DNA Barcoding Exposes Scary Data for Herbal Products

  • Americans spend $5 billion annually for products with unproven benefits
  • Recent studies show majority of herbal products are contaminated or inauthentic
  • Dietary supplements account for ~20% of drug-related liver injuries
  • What can be done to protect consumers?

In August 1873, Darwin had ‘a fit’, in which he temporarily lost his memory and could not move. He recovered, and busied himself in work on the cross- and self-fertilization of plants. Published in 1876, Effects of Cross and Self Fertilisation in the Vegetable Kingdom provided ample proof of his belief that cross-fertilized plants produced vigorous off-spring that were better adapted to survive. Taken from otago.ac.nz via Bing Images

Since this post comes on the heels of Charles Darwin Day (February 12th ), it’s apropos to recognize Darwin’s plant-related scientific contributions dealing with plant movement and cross-fertilization—largely as the result of his keen observations and powerful reasoning that we should strive to emulate in our scientific pursuits. Darwin’s sense of scientific integrity seems to have been lost on the world of herbal products according to several recent studies and reports. It appears this industry is in need of some policing and compliance that may well come from DNA barcoding.   

According to Anahad O’Connor’s sobering article in the NY Times, Americans spend $5 billion a year on unproven herbal supplements—promising everything from fighting colds to boosting memory. Given the size of this market, it’s alarming that these products are oftentimes contaminated and/or mislabeled. Hmmm, sound familiar?  You may be recalling one of my posts from last year that explored analogous DNA-based findings for meat and fish products indicating global gross negligence or fraud. To quote Yogi Berra, “it’s like déjà vu, all over again”. Before delving into the details of this unsettling—if not downright scary—situation, let’s briefly discuss some background concepts that will enable us to understand how DNA barcoding may help bring honesty and transparency to the herbal products industry.

What is DNA Barcoding?

Since the herbal products exposé described below involves use of DNA barcoding, some readers may want to know what this methodology involves. Fortunately, Cold Spring Harbor Laboratory provides a website called DNA Barcoding 101 that provides the following snippets of information, as well as detailed “how to” protocols in a downloadable PDF.

Taxonomy is the science of classifying living things according to shared features. Less than two million of the estimated 5-50 million plant and animal species have been identified. Scientists agree that the yearly rate of extinction has increased from about one species per million to 100-1,000 per million. This means that thousands of plants and animals are lost each year, and most of these have not yet been identified.

Classical taxonomy falls short in this race to catalog biological diversity before it disappears. Specimens must be carefully collected and handled to preserve their distinguishing features. Differentiating subtle anatomical differences between closely related species requires the subjective judgment of a highly trained specialist—and few are being produced in colleges today.

Now, DNA barcodes allow non-experts to objectively identify species—even from small, damaged, or industrially processed material. Just as the unique pattern of bars in a universal product code (UPC) identifies each consumer product, a “DNA barcode” is a unique pattern of DNA sequence that identifies each living thing. DNA barcodes, about 700 nucleotides in length, can be quickly processed from thousands of specimens and unambiguously analyzed by computer programs. Barcoding relies on short, highly variably regions of the genome. With thousands of copies per cell, mitochondrial and chloroplast sequences are readily amplified by PCR, even from very small or degraded specimens, to enable DNA sequencing of the barcode.

Herbal product DNA barcodes are, in principle, similar to those pictured below that exemplify unique characterization of two different cryptic species of a butterfly, which appear visually to be nearly identical, and two genera of owl that are visually distinct.

Four-color DNA sequencing trace showing sequence of T (red), C (blue), G (black), and A (green) bases that comprises a barcode and can be redrawn as depicted below (taken from srmgenetics.info via Bing Images).

Four-color DNA sequencing trace showing sequence of T (red), C (blue), G (black), and A (green) bases that comprises a barcode and can be redrawn as depicted below (taken from srmgenetics.info via Bing Images).

DNA barcodes identify all living things (taken from boomersinfokiosk.blogspot.com via Bing Images).

DNA barcodes identify all living things (taken from boomersinfokiosk.blogspot.com via Bing Images).

