Means adjusting the herbal drugs preparation to a define content of a constituent or a group of substances with known therapeutically activity respectively by adding excipients or by mixing herbal drugs or herbal drug preparation.
STANDARDIZATION OF HERBAL DRUGS
Means adjusting the herbal drugs preparation to a define content of a constituent or a group of substances with known therapeutically activity respectively by adding excipients or by mixing herbal drugs or herbal drug preparation.
It is whole, fragmented or cut, plants, parts, algae, fungi, lichen in an unprocessed state, usually in dried form but sometimes fresh. Certain exudates that have not been subjected to a specific treatment are also considered to be herbal drugs.2
The test procedures being described under this chapter are mostly related to starting materials in phyto-pharmaceutical preparation. The starting materials usually consist of fresh plants or their parts which are subjected to other operation like drying, preserving etc. The processing of this fresh plant or parts therefore to the dried crude drug has been described numerous technical publications and monograph which is considered under the province of pharmaceutical phytology. Different phyto-constituents of hydrophilic nature such as alkaloids, saponins, tannins etc. come into the cell fluid through a specific way.
1Formed in the protoplast of the living plant cell
1Migrate through the inner plasma membrane
2Migrate through the tonoplast
3Come into the vacuoles containing cell fluid.
Beside this there are some other phyto constituents like cellulose, which does not follow this route and is secreted through the outer plasma membrane (plasma lemma) to the exterior to form the basic structure of the cell membrane. Similarly, the lipophilic active substances e.g., oils, balsams, resins etc. are formed in the same way in plasma. They can even be dispersed in the plasma, being gradually converted into ethereal oil, which more or less completely fills the cell space. Example – Rose oil in rose petal, oil cells of the Lauraceae and Zingiberaceae family. In some families the lysigenic excretion vacuoles are formed which expands further by dissolution of the cell walls, e.g. crude drugs of Rutaceae family Schizogenic excretion vacuoles, which are formed by the expansion of intercellular spaces through cell division in umbelliferae family cause the formation of ethereal oil – which is secreted from the actively secreting adjacent cells. The hydrophilic active substances can be stored to a considerable extent in the aqueous cell fluid whereas the lipophillic active substances are rarely present in the hydrophilic plasma.
Hydrophilic active substances, such as alkaloids, glycosides, tannins etc. are formed in the protoplast of the living plant cell. Lipophilic active substances, particularly ethereal oils, balsams and resins are likewise formed in the plasma. In the simplest case, they can be dispersed in the plasma.e.g. In rose-petals and in the flowers of the lime tree.
Most of the enzymes in the plant need adequate water to act. The water content in dry drug used is so low that decomposition reactions do not usually occur at all, or are retarded to such an extent that a certain degree of storage stability is guaranteed. It should not be forgotten that many enzymes survive the drying process and are reactivated when water/moisture is once again available. The most important enzymes, which break down substances for the production of drugs, are
Oxidases/peroxides, which mainly oxidize phenols, unsaturated fatty acids, terpenes
Hydrolyses, which cleave esters and glycosides and break down polysaccharides
Isomerases, which for example isomerizes alkaloids or other optically active substances
There are many reasons why the raw materials play a major role in the production of phyto pharmaceuticals of standard quality. ‘Associated with this are the problems encountered during manufacturing, which include
a)Very little specific standards are mentioned in the official monographs considering the huge resources of herbal medicines
b)Even where the standard is specified, a range of variations occur which do not correspond to those stated in the pharmacopoeia
c)The minimum content requirements for certain constituents cannot be fulfilled for quantity sufficient to met the demand because of the large geographical variations
d)The crude drug rendered unfit for use through infestation or microbial contamination
e)Pesticide or preservative residue exceeds the permitted levels
f)The large demand for herbal drugs of pharmacopoeial quality make it difficult to maintain it’s quality and purity 3
What is a Standardized Herbal Extract? It is not a Herb!
What is the difference between a "Standardized Herbal Extract" and a "Herbal Extract"? There is allot of confusion between the two terms "standardized herbal extracts" and "herbal extracts".
An "herbal extract" is sometimes also referred to as a tincture, or liquid herbal extract. This is a preparation where a whole herb is steeped in alcohol, water or a combination.
A "standardized herbal extract" is a measurable marker substance that is extracted from the herb. This marker may be an active ingredient, or just one that is easily determined, but often, it is a compound that has been used in scientific research. Sometimes the wrong marker chemical is identified and used as the "active" ingredients. For example, St Johns Wart is usually measured for hypericin, though it is now thought that hyperforin is the more active substance.
These products have generated both controversy and confusion among consumers and professionals. Unfortunately herbal manufacturers have done more than anyone to cloud the issue with new jargon and proprietary names, In fact standardization means different things to different people. The word "extract" is also confusing, since this term traditionally is associated with fluid extracts, which are highly concentrated tinctures made from the whole plant.
Is a Chemical a Herb?
One well-known manufacturer tells us in its consumer literature that in standardized extracts "active compounds are natural compounds found in an herb that is proven to be responsible for its healthy benefits." Morphine is from opium, but it is not a herb! Citric acid is not an orange, and isoflavones are not a soy bean! An active compound is in fact an isolated chemical, and no longer belongs to the plant kingdom, but to the molecular world. Unfortunately, cheaper brands, like those routinely available now in pharmacies, corner stores and even airports, are usually standardized, yet do not contains whole herbs.
