QA/QC: Phytochemical Testing
Table of Contents
Extracts in the making
Strength in numbers
Screen testing
Marking time
Stability statistics
Setting the standard
Formulating with phytochemicals
Regulatory developments
Plants are an essential part of our diet. They contain vitamins, minerals and many other chemicals needed to stay healthy. Over the eons, plants have developed these chemicals as a means of protecting themselves from insects, fungi, bacteria, viruses and animals. When we ingest these plant chemicals, or phytochemical extracts, they offer us some of that same strong protection.
Throughout the ages, people have found that consuming certain plants, or parts of plants, relieves aches and pains. Most medicinemen and physicians in ancient cultures were experts on plants. American Indians chewed on willow leaves or bark to relieve aches and pains. These contain salicin, a compound very similar to the active ingredient in aspirin.
We are now on the forefront of explosive growth within the herbal-supplement industry, as people are using more and more natural products to promote health and longevity. The herbal industry has experienced some growing pains, however, as manufacturers rush herbal products to a market that is not strictly controlled by FDA. This presents its own set of problems, as the herbal industry grapples for cohesiveness in testing procedures, and with determining proper dosages, possible side effects and what chemical or chemicals within the plant material are producing the desired effect. Manufacturers, naturally, also want to build consumer confidence in their product and the safety of those products.
Typically herbs are imported into this country from China, South America, Central America, France and Italy, to name a few sources. They are also wildcrafted (picked in the wild) here in the United States, as well as cultivated both organically and non-organically. These herbs can come in fresh or dried whole forms; fresh or dried portions of flowers, leaves, seeds or roots; and in whole powdered form. Generally, however, only specific portions of the plant are used, depending on where the specific active "marker" compound or compounds are most concentrated. A marker compound is an identified phytochemical known to have some relationship to biological activity.
Herbals are processed by vat extraction, after crushing the plant material to a pulp. They are typically soaked in a solvent solution composed of 50% to 90% ethanol in water, which leaches the marker compounds from the plant material. Time spent in the solvent varies. After soaking, the solution is spray-dried or pan-dried, leaving a dry extract; or, it can be distilled using heat and pressure, resulting in a concentrated liquid form. Dried extracts are screened to a uniform particle size. These can then be microencapsulated or put onto carriers, such as maltodextrin or starch, for use in food products; or formulated with excipients such as maltose, mannitol, sorbitol and cellulose, then formed into tablets or put into capsules.
Strength of herbal extracts is generally expressed in one of two ways. If the extracts contain known marker compounds, strength is commonly expressed in terms of the content of these active compounds. Marker compounds are used to standardize the extract's concentration. This method generally works best, because it gives a processing parameter to follow during production, so that the finished product is consistent time after time. Stating the content of active constituents also allows dosage to be based on the activity of the known chemical or chemicals.
Alternately, strength is expressed in terms of concentration. A 4:1 concentration means that one unit of measure of the extract is equivalent to, or derived from, four units of the crude herb. For example, one gram of a 4:1 extract is concentrated from four grams of crude herb.
An increasing number of herbal manufacturers are instituting rigorous quality-assurance programs to ensure that the product they are manufacturing is indeed what the label indicates. Many have adapted the GMPs (good-manufacturing-practices) prevalent in the pharmaceutical and food industries to get a better handle on the quality and consistency of their product. Testing herbals is a necessary part of the overall quality program, due to growing and harvesting variables that can affect the chemical content of the plant materials used. Just as grapes used for winemaking vary from year to year and crop to crop, herbs vary year to year, crop to crop, and batch to batch.
Initially, incoming whole herbs and powders are physically examined by eyesight and microscopy for botanical identification. They are also checked for color, extraneous matter, moldy pieces, insects and insect excreta, as well as rodent excreta. Sometimes, making a quality judgment is difficult. "You can't make a botanical identification very readily on a powder. You can do a microscopic analysis which helps you identify things, but it's much easier to do it with the naked eye when you have whole plant pieces. You can compare it back to a voucher specimen. You also can make sure that you don't have any undesirable ingredients present," says Richard Cech, director of quality control, Herb Pharm, Williams, OR.
Other physical-testing procedures, such as ash, acid-insoluble ash and heavy-metal testing, as well as ethylene-oxide and pesticide-residue tests, are also done on the raw ingredient. Cech explains that buying certified-organic herbals eliminates the need to test for residual ethylene oxide and pesticides, because the ground itself has been certified. Organoleptic and sensory evaluations can also be done. No official standards have been set for microbiological testing of dietary supplements, however, so it's up to the manufacturer to set these standards. Typical microbiological procedures include aerobic plate counts and tests for coliforms, E. coli, yeast and mold, and salmonella.
Sophisticated techniques such as high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas chromatography (GC), Fourier transform infrared radiation (FTIR), mass spectroscopy (MS), UV-visible spectroscopy (UV-vis) and capillary electrophoresis (CE) further identify herbals.
