the growing years, as evidenced by a lack of lipofuscin (cellular debris) formation; (b) progressive aging once childhood growth is completed in males or menopause is approached by females, resulting in accumulation of minerals, largely calcium, copper and iron, which “rusts and calcifies” tissues and organs, resulting in dysfunctional lysosomes and mitochondria; (c) a third stage of aging where the rate of aging subsides, which has been correlated with reaching a steady-state of iron (ferritin).  There is no other explanation given as to why humans age at three different speeds.  It is the minerals that then switch the genes.

Bill Sardi, managing partner,
Resveratrol Partners LLC  © 2010

According to The Alzheimer’s Society, clusterin levels were linked with the occurrence, progression and severity of the disease (shrinkage of the brain and memory impairment).

This “news story” is not new.  It was announced that clusterin is associated with Alzheimer’s disease last year (2009).

While today’s news story about clusterin does not give any hint of ways to block formation of clusterin, of interest is a 2007 study which reported that resveratrol (rez-vair-ah-trawl), known as a red wine molecule, blocks the production of this protein.  © 2010 Bill Sardi, Resveratrol News July 2010

Publication of the first small studies involving resveratrol pills in humans appear to be encouraging, but obviously lack conclusiveness.  It will take a few decades to convincingly prove a pill can reliably extend human life.

One of the markers of aging is inflammation.  In fact, aging has been called “inflammaging.”  Researchers at the State University of New York found that 40 milligrams of resveratrol taken daily by young adults for six weeks significantly reduces markers of inflammation (tumor necrosis factor-TNF, C-reactive protein – CRP) as well as measures of oxidation.  An abstract of the study can be viewed online here.

The study employed a 20% extract of Giant Knotweed (botanical name, Polygonum cuspidatum) marketed by Pure Encapsulations which researchers said “was the purest preparation available at the time the study was conducted.” But this isn’t quite accurate.  Extracts of Giant Knotweed ranging from 20% to 98% were available as early as 2004.  A 20% extract would contain a significant amount of emodin, a molecule that induces loose stool in some subjects, and would be undesirable for human use.  Extracts exceeding 80% contain little emodin and avoid the undesirable effect on bowel regulation.

The research team claimed their study was the first to demonstrate antioxidant and anti-inflammatory action humans, but actually a prior study conducted at Appalachian State University in 2008, employing an 80 mg dose of resveratrol combined with quercetin and rice bran IP6, produced similar results.

Another recently published study conducted in Great Britain employed 250 mg and 500 mg doses of resveratrol (Biotivia) among 22 healthy young adults.  The study did show that resveratrol enhances markers consistent with improved blood flow to the brain and described the study as a success.  But they failed to note that increased delivery of oxygen to the brain also increases oxidation.  No improvement in cognition (thinking) was exhibited, but this was likely due to selection of relatively young adults for the study group.  It was a failed study, but the manufacturer of the product selected for this study has irresponsibly chosen to describe the study as a success and that it improved cognition.

Yet another recent study shows that mega-dose resveratrol (4000 mg/day, taken in two divided doses of 2000 mg -Biotivia) produced loose stool among the majority of subjects ( 6 of 8 ) and there was one report of a skin rash, a sign of over-dosage.   The loose stool may be indicative of a significant amount of emodin within the product, which often induces this problem.  The manufacturer of the pill makes claims of product purity which appears to be specious.  The co-administration of wine with the resveratrol pills did not enhance absorption.  The dosage employed in this human study should be deemed to be potentially harmful as other studies indicate doses above 350 mg per day in humans may induce cell death and worsen tissue damage in the heart in the event of a heart attack.  An abstract of the study is available to view here.  Again this is an example of irresponsible marketing of a dietary supplement.  Long-term use of this type of supplement is potentially problematic.  © 2010 Bill Sardi, Resveratrol News

The dose of resveratrol used in this study is beyond practicality in humans – 200 milligrams per kilogram (2.2-lbs) of body weight per day, or the human equivalent of 14,000 milligrams per day for a 70-kilogram (160-lb) human.

This animal is particularly interesting to study because it exhibits a strong seasonal onset of obesity in winter (short-days) compared to summer (long-days), which were mimicked in the laboratory. Resveratrol supplementation had no effect upon this seasonal change. Calorie restricted lemurs, their diet reduced in calories by 30% compared to animals given a standard calorie diet, exhibited the anticipated lean body mass seen in prior experiments. Resveratrol had no effect upon body mass but did rev up metabolism.

Prior studies have shown that a much lower dose of resveratrol partially mimics calorie restriction and that long-term calorie restriction is required to produce a genomic response affecting 823 genes in heart tissue. However, another prior study showed that relatively low dose resveratrol, when provided with other small molecules (quercetin, rice bran phytateIP6) significantly affected 1711 genes in the short-term, mimicking what long-term calorie restriction would produce.

In conclusion, after only 1 year of treatment, it cannot be proposed that the hormesis theory is the most appropriate hypothesis to explain the effects observed in a non-human primate exposed to CR or RSV supplementation. CR and RSV might be two different stressors taking a different path to produce the same effects. Indeed,

Several differences were observed between calorie restriction and resveratrol-fed animals suggesting similar but different mechanisms and gene pathways. – © 2010 Bill Sardi, Resveratrol News.com

Source: Caloric restriction or resveratrol supplementation and ageing in a non-human primate: first-year outcome of the RESTRIKAL study in Microcebus murinus, Age, early online publication June 9, 2010.

Pterostilbene is alleged to be superior to resveratrol and the commercial availability of a pure form of pterostilbene (pTeroPure) is creating some manufacturer-driven hype that consumers should opt for it over resveratrol.