Many herbal supplements contain "standardized herbal extracts" which is the chemical market that has been extracted using solvents such as acetone. These chemical derived compounds may contain residues which themselves could act as toxins on the body. "Standardized herbal extracts" are normally listed as : Blueberry Leaf Extract, Green Tea Extract, Bilberry Extract or Grape Seed Extract.
Herbal-Medi-Care does not use standardized herbal extracts, as we believe that it is best to use the whole herb. There are hundreds if not thousands of active ingredients in the whole herb that have benefits researchers still do not fully understand. A herbal extract is an isolated component of only one of these compounds present in the whole herb.
Standardized Herbal Extracts
The advent of herbal products in the form of standardized extracts was initiated in 1992.Since that time; proponents have heralded standardized herbal extracts as a major historical advance, allowing both consumers and medical doctors to use herbal products with greater confidence and more consistent results. What most don’t realize, however, is that the majority of these advocates consist of
1. Academics and medical doctors who often have little personal or clinical experience with herbs
2. Researchers whose work is funded by drug companies that manufacture the standardized extracts used in their clinical trials, and
3. Naturopathic physicians who have financial ties with the companies that produce these products.
One group noticeably lacking from the chorus of standardized extract enthusiasts is professional clinical herbalists who rely on herbs as their primary healing modality. While not categorically condemning standardized extracts, clinical herbalists agree that just because an herb is biochemically standardized, it is not automatically more potent or efficacious than a non-standardized extract.
Standardized Herbal Extracts – What are they?
Standardized herbal extracts are of two main types an active constituent extract where there is a known and accepted active biochemical principle, and a marker extract where the active biochemical principle is not known and a characteristic compound is used as a "marker," which signifies the presence of the other biochemical compounds that give the herb its therapeutic properties.
In an active constituent extract, the known biochemical compound is isolated from the herb and concentrated to an amount not naturally found in the plant. Think of caffeine from coffee or morphine from the opium poppy. This type of extract tends to be more drug-like, potentially having undesirable side effects not normally present in the herb or its non-standardized extract.
From the herbalist’s perspective, this type of extract, while stronger in intended action is more of an herbal drug or "phytopharmaceutical" as it is called in Europe. As such, when the isolate is manipulated at the expense of the whole herb’s constituents, one may lose other properties contained within the herb, as well as buffering compounds that may lessen adverse reactions.
Examples of herbs where the known active principle is treated as an isolate include:
gingko (24% flavoglycosides), milk thistle (80% silymarin), grape seed (95% polyphenols), turmeric (95% curcumin), saw palmetto (90% free fatty acids), green tea (60% catechins), cascara sagrada (20-30% anthraquinones), bilberry (25% anthocyanosides), pygeum (12% phytosterols) and kava (30-40% kavalactones).
In a marker extract, no single active constituent is known, so the entire extract is treated as active and all plant constituents are present. With this type of extract, the caffeine in the above example would not be used as the marker compound because it is not unique enough to any one plant. When the ginsenosides of ginseng, for example, are standardized from 5 to 15 percent, all other properties of the herb are present in a marker extract. In the case of ginseng, however, the herb’s strength depends greatly on its age and growing conditions. So the mere presence of a fixed percentage of ginsenosides does not guarantee the tonic properties of a well-aged root. In fact, since ginsenosides are also found in the cheaper leaves, some standardized ginseng extracts are made only with ginseng leaf rather than the root.
Examples of herbs where the active principle is either not known or not treated as an isolate include: artichoke (2-5% cynarin), chamomile (1.2% apigenin/0.5% essential oil), devil’s claw (5% harpogosides), echinacea (4% echinacosides), ephedra (6-8% ephedrine/pseudoephedrine), feverfew (2.6% parthenolides), ginseng (5-15% ginsenosides), goldenseal (5% hydrastine), horsechestnut (20% aescin), uva ursi (20% arbutin), gotu kola (10% asiaticosides), green tea (20-50% polyphenols), licorice (12% glycyrrhizin), St. John’s wort (0.3-0.5% hypericin), schisandra (2.6-4% schisandrins), valerian (0.8-1% valerenic acid) and willow (8% salicin).
While these represent the most widely used categories, science continues to develop further methods. One created by PharmaPrint Inc. is able to identify and standardize several active constituents. Their process represents the cutting edge of standardization, but with a cost of more than $500,000 per herb is only feasible for the most vested of pharmaceutical companies. They ultimately plan to make pharmaceutical-grade herbal products for the use of medical doctors and pharmacists.
Science versus Tradition
Traditional clinical herbalists use herbs not so much to treat named diseases, but to implement a shift in underlying physiological processes so the body can heal itself. The body’s complex processes can be affected by herbs, drugs, foods, emotional experiences and therapeutic exercises. This alone delineates a fundamental difference between holistic herbal medicine and the phytotherapeutic or symptomatic drug-like approach of phytotherapy. While neither is fully exclusive of the other, the difference is in the intention of the final therapeutic goal.
Does this mean that one shouldn’t try an herb such as St. John’s wort for mild depression? Certainly not, but herbal medicine’s strength is its capacity to effectively treat based on the individual needs of each patient. The phytotherapist, on the other hand, uses herbs to treat specific named diseases. For this purpose, standardized extracts based on identified chemical constituents are appropriate. However, the fact that standardized garlic is sold for reducing cholesterol and hawthorn for reducing hypertension does not make them the best herbs in all cases. If you are not using the right herb for an individual’s condition and constitution, it makes no difference whether it is a high-priced standardized extract of guaranteed potency or a more traditional preparation of the same herb.