Selling Herbal Products is BIG Business…But Who’s Watching Out for You?

The international trade in herbal products is a major force in the global economy and the demand is increasing in both developing and developed nations. According to a recent report, there are currently more than 1,000 companies producing medicinal plant products with annual revenues in excess of $60 billion. Notably, medicinal herbs now constitute the most rapidly growing segment of the North American alternative medicine market, with over 29,000 herbal substances generating billions of dollars in trade. For those of you who may be interested, I found this list of the top 10 best-selling herbal supplements in the US, among which are Ginseng (#9), Purple coneflower (#6), Ginkgo (#4), Cranberry (#2), and—surprising to me—Soy (#1).

Ginseng berry growth is restricted to certain areas of the world due to climate and weather conditions, making it difficult to cultivate. When this rare berry is picked, it is after a three- to four-year wait, and then the harvest must occur during a short, two-week period. Ginseng berry contains potent antioxidants called ginsenosides—such as Ginsenoside Rg1 pictured here—that are more abundant and different than those found in the root of the ginseng plant. These nutrients are believed to enhance the body's natural defense system to aid against invading free radicals (taken from eexcel.net via Bing Images and Wikipedia.org).

Ginseng berry growth is restricted to certain areas of the world due to climate and weather conditions, making it difficult to cultivate. When this rare berry is picked, it is after a three- to four-year wait, and then the harvest must occur during a short, two-week period. Ginseng berry contains potent antioxidants called ginsenosides—such as Ginsenoside Rg1 pictured here—that are more abundant and different than those found in the root of the ginseng plant. These nutrients are believed to enhance the body’s natural defense system to aid against invading free radicals (taken from eexcel.net via Bing Images and Wikipedia.org).

Although soy has been a staple of Asian cuisine for centuries, Westerners have only recently become aware of its valuable health benefits. Soy is a source of easily digestible protein and contains no saturated fat or cholesterol. Scientists have demonstrated through over 40 studies, spanning a 20-year period that eating 25 grams of soy per day helps reduce the risk of America's number one killer—heart disease. According to the US Food and Drug Administration (FDA), eating 25 grams of soy per day as a part of a low fat, low-cholesterol diet may reduce the risk of coronary heart disease (taken from eexcel.net via Bing Images).

Although soy has been a staple of Asian cuisine for centuries, Westerners have only recently become aware of its valuable health benefits. Soy is a source of easily digestible protein and contains no saturated fat or cholesterol. Scientists have demonstrated through over 40 studies, spanning a 20-year period that eating 25 grams of soy per day helps reduce the risk of America’s number one killer—heart disease. According to the US Food and Drug Administration (FDA), eating 25 grams of soy per day as a part of a low fat, low-cholesterol diet may reduce the risk of coronary heart disease (taken from eexcel.net via Bing Images).

Unfortunately, product adulteration and ingredient substitution is not uncommon in the medicinal herb and dietary supplement markets, as species of inferior quality are often substituted for those of a higher value. This practice constitutes not only product fraud, but according to the World Health Organization (WHO), it is a serious threat to consumer safety, as commented on below.

Currently, there are no best practices in place for identifying the species of the various ingredients used in herbal products. This is because the diagnostic morphological features of the plants cannot typically be assessed from powdered or otherwise processed biomaterials. As a result, the marketplace is prone to contamination and possible product substitution, which dilute the effectiveness of otherwise potentially useful remedies. Fortunately, DNA barcoding can now be used to combat this serious situation.

Report Reveals Rampant Contamination and Substitution

Dr. Steven G. Newmaster, Associate Professor, Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada (taken from uoguelph.ca).

Dr. Steven G. Newmaster, Assoc. Prof., Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Ontario, Canada (taken from uoguelph.ca).

In October of 2013, a team of Canadian and Indian scientists led by Dr. Steven G. Newmaster published in BMC Medicine a report entitled DNA barcoding detects contamination and substitution in North American herbal products that has received widespread media attention. This study utilized blind sampling of commercially available herbal products, which were tested for authentication of plant ingredients using a Standard Reference Material (SRM) herbal DNA barcode library. The research questions focused on the following three areas. (1) Authentication: is the herbal species on the label found in the product? (2) Substitution: is the main herbal ingredient substituted by other species? (3) Fillers: are any unlabeled fillers used?