For initial raw-product testing, approximately 90% of assays rely on HPLC methods, and the remaining 10% on TLC. HPLC methods quantitate marker compounds against a standard, while TLC methods are used as a qualitative measure. TLC separates and identifies the chemical compounds individually, to make sure that the botanical has not been adulterated and that it is identical to a predetermined standard. The evaluated shipment is either accepted or rejected based on the outcome of the initial quality screening.
During production of the extract, samples are evaluated for the marker compound(s) present using HPLC and other quantitative methods. This information is used to help control the manufacturing process, during which botanical extracts are sometimes blended to reach the required marker-compound levels. Finished products are tested for solvent residue, and other final quality-control checks for the marker compound's activity are performed. Following is a more-detailed description of the tests often used for evaluating phytochemicals:
HPLC uses a mobile liquid phase flowing past a stationary material to separate samples into their individual components. These components produce characteristic peaks that can be identified and quantified against a reference standard.
TLC is used to quickly screen for product purity. The technique involves spotting a drop of test solution onto a small plate of glass that is coated with a fine powder. The bottom edge is then placed into solvent. The solvent pulls the sample up the plate by capillary action, separating the solution into smaller spots, each representative of a component of the original solution. The result can be compared with a standard for identification of compounds. The final number of spots and their relative sizes give an indication as to the number of components and their amounts in the original solution.
GC consists of a form of column chromatography in which separation occurs due to the interaction between the sample components and the stationary phase, typically coated on the inside of a capillary column, and a gas mobile phase. For botanicals, GC is most commonly used to quantitate volatile marker compounds, active ingredients or residual solvents.
FTIR measures the sample's absorption of infrared radiation as a function of wavelength. The resulting spectrum, or "fingerprint," is most often used to identify the sample based on the characteristic absorption bands of specific functional groups.
MS measures the mass of sample molecules that have been converted to charged species in the instrument's ion source. This measurement is done in a vacuum; the ions are separated according to their mass-to-charge ratio. MS is a powerful analytical technique for identifying unknown compounds and for quantifying sample components. MS is commonly linked to chromatographic techniques (e.g., GC/MS and LC/MS) to provide a sensitive detector that also can assist in the identification of the chromatographic peak.
UV-vis measures the sample's absorption of ultraviolet or visible light as a function of wavelength. The resulting spectrum can be used in the identification of the sample and in the determination of the sample components.
CE separates sample components based on differential migration of solutes in an electrical field within a buffer-filled, narrow-bore capillary column. CE offers excellent separating power for the analysis of components in a complex matrix. For botanicals, CE is most commonly used to quantitate marker compounds.
Product stability is a very important aspect to consider in an overall quality program for botanical products. Establishing credible shelf-life data requires testing to determine stability.
"Generally an accelerated study in a stability chamber is done to expose the material to high temperature (408C) and relative humidity (75%) purposely in an attempt to degrade the material," says Mark Lange, Ph.D., science director, Institute for Nutraceutical Advancement, Denver. "You watch the degradation over a period of months. Usually 6 to 9 months is needed to gather preliminary data to help give you a predicted shelf life. Product is checked for color, appearance, moisture content, pH, dissolution rate, friability and hardness. Data gathered from preliminary tests can be used to get your product onto the marketplace, but you also need to follow up by immediately or simultaneously running a degradation study at room temperature (258C) and at lower relative humidity (60%) to give you a real-time study. You carry that out for a period of two years. Once that data has been gathered and analyzed, you then adjust your initial shelf-life dates accordingly."
Michael Tempesta, Ph.D., vice president of research and development, PharmaPrint, Inc., Irvine, CA, also notes that: "Many herbs have stability problems. They are very complicated mixtures of sometimes very fragile chemicals. They need to be treated and formulated very well, otherwise they will not have a very long shelf life."
Germany has developed medicinal regulatory guidelines called the "German Commission E Monographs." These one-page therapeutic usage guides for herbs - which include nomenclature, plant composition, chemical constituents, uses and indications, dosage and medicinal action - were developed over a period of nine years. The guides, resulting from thorough clinical literature searches of botanicals, are currently being translated into English. This type of herb-safety and -efficacy information helps improve the credibility of using herbals to treat common ailments. Although the monographs do not contain quality-control methods or in-depth botanical information, this information will be an invaluable tool for groups currently developing standards.
Other European countries, and China, have developed their own standards for product quality and testing methods. The European Pharmacopoeia is the official standard of herbal quality control methods in 28 European countries. There is also a Chinese Pharmacopoeia. These countries have a jump on the U.S. herbal market when it comes to identifying botanicals, types of testing methods used and efficacy and usage of botanicals. Right now, U.S. herbal companies, as well as several non-profit organizations and institutions, are working on ways to collectively evaluate the many analytical methods currently used within the industry, as well as the methods used in Europe and China. The goal is to set up "official" specific quality marker compounds and standardized testing methodologies and reporting, as well as therapeutic recommendations.