There is no universally accepted "standard" for the manufacture of standardized herbal extracts. Companies’ manufacturing methods may vary so widely their finished products hardly resemble each other. Extracts may not be consistently standardized to one marker. For instance, nettle root is standardized by one company to 5% amino acids, by another to 8% sterols, and yet a third to 35ppm (parts per million) scopoline. Echinacea can be standardized to three different constituents: echinocosides, polysaccharides or polybutylides. And what is considered an active compound for any given herb may change in time, such as the hyperforin of St. John’s wort recently understood to be more active than its previous marker, hypericin.
As with decaffeinated coffee, the manufacture of high isolate standardized extracts may also involve highly toxic solvents such as hexane, benzene, methyl chloride or acetone. Besides leaving minute residue in the finished product, these solvents have hazardous effects on the environment.
Finally, on average, the comparison for the cost of standardized extracts over their non-chemically standardized counterpart is more than double that of the standardized version of the same herb.
Pharmaceutical Takeover of the Herbal and Vitamin Industry
With the development of standardized extracts, pharmaceutical companies are able to obtain exclusive international patents on isolated herbal constituents, as well as on the process of manufacturing them. Standardization may not mean better herbal medicine, but it does mean higher costs and more profits to pharmaceutical companies who can afford research that will guarantee them exclusive rights to these extracts.
This issue of error and deception has been reported down through the ages. According to veteran herbalist James Duke, herbal extracts have been "spiked" to deceptively indicate the presence of a specific marker with little or none of the herb’s associated constituents. In other words, except for the marker compound, there may be a completely different herb or no herb at all present. Therefore, while standardized extracts may offer a degree of assurance that the product is what it claims to be, it is still possible to be deceived.
Science does not exist in a vacuum, as medical and herbal research is dependent upon funding from business and industry. As long as science is influenced by the bottom line, truth will be threatened. The danger for herbs is that the public will be misled to accept primarily scientifically manufactured products that will eventually be superseded by "more effective chemical drugs."
So the questions remain: Is the exclusive sale of herbs in the form of standardized extracts a Trojan horse, bringing increased adverse reactions, increased governmental regulation and intervention, and ultimately the availability of only a few herbs deemed financially expedient to standardize by multinational pharmaceutical companies?
One thing that all sides agree on is that labeling an herb as a standardized extract is good for business. With projected 4½ billion dollar sales of herbal products for 1999, standardized extracts are increasingly playing a significant role in the popular acceptance of herbs. Yet, does this really promote herbalism?
As one herbalist-manufacturer glibly commented: "One way I can tell how powerful St. John’s wort is for depression is how good it instantly makes me feel when I look at my sales figures." Today, St. John’s wort standardized to 0.3% hypericin easily outsells its pharmaceutical rival, Prozac, and its derivatives. While it is certainly preferable to substitute the herb for the pharmaceutical, only using St. John’s wort may not fully address the patient’s issues and fails to reflect the high standards of clinical herbal medicine.
There are literally thousands of medicinal herbs growing worldwide – most of them are not, and may never be, standardized. In the North Hawaii Community Hospital on the big island of Hawaii, the focus is on integrating alternative and complementary medicine with conventional medicine. A considerable number of their patrons are of native Hawaiian heritage. Unfortunately, the hospital can’t endorse the use of their time-honored native herbs because, aside from being a long way from standardization, there is no accepted research on them.
Researchers and academics claim scientific separation from industry when it comes to the marketing and sales of the final herbal product. However, a deluge of articles and books currently recommend that consumers purchase only standardized herbs. Any publicized research attesting to the efficacy of an herb is, with few exceptions in the West, fully supported and paid for by vested manufacturers who are increasingly pharmaceutical companies. It is even possible in today’s world to hire a scientist, as if soliciting paid favors from the local brothel, to prove a case for anything from algae to oregano.
From a herbalist’s perspective, the empirical traditions of East and West based on "what works" are of the few sources for impersonal evaluation of an herb’s uses. Herbal products made based on these observations have proven their therapeutic effects for thousands of years without the need for biochemical standardization. Because certain standardized extracts, such as milk thistle extract with 80% silymarin, have been proven through funded research to be effective against liver poisons and toxins, this does not mean that the whole non-standardized herb may not have equally beneficial properties. In fact, there are traditional uses for milk thistle seeds, such as for enlarged spleen, menstrual irregularities and varicose veins, for which the standardized extract is not as suitable.
At this developmental stage of standardization, herbalists agree that most standardized extracts have significant shortcomings and should not be exclusively relied upon for all herbal needs.
Alternatives to Standardized Extracts
One effective alternative to standardization is called "fingerprinting." This involves a chromatographic analysis of an herb for all of its constituents, not just one. This colored graph of valleys and peaks ensures that the intended herb is present in an extract, thus identifying bogus products.Companies should also hire qualified herbalists to supervise the timing and harvest of herbs, such as fully ripened saw palmetto berries for extracts used in research. If following GMP standards, as many outstanding herb companies do, why would an extract made from a large sampling of the finest quality herb be unsuitable for double blind clinical trials and research?