They tested the authenticity of 44 herbal products (41 capsules; 2 powders; 1 tablet) representing 12 companies. The samples were collected in the greater Toronto area in Canada, with several samples mailed from distributors in the US. All products are available to consumers in both Canada and the US. The herbal product samples represented 30 herbal species that were each represented by 2 or 3 different companies. The samples were submitted in a blind test for authentication (product no. label only) using PCR-BigDye® Sanger sequencing-based DNA barcoding at the Centre for Biodiversity Genomics within the Biodiversity Institute of Ontario, University of Guelph.

The following are some of their sobering findings:

  • Only 2 of the 12 companies tested provided authentic products without substitutions, contaminants or fillers.
  • Nearly 60% of the herbal products contained plant species not listed on the label.
  • Product substitution was detected in 32% of the samples.
  • More than 20% of the products included fillers such as rice, soybeans and wheat not listed on the label.

This graphical summary of the results taken from the NY Times speaks for itself.

1105-HERBAL-FOR-FRONT-popup

In a follow-up press release from the University of Guelf, Dr. Newmaster said that “[c]ontamination and substitution in herbal products present considerable health risks for consumers.” He added that “[w]e found contamination in several products with plants that have known toxicity, side effects and/or negatively interact with other herbs, supplements and medications.” The statement added that one product labeled as St. John’s Wort contained Senna alexandrina, a plant with laxative properties—not intended for prolonged use, as it can cause chronic diarrhea and liver damage, and negatively interacts with immune cells in the colon. Also, several herbal products contained Parthenium hysterophorus (feverfew), which can cause swelling and numbness in the mouth, oral ulcers and nausea. It also reacts with medications metabolized by the liver.

Furthermore, one ginkgo product was contaminated with Juglans nigra (black walnut), which could endanger people with nut allergies. Unlabeled fillers such as wheat, soybeans and rice are also a concern for people with allergies or who are seeking gluten-free products, Newmaster said. “It’s common practice in natural products to use fillers such as these, which are mixed with the active ingredients. But a consumer has a right to see all of the plant species used in producing a natural product on the list of ingredients.”

Dietary Supplements Account for ~20% of Drug-related Liver Injuries

Consumer safety is not a theoretical concern. According to Anahad O’Connor’s follow-on and lengthier Dec 22nd 2013 NYTimes article entitled Spike in Harm to Liver is Tied to Dietary Aids, “[d]ietary supplements account for nearly 20 percent of drug-related liver injuries that turn up in hospitals, up from 7 percent a decade ago, [that] according to an analysis by a national network of liver specialists. The research included only the most severe cases of liver damage referred to a representative group of hospitals around the country, and the investigators said they were undercounting the actual number of cases.” The article features a 17 year-old male who suffered severe liver damage after using a concentrated green tea extract he bought at a nutrition store as a “fat burning” supplement. The damage was so extensive that he was put on the waiting list for a liver transplant. Fortunately he recovered and the transplant wasn’t necessary.

This NYTimes article is well worth reading, and provided further evidence that tighter regulations are needed.

Conclusions

Given the sobering significance of the above study by Newmaster and coworkers, I thought it was better to directly quote concluding remarks by these authors, rather than selectively paraphrase and oversimplify the situation:

“Currently there are no standards for authentication of herbal products. Although there is considerable evidence of the health benefits of herbal medicine, the industry suffers from unethical activities by some of the manufacturers, which includes false advertising, product substitution, contamination and use of fillers. This practice constitutes not only food fraud, but according to the WHO, serious health risks for consumers. A study of health claims made by herbal product manufacturers on the internet found that 55% of manufacturers illegally claimed to treat, prevent, diagnose or cure specific diseases. Regulators such as the FDA and Canadian Food Inspection Agency (CFIA) may not have the resources to adequately monitor the dietary supplement manufacturers and their advertising claims, and there are concerns that the current regulatory system is not effective in protecting consumers from the risks associated with certain herbal products. Chemical research studies have documented poor quality control and high content variability of active ingredients among products from a variety of manufacturers of herbal supplements. This is partly because herbs contain complicated mixtures of organic chemicals, the levels of which may vary substantially depending upon many factors related to the growth, production and processing of each specific herbal product. Although many manufacturers provide products with consistent levels of active ingredients through a process known as chemical standardization, this technique has uncertain effects on the safety and efficacy of the final product.”