The actual development of pterostilbene as a raw material for dietary supplements began with research conducted by US Department of Agriculture scientists.  Pterostilbene is derived from blueberries.  It’s being imported from India as a nearly pure extract.

Technically, pterostilbene is methylated resveratrol and another molecular cousin, piceatannol, is hydroxylated resveratrol.  This means that a methyl group or hydroxyl group is added to the molecule.  Methylated resveratrol (pterostilbene) is said to be better absorbed and more “bioavailable.”

The research surrounding pterostilbene is promising, however, like resveratrol, human studies have yet to be launched and while some studies indicate pterostilbene may reduce elevated cholesterol and blood sugar levels, it has been demonstrated to do so only at very high doses – 2800 milligrams for blood sugar and 1750 mg to lower cholesterol.  Both of these doses may be potentially harmful.  It is possible that lower doses of pterostilbene will work.  Better dosing studies are needed.  (This is problem that the Federal Trade Commission has with dietary supplement makers – they often quote published studies that employed far higher doses than offered by their products.)

As we are all learning about resveratrol, higher dosage may not be optimal.  Lower, not higher-dose resveratrol has been found to exert biological action similar to a calorie restricted diet, and lower, not higher doses protect the heart.  So there may be diminishing returns in any attempt to build a more potent form of resveratrol.

The impetus to bring pterostilbene to the nutriceutical marketplace is based upon some widely disseminated misconceptions — that resveratrol is not readily absorbed and very little of it is biologically available (bioavailable) for use by the body.

Pterostilbene advocates claim resveratrol is poorly absorbed, but the research they cite in that regard says otherwise.  Researchers have previously shown that more than 70% of plain resveratrol is absorbed in the digestive tract.  So any increase in absorption brought about by pterostilbene would only improve absorption by a marginal amount.

For example, if pterostilbene improves oral absorption from let’s say 70% to 85%, then 115-125 milligrams of plain resveratrol would equal 100 milligrams of pterostilbene, absorption-wise.

Pterostilbene is lipophilic, that is, it dissolves in fats and oils better than in water.  Since the lining of the intestinal tract is composed of tightly-packed lipids (fats) and proteins, lipophilic molecules like pterostilbene penetrate this barrier better.  But this doesn’t mean plain resveratrol is not well absorbed.  It is a small molecule (228 Daltons molecular weight) and is readily absorbed.

Some makers of resveratrol pills have micronized their products (reduced the average diameter of a solid material’s particles) and folded resveratrol into plant starches or dextrins (microencapsulation) to improve absorption.  So methylation is not the only way to improve absorption.

Bioavailability refers to the passage of resveratrol or pterostilbene through the liver.  As these small molecules navigate through the liver they are detoxified in different ways.  One way is to attach them to detox molecules, namely sulfate and glucuronate.  Once bound to these other molecules, resveratrol is unable to pass through cell walls and interact independently.  So it has been temporarily rendered “non bio-available.”

Methylated resveratrol makes more passes through the liver before it is attached or complexed with sulfate and glucuronate, making it more immediately bioavailable.  This can also be accomplished by delivering quercetin, another red wine molecule, with resveratrol.

The problem is that resveratrol has a relatively short half-life, ~1-3 hours, circulating the body on its own, as a free unbound molecule.  Methylated resveratrol is said to have a longer half life.  However, as resveratrol naturally attaches to glucuronate, this will extend its half-life to ~9 hours.  So nature does for resveratrol what pterostilbene is advertised to do.

Whether methylated or glucuronidated or sulfated, resveratrol must become a free, unbound molecule to pass through cells walls and exert influence over intracellular genetic machinery.  This is demonstrated in the following chart.

The above chart shows various concentrations of resveratrol, MET-1 and MET-2 being resveratrol plus liver metabolites (glucuronate or sulfate) and RES as unbound, free resveratrol.  The height of the bars indicates viral-infected cell viability.  Only at higher concentrations and in its unbound form did resveratrol demonstrate an ability to kill viruses (cytotoxic) in combination with an antiviral drug.

Nature takes care of the problem of bioavailability by producing glucuronidase enzyme, which is 16-times more abundant at sites of inflammation, infection and malignancy, which in turn unzips or releases resveratrol from glucuronate, therefore facilitating the delivery of resveratrol at the right time and place.

The same holds true for resveratrol when has been conjugated (attached) to sulfate in the liver.  Sulfatase enzyme activity unlocks resveratrol from sulfate, making it bioavailable.

This is nature’s drug delivery system.  Some drug molecules are pre-glucuronidated to facilitate delivery.  So it is a mistaken idea that resveratrol, once metabolized in the liver, is not bioavailable.

Most certainly, resveratrol by itself has been documented to produce a profound biological effect throughout the body – brain, eyes, heart, liver, kidneys – not just in the digestive tract where it is deposited via oral intake.  Just why it has been said that resveratrol is not bioavailable is inexplicable.  Pterostilbene is attempting to solve an imagined problem.  Researchers are creating science to fit their own commercial interests.

Another alleged health benefit for pterostilbene is that it exhibits ability to lower cholesterol in a superior manner to fenofibrate drugs.  While this has been demonstrated in animals, this research chases another long-standing falsehood – that cholesterol reduction saves lives.  Cholesterol-lowering drugs have never been shown to reduce coronary artery disease mortality rates and cholesterol is not the primary plaque that occludes arteries – calcium is.  Fenofibrate drugs reduce cholesterol absorption from the diet, but the diet provides less than 20% of circulating cholesterol while the liver naturally produces the other 80%.  Cholesterol reduction is a straw man argument.  So this health benefit ascribed to pterostilbene is specious. ### © 2010 Bill Sardi