As herbal medicine extends further into the mainstream, science and industry must not forget that there is an as yet unrecognized profession of highly qualified and experienced clinical herbalists. The American Herbalists Guild (435-722-8452), founded in 1989, represents the emerging herbalist profession.As science becomes more involved with herbal medicine, which is much needed in one sense, those who are attempting to integrate traditional medicines with holistic medical facilities, such as the native Hawaiians, must not be disempowered. Herbal medicine has always been a medicine by and for the people. Along with the discussions of biochemistry, standardized extracts, phytopharmaceuticals, and the inevitable involvement of pharmaceutical companies, those dedicated to the use of medicinal herbs must not forget their "roots" – an earth-centered awareness that affirms our interdependence with all life, and our relationship with the plant kingdom.4
WHO Guidelines for Quality Standardized Herbal Formulations
Quality control of crude drugs material, plant preparations and finished products.
Stability assessment and shelf life.
Safety assessment; documentation of safety based on experience or toxicological studies.
Assessment of efficacy by ethnomedical informations and biological activity evaluations.
The bioactive extract should be standardized on the basis of active principles or major compounds along with the chromatographic fingerprints (TLC, HPTLC, HPLC and GC).5 The Standardization of crude drug materials includes the following steps:
1. Foreign matter (herbs collected should be free from soil, insect parts or animal excreta, etc.)
Medicinal plant materials should be entirely free from visible signs of contamination by moulds or insects, and other animal contamination, including animal excreta. No abnormal odour, discoloration, slime or signs of deterioration should be detected. It is seldom possible to obtain marketed plant materials that are entirely free from some form of innocuous foreign matter. However, no poisonous, dangerous of otherwise harmful foreign matter or residue should be allowed.
During storage, products should be kept in a clean and hygienic place, so that no contamination occurs. Special care should be taken to avoid formation of moulds, since they may produce aflatoxins. Macroscopic examination can conveniently be employed for determine the presence of foreign matter in whole or cut plant materials. However, microscopy is indispensable for powdered materials. Any soil, stones, sand, dust and other foreign inorganic matter must be removed before medicinal plant materials are cut or ground for testing.
2. Macroscopic and microscopic examination: Medicinal plant materials are categorized according to sensory, macroscopic and microscopic characteristics. An examination to determine these characteristics is the first step towards establishing the identity and the degree of purity of such materials, and should be carried out before any further tests are undertaken. Wherever possible, authentic specimens of the material in question and samples of pharmacopoeial quality should be available to serve as a reference.
Visual inspection provides the simplest and quickest means by which to establish identity, purity and, possibly, quality. If a sample is found to be significantly different, in terms of colour, consistency, Odour or taste, from the specifications, it is considered as not fulfilling the requirements. However, judgment must be exercised when considering odour and taste, owing to variability in assessment from person to person or by the same person at different times.
Macroscopic identity of medicinal plant materials is based on shape, size, color, surface characteristics, texture, fracture characteristics and appearance of the cut surface. However, since these characteristics are judged subjectively and substitutes or adulterants may closely resemble the genuine material. It is often necessary to substantiate the findings by microscopy and/or physicochemical analysis.
Microscopic inspection of medicinal plant materials is indispensable for the identification of broken or powdered materials; the specimen may have to treated with chemical regents. An examination by microscopy alone cannot always provide complete identification, though when used in association with other analytical methods it can frequently supply invaluable supporting evidence. Comparison with a reference material will often reveal characteristics not described in the requirements which might otherwise have been attributed to foreign matter, rather than normal constituents.
Any additional useful information for preparation or analysis should also be included in the test procedures for individual plant materials, for example, the determination of vein islets and the palisade ratio.
3. Thin layer chromatography: Thin layer chromatography is particularly valuable for the qualitative determination of small amounts of impurities. The principles of thin layer chromatography and application of the technique in pharmaceutical analysis are described in volume 1 of The international pharmacopoeia. As it is effective and easy to perform, and the equipment required is inexpensive, the technique is frequently used for evaluating medicinal plant materials and their preparations.
The following parameters should be determined on the basis of published pharmacopoeial monographs or established experimentally for the analysis of each individual plant material:
Type of adsorbent and method of activation; if no information on the latter can be obtained, heat at 110c for 30minutes;
Method of preparation and concentration of the test and reference solutions;
Volume of the solutions to be applied on the plate;
Mobile phase and the distance of migration;
Drying conditions (including temperature) and method of detection;
For the spots obtained:
number and approximate position, or the Rf values if necessary and
fluorescence and colour.
Two thin layer chromatography methods are described below; the classical method and the micromethod, which uses different sizes of plates and hence different quantities of solvents.
4. Determination of ash: The ash remaining following ignition of medicinal plant materials is determined by three different methods which measure total ash, acid-insoluble ash and water-soluble ash.
The total ash method is designed to measure the total amount of material remaining after ignition. The includes both “physiological ash”, which is derived from the plant tissue itself, and “non-physiolgical” ash, which is the residue of the extraneous matter adhering to the plant surface.
Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth.
Water soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.
5. Determination of extractable matter: This method determines amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for materials for which as yet no suitable chemical or biological assay exists.
6. Determination of water and volatile matter: An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and deterioration following hydrolysis. Limits for water content should therefore be set for every given plant material. This is esecially important for material that absorb moisture easily or deteriorate quickly in the presence of water.
The azeotropic method gives a direct measurement of the water present in the material being examined. When the sample is distilled together with an immiscible solvent, such as toluene R or xylene R, the water present in the sample is absorbed by the solvent. The water and the solvent are distilled together and separated in the receiving tube on cooling. If the solvent is anhydrous, water may remain absorbed in it leading to false results. It is therefore advisable to saturate the solvent with water before use.