“Many of the dangers of commercial plant medicine have been brought to light by DNA technology based studies that have identified contamination of herbal products with poisonous plants. Eroding consumer confidence is driving the demand for a product authentication service that utilizes molecular biotechnology. One approach to vetting herbal product substitution and contamination is product authentication using DNA barcoding. Research studies such as ours and others reinforce the importance of using DNA barcoding in the authentication of socioeconomically important herbal species. We suggest that the herbal industry should voluntarily embrace DNA barcoding for authenticating herbal products through testing of raw materials used in manufacturing products, which would support sovereign business interests and protect consumers. This would be a minor cost to industry with a limited amount of bulk product testing, which would certify a high quality, authentic product. If the herb is known to have health benefits and it is in the product, then this would provide a measure of quality assurance in addition to consistent levels of active ingredients. Currently we are building an SRM DNA barcode library for commercial herbal species and standard testing procedures that could be integrated into cost effective ‘best practices’ in the manufacturing of herbal products. This would provide industry with a competitive advantage as they could advertise that they produce an authentic, high quality product, which has been tested using DNA-based species identification biotechnology, therefore gaining consumer confidence and preference. This approach would support the need to address considerable health risks to consumers who expect to have access to high quality herbal products that promote good health.”

I for one would gladly pay a premium for herbal products that are “DNA Barcode Certified”.

Wouldn’t you?

As always, your comments are welcomed.

Postscript

After I finished writing this post, I went back to the aforementioned BMC Medicine article by Newmaster and coworkers to use its Google-based link to blogs citing this publication. While most were recaps of the report, one entitled It’s Now Herbal Products’ Turn to (Unfairly) Wear the Scarlet Letter caught my attention. I won’t provide a synopsis here, but you may want to read the blogger’s rather different, über critical commentary that seems (unfairly) biased to me.

If you’re interested in all facets of DNA barcoding, I highly recommend visiting the DNA Barcoding blog by Dirk Steinke at Guelf University that has new posts daily.

For those of you who are educators, the BioBelize website provides an example of how secondary education programs in New York City and Belize came together to define and implement DNA Barcoding curricula to encourage young student interest and involvement in this aspect of science.

Meat Adulteration Runs Rampant Around the Globe

DNA-based food tests reveal there’s a good chance the meat and fish you eat contain something other than you expected and paid for. horsehamburger

DNA-based food identification and traceability are available but seemingly not adequately deployed, as demonstrated by some new reports emerging from several countries recently.  DNA tests reveal 50,000 tons of beef in Europe, 22 tons of mutton in China, 33% of the fish in the US and 75% of game meats in South Africa involved incorrect or dishonest labeling.  Naturally, this led me to wonder if we’re really getting what we think we order in restaurants or buy at the market.  Read on for shocking details and statistics about meat and fish mislabeling around the world.

Horse Burger:  Mistake or Misdeed?

In March, the NY Times reported that ‘the discovery of horse meat in products labeled as beef in the European Union has raised serious questions, not just about food labeling, but also about food safety and the working of the somewhat opaque, global horse meat industry.’ While horse meat is edible—and indeed a culinary tradition in countries such as France and Italy—investigations to prevent mislabeling or contamination are continuing, and may reveal illegal activities in oftentimes very complex food supply chains. A recent BBC News report stated that 50,000 tons (!) of meat sold as beef by two Dutch trading companies may have contained horse meat, and that these two companies are owned by one man who has previously been investigated by food safety officials. Click here for more information on this so-called ‘2013 meat adulteration scandal.’