Roy Upton, executive director of the American Herbal Pharmacopoeia (AHP), Santa Cruz, CA says that AHP is currently working on 25 monographs that will combine quality-control methods, therapeutic monographs and safety information that will be peer-reviewed by experts around the world. He says, "Our goal is to standardize the many testing methods, beginning with international harmonization, so that we are in step with at least the European Pharmacopoeia. This will help us all have comparable results. The heart and soul of the monographs will be to standardize analytical procedures, both qualitative and quantitative, as well as give the absolute most accurate therapeutic information so that health professionals, consumers, or whomever can look at the data and feel confident about using the botanical."
The Institute of Nutraceutical Advancement, a non-corporate division of Industrial Laboratories, Denver, sponsors a group called the Methods Validation Program (MVP). This program is also working cooperatively toward the common goal of improving quality standards, while opening up the lines of communication between scientists, trade organizations, the media and private industry here in the United States. Once methods have been validated and shown to be useful, they will be submitted to AOAC and the United States Pharmacopoeia (USP) for publication consideration.
PharmaPrint Inc. is taking testing one step further by adopting methods used in the pharmaceutical industry. The company is running herbs through mechanism-indicating in vitro bioassays, which include test-tube, microtiter, enzyme and receptor assays, as well as others. The activity of these assays gives a good idea of how the herbal constituents work in people - that is, how the active chemicals interact with brain pathways and how the body responds to the chemicals. Active compounds responsible for the desired medicinal effect are isolated and extracted from herbal sources. These compounds, which are in their pure form, are used to prepare the final extract. "Industry should adopt some form of biological testing as a release criteria," says Tempesta. "There should be wider analysis of herbal compounds as more compounds are recognized as having biological activity. By purifying these active compounds, it may be possible to market an herbal as a drug. It will increase the confidence level for consumers and physicians."
Herbals in food systems are becoming more commonplace, as evidenced by the grocery shelves of your neighborhood store - herbal teas, cereals fortified with echinacea and juices with added herbals - the list goes on and on. Botanicals in these products are found as extracts, where the botanical is on a carrier or is microencapsulated, or as concentrates and liquids.
What are some of the things food product designers should consider when working with botanicals? Randy Kreienbrink, marketing and sales manager, <%=company%> Fine Ingredients Division, Columbus, OH, explains that product developers often have to deal with the bitterness of botanicals - bitterness greatly affects flavor, especially when using the botanical in a juice product.
Also, because a lot of botanicals may precipitate when going through a flash-pasteurization process, the correct form of an herbal for the food system and process is necessary. "For label claims, the product is typically tested after heat treatment to make sure that the active components in the botanical are still there," Kreienbrink says. "Most botanicals are fairly stable to use for processed products, where as vitamins and enzymes require special formulations to meet label claims. They are affected by heat and pH, so you need to really watch the activity levels of vitamins." Tempesta also notes that the process stability of herbals is dependent upon which herbal is used. Food developers should look very closely at what processing does to an herb, because this may allow picking one herb over another to give desired health benefits.
As of March 23, 1999, dietary supplements (vitamins, minerals, herbs or other botanicals, amino acids and metabolites) must be labeled according to the requirements set forth by the 1994 Dietary Supplement Health and Education Act (DSHEA) and the FDA. If product does not conform to the conditions set forth by this act, the product will be misbranded and subject to recall. Also, the product can be subject to recall if FDA considers it a significant or unreasonable risk for illness or injury under ordinary conditions of use.
In all cases, however, the burden of proof rests on the FDA to show that the dietary supplement is an adulterant. Herbal manufacturers are cooperatively working with the FDA to get any harmful products off the shelves as quickly as possible. The herbal industry is aggressively trying to get quality-control methods standardized, so that any type of adulteration - such as plantain contaminated with Digitalis lanta - does not happen; but if it does, they will take responsible action to see that the product is quickly retrieved. This cooperative action will help heighten consumer confidence in herbal products.
MedWatch, the FDA Medical Products Reporting Program, covers drugs and dietary supplements. Its primary function is educating health professionals about the importance of being aware of, monitoring for, and reporting adverse events and problems to the FDA and/or the manufacturer. The FDA has plans to expand the scope of this program to include botanicals.
While numerous reports of adverse events related to herbal use appear in the media, further research shows that there is not always a direct correlation between the botanical and the adverse event. Conversely, numerous adverse events likely go unreported, because consumers and health professionals do not know where to file such a report, or they do not make a correlation between the event and the botanical. According to Upton, an "active" reporting system needs to be set up by FDA that will help ensure consumer safety and confidence in herbal products. Many in the herbal industry agree with Upton, and they are in the process of coming up with a collective, workable solution.
Carol Vandell is a freelance technical writer living and working in the Pacific Northwest. She has a bachelor's degree in food technology and 11 years of experience in the food industry. Her specialties include quality assurance/quality control, R & D and food-safety fields.
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