The test for loss on drying determines both water and volatile matter. Drying can be carried out either by heating to 100-105oc or in a desiccator over phosphorus pentoxide R under atmosheric or reduced pressure at room temperature for a specified period of time. The disiccation method is especially useful for materials that melt to a sticky mass at elevated temperatures.
7. Determination of volatile oils: Volatile oils are characterized by their odour, oil like appearance and ability to volatilize at room temperature. Chemically they are usually composed of mixtures for example, monoterpenes, sesquiterpenes and their oxygenated derivative. Aromatic compounds predominate in certain volatile oils.Because they are considered t be the "essence" of the plant material and a often biologically active, they are also known as "essential oils". Then tern "volatile oil" is preffered because it is ore specific and describes the physical properties.In order to determine the volume of il, the plant material is distilled with water and the distillate is collected in a graduated tube. The aqueous portion separates automatically and is returned to the distillation flask. If the volatile oil possses a mass density higher than or near to that of water, or are difficult to separate from the aqueous phase owing to the formation of emulsions, a solvent with a low mass density and a suitable boiling point may be to the measuring tube. The dissolve volatile oils will then float on top of the aqueous phase.
8. Determination of bitterness value: Medicinal plant materils that have a strong bitter taste are employed therepeutically, mostly as appetizing agents. Their bitterness stimules secretions in the gastrointestinal tract, especially of gastric juice.Bitter substances can be determined chemically. However, since they are mostly composed of two or more constituents with various degrees of bitterness, it is first necessary to measure total bitterness by taste.
The bitter properties of plant material are determined by comparing the threshold bitter concenration of an extract of the materials with that of a dilute solution of quinine hydrochloride R. The bitterness value is expressed in units equivalent to the bitterness of a solution containing 1 gm of quinine hydrochloride r in 2000ml.Safe drinking water should be used as a vehicle for the extraction of plant materials and for the mouth wash after each tasting. Taste buds dull quickly if distilled water is used. The hardness of water rarely has any significant influence on bitterness.
9. Determination of haemolytic activity: Many medicinal plant materials, especially those derived from the families Caryophyllaceae, Araliaceae, Sapinaceae, Primulaceae, and Dioscoreaceae contain saponins.The haemolytic activity of plant materials, or a preparation containing saponins, isdetermined by comparison with that of a reference material, saponin R, which has a haemolytic activity of 1000 units per gm, A suspension of erythrocytes is mixed with equal volumes of a serial haemolysis is determined after allowing the mixtures to stand for a given period of time.A similar test is carried out stimultaneously with saponin R.
10. Determination of tannins: Tannins are substances capable of turning animal hides into leather by binding proteins to form water insoluble substance that resistant to proteolytic enzymes. This process, when applied to living tissue, is known as an "astringent" action and is the reason for the therapeutic application of tannins.Chemically, tannins are complex substances; they usually occur as mixtures of polyphenols that are difficult to separate and crystallize. They are easily oxidized and polymerized in solution; if this happens they lose much of their astringenteffect and are therefore of little therapeutic value.
11. Determination of swelling index: The swelling index is the volume in ml taken up by the swelling of 1 gm of plant material under specified conditions. Its determination is based on the addition of water or a swelling agent as specified in the test procedure for each individual plant material. Using a glass stoppered measuring cylinder, the material is shaken repeatedly for 1 hour and then allowed to stand for a required period of time. The volume of the mixture is then read.The mixing of whole plant material with the swelling agent is easy to achieve, but cut or pulverized material requires vigorous shaking at specified intervals to ensure even disribution of the material in the swelling agent.
12. Determination of foaming index: many medicianl plant materials contain saponins that can cause a persistent foam when an aqueous decoction is shaken. The foaming ability of an aquousdecoction of plant materials and their extracts is measured in termsof a foaming index.
13. Determination of pesticide residues: Many medicinal preparations of plant origin the taken over long period of time, limits for pesticide residues should be established following the recomendations of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) which have already been established for food and animal feed(9). These recommendations include the analytical methodology for the assessment of specific pesticide residues.
14. Determination of arsenic and heavy metals: Contamination of medicinal plant materials with arsenic and heavy metals can be attributed to many causes including environment pollution and traces of pesticides.
15. Determination of microorganism: Methods for decontamination are restricted. For example, the use of ethylene oxide has been forbidden within countries of the European Union. Treatment with ionizing irradiation is also for bidden or requires a special registration procedure in some countries. In addition, the presence of aflatoxins in plant material can be hazardous to health if absorbed even in very small amounts. They should therefore be determined after using a suitable clean up procedure.
16. Radioactive contamination : Microbial growth in herbals are usually avoided by irradiation. This process may sterilize the plant material but the radioactivity hazard should be taken into account. The radioactivity of the plant samples should be checked accordingly to the guidelines of International Atomic Energy (IAE) in Vienna and that of WHO.
In order to obtain quality oriented herbal products care should be taken right from the proper identification of plants; season and area of collection, extraction, isolation and verification process.
Chemical and instrumental analyses are routinely used for analyzing synthetic drugs to confirm its authenticity. In the case of herbal drugs, however the scene is different especially for polyherbal formulation, as there is no chemical or analytical methods available. Therefore biological-screening methods can be adopted for routine checkup of herbal drugs and formulations. In the case of herbal drugs, the quality of raw materials and products can be furnished by regular pharmacognostic identifications and phytochemical analysis. The herbal formulations in general can be standardized schematically as to formulate the medicament using raw materials collected from different localities and a comparative chemical efficacy of different batches of formulation are to be observed. The preparation with better clinical efficacy are to be selected. After all the routine physical, chemical and pharmacological parameters are to be checked for all the batches to select the final finished product and to validate the whole manufacturing process.