If horsemeat is a culinary tradition to some, you may wonder why horsemeat contamination is a food safety issue.  It’s due to the possible presence of residual amounts of veterinary or other drugs.  According the NY Times article I mentioned above, horses sent to slaughter are often not raised as a food source and thus are given prohibited substances.  For example, it’s estimated that each year 160,000 horses from US horse tracks and pastures are taken to Canada and Mexico to be slaughtered, and most of that meat is sent to Europe and Japan.  In further researching this concern, I found a list of 115 banned and dangerous substances commonly given to horses sent to slaughter that pose potential problems from ingestion of residue or metabolites.

Sushi bars now serving Tapper, Colmon, Talatail?

Tilefish sold as snapper, cod sold as salmon, tilapia as yellowtail?  DNA tests show it’s happening frequently.  Unfortunately, the mislabeling isn’t limited to Europe.  Alarmingly, another NY Times article reports that similar problems apply to fish in US restaurants, markets and sushi bars. In this study, about one-third of the 1,215 fish samples spanning 28 different species of fish were mislabeled.

Which fish did you actually eat?

Which fish did you actually eat?

A common pattern was said to be economic gain at the consumer’s expense, “with understudy fish—tilapia, in particular—often substituting for a menu’s star, without a word to the audience.” The study also contained surprises about where consumers were most likely to be misled—sushi bars topped the list in every city studied—while grocery stores were most likely to be selling fish honestly; restaurants ranked in the middle.

mislabeling

Image from Daily Postal

Endangered Species Labeled As Game Meat in South Africa

Further evidence for deficiencies and/or dishonesty in food labeling comes from a recent study that used so-called “DNA barcodes” to identify source animals in commercially sold meat in South Africa, and found that the majority of game meat samples contained animals other than those described in product labeling.

As reported in the journal, Investigative Genetics, sequences from two mitochondrial genes—cytochrome c oxidase subunit I and cytochrome b—were used to barcode almost 150 commercial meat samples from South Africa, focusing on beef and wild game products. The beef samples all checked out, but results for game products were much more variable, and revealed rampant inaccuracies in food labeling—more than 75% of the game meat samples had sequences from animals not mentioned in product labels. In one instance, barcode sequences suggested that some of the substituted meat came from cape mountain zebra, an endangered species.

Since most of these types of articles that I read simply referred to use of “DNA tests,” I did some searching to learn about meat and other food identification methodology. These methods include PCR-sequencing, PCR of random amplified polymorphic DNA, PCR-restriction fragment length polymorphism (RFLP), PCR using species-specific primers, real-time PCR, DNA microarrays, short tandem repeat (STR) markers, and single nucleotide polymorphisms (SNPs). If you’re interested in a comprehensive and up-to-date free via Google Books—check out “DNA-based Methods for Authentication of Meat and Meat Products” by Fajado et al.   If you’re not familiar with SNP detection by single-base primer extension, a short description is available at TriLink’s web page for mtDNA SNP Assay Primers.

As depicted below, STR markers are polymorphic DNA loci containing repeated nucleotide sequences, typically from 2 to 6 nucleotides per unit. The length of the repeated unit is the same for the majority of the repeats within a locus; the number of repeats for a specific locus may differ, resulting in alleles of varying length that, in effect, identify a particular individual in a given species. STR analysis includes multiplexed PCR amplification of a number of STR loci using fluorescently (blue, green, etc.) labeled primers; labeled PCR products are then analyzed by electrophoresis to separate the alleles by size that is measured relative to size-standards (red).

STRsequencing

Quantitation is another aspect of food identification for which DNA analysis is used. In some instances this involves checking whether, say, a product labeled “all beef” is actually 100% beef, free of detectable levels of, say, horsemeat. Alternatively, whether a sausage product labeled as a certain mixture of meats is in fact that. Given the widespread applicability of real-time PCR for simultaneous identification and quantitation, it’s not surprising to find qPCR used for food analysis. For one example of this, see Koppel et al. 2012 wherein tetraplex real-time PCR was used for quantitation of beef, pork, sheep (mutton), and horsemeat proportions in sausage. Even more impressive multiplexing can be found in Koppel et al. 2009 wherein the fractional proportion of each of seven common types of meats—pork, beef, chicken, turkey, horse meat, sheep (mutton) and goat—was simultaneously measured between 2 and 100% by heptaplex real-time PCR.