The stability parameters for the herbal formulations which includes physical parameters, chemical parameters, and microbiological parameters.
Physical parameters include color, appearance, odor, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flowability, flocculation, sedimentation, settling rate and ash values.
Chemical parameters includes limit tests, extractive values, chemical assays, etc.
Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC and GC, UV, Fluorimetry, GC-MS, etc.
Microbiological parameters include total viable content, total mold count, total enterobacterial and their count. Limiters can be utilized as a quantitative or semiquantitative tool to ascertain and control the amount of impurities like the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels, impurities from the solvents, etc.
Chemical decomposition of substances present in the formulation also produces several toxic or impure compounds during storage in undesirable conditions. Contaminants may come directly from the atmosphere also. This include mainly dust, sulfur dioxide, H2S, CO2, Arsenic, moisture, etc.6-11
The guidelines set by WHO can be summarized as follows:
A.Reference to the identity of the drug. Botanical evaluation – sensory characters, foreign organic matter, microscopical, histological, histochemical evaluation, quantitative measurements, etc.
B.Reference to the physiochemical character of the drug. Chromatographic profiles, ash values, extractive values, refractive index, polarimetric readings, moisture content, volatile oil content, etc.
C.Reference to the pharmacological parameters. Biological activity profiles, bitterness values, haemolytic index, astringency, swelling factor, foaming index, etc12.
D.Toxicity details – heavy metals like cadmium, lead, arsenic, mercury, etc. Pesticide residues.
Maximum residue limits =
Acceptable daily index x body weight x extraction factor
--------------------------------------------------------------- x Therapeutic doses
Mean daily intake of drug x safety factor x 100
E.Microbial contamination – Total viable aerobic count, pathogenic bacteria like enterobacteria, E. coli, salmonella, Pseudomonous aeruginosa, Staphylococcus aureus, etc. and presence of afflatoxins etc.
F. Radioactive contamination.13-14
Modern herbal ayurvedic monographs
In the modern herbal ayurvedic monographs the standardization parameters are discussed in a comprehensive way. According to the modern ayurvedic monograph the quality control protocols include the following:
Title, synonyms, publications related to that plant, constituents present, analytical methods. Descriptive evaluation: Description of the drug, phytomorphological, microscopical, organoleptic evaluations, foreign matter, foreign minerals, etc.15
Identity: Physical and chemical identity, chromatographic finger prints, ash values, extractive values, moisture content.
Strength: Ethanol and water extractive values, volatile oil and alkaloidal assays, quantitative estimation protocols, etc.
Biological Activity Evaluation
Bitterness values, astringency, swelling factor, form index, hemolytic index, etc.
Pesticide residues, heavy metals, microbial contamination like total viable aerobic count, pathogens like E. coli, Salmonella, P. aeruginosa, S. aureus, Enterobacteria, etc.
The presence of aflatoxins can be determined by chromatographic methods using standard aflatoxins B1, B2, G1, G2 mixtures. Aflatoxin is a product of the microbial strain Aspergillus flavus.
Classical Evaluation as per Ayurvedic Literatures
Classical therapeutical attributes like Rasna, Guna, Virya, Vipaka and Karma classical formulations, doses, storage conditions.
The quality of the raw materials can be tested according to the following format:
•Name of the drug (English, Regional names, Exact botanical nomenclature)
•Part of the plant used
•Area of collection
•Season of Crop
•Time and year of collection
•Pesticide and insecticides
•Condition of the drug (fresh or dry)
•Form of the drug (powdered or intact or cuttings like etc.)16-17
The importance of standardization techniques for herbal medicines officially recognized standards
Dr Keith Helliwell, William Ransom, UK, referred to the categorisation of three types of extract according to correlation with their clinical activity. These are (1) herbs such as senna, where the characterised constituents of HMPs are solely responsible for the therapeutic and clinical effects with a dose related response to quantified constituents, (2) herbs such as St John's wort, where the characterised constituents are not solely responsible for the therapeutic and clinical effects, and (3) those other herbal materials, such as valerian, where there were no characterised constituents responsible for the therapeutic and clinical effects.
He differentiated therapeutically active herbal materials, with examples, within five chemobotanical families: anthraquinone-based (eg, senna), a few solanaceous alkaloids, certain other alkaloids (such as cinchona and opium), tannin-containing plants, and a miscellaneous category that included digitalis and capsicum. For quantification, Dr Helliwell exemplified three alternative methodologies used in European Pharmacopoeia monographs: solvent/solvent extraction followed either by titration [as for Belladonna] or by spectrophotometry [cascara, cinchona] or using liquid chromatography [capsicum, opium]. He proposed five "ideals" for standardisation:
1. The quantification method should be applicable equally to initial herbal materials, to the primary extraction product and to the final dosage form
2. Methodology as simple as possible to achieve the required conditions
3. Realistic levels and limits of quantification
4. Methods to be stability-indicating
5. The results of the quantification should reflect the therapeutic effect18
He illustrated the applicability of his five ideals to plant sources, and extract and dosage form, of four typical families of herbal material. For ipecacuanha, the solvent method for the various herbal starting materials differs from PhEur and British Pharmacopoeia monographs for the liquid extract and the tincture, and from that used by industry for the final dosage form. The method was suited to realistic levels, and was stability indicating, but the reproducible quantization does not necessarily match therapeutic activity because alkaloids are measured in total and there could be a significant difference from batch to batch in the ratio of the two main alkaloids, emetine and cephaeline.
For cascara, the official assay, which comprises extraction/hydrolysis/extraction followed by color development and spectrophotometric measurement, is tedious and needs careful training; however, it eliminates most congeners and there is a reasonable correlation with therapeutic activity.
He instanced three distinct approaches to the assay of capsicum fruit: (i) total pungent constituents (capsaicin, dihydrocapsaicin and nordihydrocapsaicin), (ii) total capsaicinoids by UV spectrophotometry (cf British Pharmaceutical Codex), and (iii) individual capsaicinoids by HPLC, as in the PhEur monograph on capsicum. These methods were reasonably stability-indicating and correlated with therapeutic effect. Explaining that capsicum fruit normally contains less than 5 per cent nonanoyl vanillylamide, he said dosage forms with much larger amounts (up to 50 per cent) of this synthetic capsaicin point to adulteration.
His fourth example was opium, which reflected a "a saga of more than 30 years". Historically, there has been diversity of assay methods for different dosage forms. Since 1993, the classic PhEur/BP gravimetric assay for medicinal opium has been superseded by HPLC, whereas for various formulations (opium tincture BP, camphorated tincture BP and squill linctus opiate BP [Gee's linctus]) that assay has been replaced by the Radulescu (nitrite/ammonia) reaction. The current PhEur method is generally applicable to all forms, has simple extraction and chromatography, reflects realistic limits of quantification, is stability indicating and provides a reasonable correlation with the therapeutic effect.
In conclusion, Dr Helliwell emphasised that:
• Herbal materials have been used for centuries for their therapeutic efficacy
• Accurately controlled dosage is important to prevent discomfort or even death
• Many current methods of standardisation were developed 50 or more years ago, and in many cases they are still the most applicable
• Modern chromatographic methods are not suitable for herbal materials where the therapeutic activity derives from a complex mixture of many closely related compounds, which could still be quantified by traditional methods19
Absence of known active components
Dr Ezio Bombardelli, president of Indena Spa, Italy, dealt with the controversial situation of acceptable standardisation of herbals with no known active components. He agreed that the main problem has been the enormous variety of plant products in several countries. He listed the top ranked 47 herbs in tonnage quantities in France, and in Germany, where more than 200 plants are used. Relatively few extracts have modern documentation in terms of safety, efficacy and chemical standardisation comparable with the registration requirements for any chemical drug. For plants with no known active components, the plant material must be identified and properly characterised, with well chosen markers belonging to at least two different classes of chemical substances, and analytical methods thoroughly validated with respect to a defined component mixture. He proposed that instrumental methods, for instance, nuclear magnetic resonance and Fourier transform-infrared spectrometry, should be used to provide a generally recognisable fingerprint.
The ratio between originating plant material and extract could be hugely variable. Moreover, for HMPs in popular demand, there are limited natural sources world-wide, sometimes with ecological restriction on harvesting, and time is needed for agronomic development to large-scale cultivation. He gave several examples of variable composition found in practice: there is a wide range of fatty acids and alcohols in extracts from Serena repens (used for benign prostatic hyperplasia); there is variable concentration of caffeic acids and amides in Echinacea spp; and Hypericum perforatum extracts include many inactive compounds, such as pseudohypericin and flavanoids, with range of hypericin wider in "wild" crops (0.053–0.3 per cent) but around 0.15 per cent in cultivated varieties from four countries. He concluded: "A perfect standardisation is essential to obtain biologically reproducible data in terms of safety and efficacy and the preparation must be consistent over time, stable and be devoid of unpredictable toxicity due to degradation or poor quality, or selection, of the plant material."20
Quality control of isoflavones in soya
Professor Maria da Graça Campos, University of Coimbra, Portugal, referred to the current interest in the role of isoflavonoids as phyto-oestrogens, particularly soya products also present in infant formula products. These are of interest in hormone replacement therapy in women and for treatment of prostatic cancer in men. Genistein is less effective for hot flush relief but no side effects have been reported other than allergenic response in asthma patients to dust from extract of soya protein.
She emphasised the need for rapid, sensitive and precise assays to analyse the compounds involved in pharmaceutical formulations, mostly tablets, with a minimum of manipulation, and to compare the results with those from extracts prepared from soya seeds. She advised that processing of the extracts may alter the distribution of the forms and can result in the loss of some isoflavones through leaching and through removal of undesirable fractions. To avoid this problem, the isoflavones were analysed using reversed-phase HPLC, with diode array detection, under gradient conditions, and without prior separation from the complementary bioactive compounds present in the tablets. She had confirmed by liquid chromatography/mass spectrometry that those bioactive impurities were eliminated in the first step of the HPLC gradient without producing any interference in the analysis. Coloured components were removed with a C18 pre-column and the isoflavone markers were stable.
The results that her group obtained with a series of tablets available on the Portuguese market suggest that there is no well-established dose/activity relationship, because the amount of isoflavones recommended is different for each product, with complex HPLC profiles exhibited by different herb species. She proposed to follow up this work with a more detailed project, involving wider collaboration — including the Lisbon national laboratory — with the objective of studying isoflavonoids obtained from soya seeds from different botanical sources, and also assessing these compounds for their pharmacological and toxicological effects in laboratory rats. Chemical studies would be performed to analyse isoflavonoid compounds from tablets and extracts from soya seeds. In parallel, pharmacological and toxicological studies, using rats, would evaluate the effects on oestrogen receptors, and the toxicity effects of these compounds in relevant tissues.21
Disadvantages of Standardized Herbs
Thousands of natural compounds and co-factors in the herb are lost.