While all of the above DNA analysis methods are scientifically interesting, I came across the following backstory for IdentiGen that was even more interesting to me because it nicely demonstrates how academic researchers can apply their scientific expertise to solve a practical problem that in turn leads to a successful commercial enterprise. The genesis of IdentiGen began—appropriately, with geneticists—at Trinity College in Dublin, Ireland, who recognized that DNA analysis methods they used to trace the origin of domesticated cattle could also be used to trace back a cut of meat to the animal and farm from which it came. IdentiGen, which is described as a “campus company,” now has a testing facility in Lawrence, Kansas—smack in the middle of the US beef industry. One outcome of this type of traceability testing—sometimes referred to as “Farm to Fork”—is that more than 10,000 restaurants in the US now serve Braveheart Black Angus Beef that is guaranteed to be from individually identified, all-vegetarian, corn-fed Angus cattle.braveheartbeef

IdentiGen supplies the proprietary DNA TraceBack® testing system to meat processing plants that supply these restaurants in order to verify the beef composition and origin.

For those interested in learning more about farm-to-store/restaurant supply-chains and the non-trivial science/statistics underlying DNA TraceBack®, I highly recommend reading a freely accessible Google Book Chapter entitled Tracing Meat Products through the Production and Distribution Chain Farm to Consumer

DNAtraceback

written by Ronan T. Loftus and Ciaran Meghen, who were involved with founding IdentiGen. They are also among the inventors of IdentiGen’s patent broadly covering “a method for identifying the animal from which a meat product is derived comprising genotyping the meat, comparing the genotype with known animal genotypes and locating any matching genotype to identify the animal from which the meat product is derived.”

Rats Passed Off as Mutton in China

If only the reports stopped at horsemeat contamination, misleading fish identities and mislabeled game meat.  Just a few days ago, a million dollar crime ring was busted in China for passing off some very unsavory creatures as lamb.   Police have arrested more than 904 suspects and confiscated over 22 tons of ‘fake or inferior meat since January of this year, including ‘one suspect who had used additives to spice up and sell rat, fox and mink meat’.  We can only hope that we’ll soon seen wider spread testing to verify that the foods we pay for and put in our bodies are really what we expect them to be.

Even your EVOO could be mislabeled!

Over the past few years we’ve seen resurgence in the popularity of olive oil with home cooks as well as high profile chefs.  While the price might cause hesitation, many of us go ahead and spend the money expecting high quality oil.  Recent studies published by the USDA, however, show that what is labeled as olive oil may actually contain less expensive canola, safflower and/or sunflower oil.  Researchers Talwinder Kahlon and coinvestigators at the Agricultural Research Service’s (ASR) Western Regional Research Center in Albany, California have developed a PCR assay to determine the true composition of oils labeled as olive.  The assay compares DNA of olive, canola, and sunflower plants from key regions of two genes—matK and psbA-trnH.  These two genes commonly occur in sources of vegetable oil, including in olive, canola, sunflower, safflower plants.

The assay has proven to be a ‘reliable basis of comparison and can be used to quickly detect the presence of the non-olive oils in an “olive oil” sample. The assay can identify the three oils at concentrations of 5 percent or higher.’

So, What Can and Should Be Done to Ensure Honest Food Labels? 

In concluding this post, I’d like to point out that various commercial products are available for DNA-based food testing. For example, DuPont Food Diagnostics offers the BAX® System for real-time PCR detection of pathogens or other organisms in food samples. Its RiboPrinter® system analyzes regions encoding not only the 16S rRNA sequence, but also the 5S and 23S sequences, plus the spacer regions and flanking genes on either side. This rich depth of information allows for highly precise differentiation among strains of the same species, even those with the same 16S sequence.   The same principles apply to any food.  As of today, routine food testing is limited and not part of the normal production process for most manufacturers.  Public pressures will hopefully lead to more companies adopting routine food testing as a normal standard of quality .

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