This may reduce their effectiveness, broad scope and subtle effects, and eliminate compounds that could be essential and valuable.
Standardized extracts may use solvents or acetone to extract the active ingredients (leaving residues) then destructive heat to evaporate them. Their nature is closer to pharmaceutical drugs than herbal medicine. They may be less gentle, with a greater tendency toward side effects. Confusion and errors occur regarding just what is the active ingredient or substance that is used as a marker for standardization. For example, St. Johns Wart is usually measured for hypericin, though it is now thought that hyperforin is the more active substance.
Products may contain only one isolated ingredient, the remainder of the product being inert filler and not the rest of the plant.
Only a small number of plants are standardized. If different standardized and traditional whole herbs are used together, the one may overshadow the other or have unpredictable results.22
Standardization means adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity by adding excipients or by mixing herbal drugs or herbal drug preparations. Botanical extracts made directly from crude plant material show substantial variation in composition, quality and therapeutic effects. Standardized extracts are high quality extracts containing consistent levels of specified compounds, and they are subjected to rigorous quality controls during all phases of the growing, harvesting and manufacturing processes.
Medicinal herbs are moving from fringe to mainstream use with greater numbers of people seeking remedies and health approaches free of the side-effects caused by synthesized chemicals. The crude medicinal herbs for this industry have long been grown and traded in many countries around the world. The herbal, raw materials are comprised of dried plant materials in the form of roots, barks, flowers, and fruits. Besides being important to consumers of herbal remedies, the quality of medicinal herbs is also vital to growers and suppliers of these crude botanicals. It is reasonable to expect that herbs of superior quality will sell for the premium price.
Alternative systems of medicine have become increasingly popular in recent years. The efficacy of some herbal products is beyond doubt, the most recent examples being Artemisia annua (i.e., artemesinin: wormwood derivative used to target cancers), Silymarin marianum (i.e., silymarin: seeds of the milk thistle effective in treating diseases of the liver) and Taxus brevifolia (i.e., taxols: pacific yew derivative that exhibits antimitotic activity and is used for treating refractory tumors). A number of recent findings have focused on the adverse effects of herbal products. Hepatotoxicity, nephrotoxicity and most critically, drug interactions with synthetic medicines are common in herbal practice. In light of above discussion, consumers and clinicians should be increasingly cautious about following the latest trends in medicinal herbs and be alert to the potential risks herbal medicines pose.23
Herbal extracts in the European Pharmacopoeia
Professor Gerhard Franz, University of Regensburg, Germany, and chairman of PhEur group 13B, said that "perhaps 75 per cent of the HMPs in Western Europe contain extracts, mainly in the dry form", but this "industrial reality is not completely reflected in the PhEur, which contains more than 130 monographs on herbal drugs but only eight examples of quality defined extracts and tinctures". As a consequence, the general monograph on extracts and tinctures was recently revised as a basic framework monograph and as a future guideline for the establishment of individual extract- and tincture monographs.
There is usually a lack information on active pharmaceutical ingredient (API) and relatively few pharmacokinetic studies but there are compendial controls on characteristic marker substances, inert materials, allergens and toxins, cellulose and lignin. Dr Franz recalled that problems largely flowed from the huge variety of HMPs on the European market, containing many different types of extracts, produced by several methods, and using a broad spectrum of solvent systems. Generally, the production of different types of extracts is outlined and specified according to the obvious needs in industrial practice, eg, great variation in batches of ginkgo, but it is possible to consolidate in an average standardised product. The PhEur "Production statement" concept controlled a "dry extraction ratio" (DER) reflecting polarity, concentration and volume of solvent, manufacturing process, time, pressure and temperature. An example is chamomile maceration, where choice of solvent varies the API found. Such "statements" would also relate to extraction water quality (eg, potable water for extraction).
In conclusion, Professor Franz observed that the increasing development of new and more effective synthetic drugs has not been reflected in HMPs; nevertheless HMPs are being better defined, and incorporation of similar concepts of safety, quality and efficacy are included in monographs acceptable to regulators.24
Preparations on the Belgian market
Dr Jozef Corthout, Belgian Medicines Control Laboratory, Brussels, described some problems they had encountered in evaluating hypericum. His laboratory undertook post-market quality control of branded drugs sold in public pharmacies, including herbal drug preparations. A general problem was the different ways that a plant could be used — harvested from a different part of the plant, or as crude drug , or different kind of extracts, not necessarily in a standardised preparation or normalised to a certain quantity of active substances or markers. A further problem was that the product could be standardised with respect to a single constituent (active or not), or to a group of metabolites — for example, hydroxyanthracene glycosides, or total cascarosides, or specifically cascaroside A.
He preferred a colorimetric assay for analysing a group of related compounds, whereas the determination of a single constituent required the separative power of a chromatographic method such as TLC, GC or HPLC, with various options for detectors. Their analytical method was then validated for linearity over a 70–130 per cent range, for precision as repeatability of 18 replicates; and for accuracy by determination of recovery of a spiked analyte with 70, 100 and 130 per cent of the nominal amount. There were also problems with labelling as to content, the form used (such as salt, ester or ether), whether they were using a drug or a crude extract, and whether it was "normalised" to a fixed content.