PROSEA, Introduction to Medicinals 2
- 1 Choice of genera/species
- 2 Quality control of herbal drugs
- 2.1 Why quality control ?
- 2.2 A practical approach to quality control
- 2.2.1 Identity, purity and content
- 2.2.2 Identity
- 2.2.3 Purity
- 2.2.4 Content
- 2.3 Anticipated developments
- 3 Authors
The general aspects of medicinal and poisonous plants have already been highlighted in the introduction of Prosea 12(1): “Medicinal and Poisonous Plants 1". These included definitions, subgrouping, role, phytochemistry, biological and pharmacological activity and therapeutical applications, botany, ecology, agronomy, harvesting and handling after harvest, processing, utilization and quality control, genetic resources and breeding, research and development, from plant to drug and prospects. The introduction to the present subvolume provides information on the choice of genera and species to be treated here and on aspects of quality control.
Choice of genera/species
The choice of the genera/species to be dealt with in each of the 3 subvolumes on medicinal and poisonous plants has been somewhat arbitrary. The importance attached to a given genus/species was based on existing information in handbooks on medicinal or useful plants for the South-East Asian region. In general, less information on the phytochemistry and pharmacology is available in the literature for the medicinal and poisonous plants to be covered in the present subvolume compared to those highlighted in Prosea 12(1). However, several genera/species covered in the present subvolume have a long-standing reputation in traditional medicine, such as Alstonia, Alyxia, Capparis, Croton, Polygala and Quassia. Other genera/species have been quite well investigated in the field of phytochemistry and may have prospects for the production of bio-active intermediates. Examples include Strophanthus and Cerbera for their cardiac glycosides, Tabernaemontana, Ipomoea and Phaeanthus for their alkaloids and Dioscorea for its steroidal glycosides.
Quality control of herbal drugs
Why quality control ?
As long as mankind has existed, people have been dependent on the diversity of plant resources for food, feed and medicine. They learned by trial and error to distinguish useful plants with beneficial effects from those which were toxic or non-active, and also which combinations or processing methods had to be used to gain consistent and optimal results. This knowledge of plant-based drugs developed gradually and was passed on, thus laying the foundation for many systems of traditional medicine all over the world. Over centuries traditional medicine evolved, depending on local flora, culture and religion (Horsten, 1995).
Nowadays, plant materials are used throughout the industrialized and developing countries as home-remedies, over-the-counter drugs and raw materials for the pharmaceutical industry. As such, they represent a substantial proportion of the global drug market (WHO, 1998). In general all medicines, whether they are of synthetic or plant origin, should fulfil the basic requirements of being efficacious and safe. Ultimate proof of these can only be achieved by some form of clinical research. This applies for instance both to the multinational pharmaceutical company conducting a multi-centre, double blind placebo-controlled study with a herbal extract, as well as to the health-practitioner in a rural village who applies a locally produced herbal mixture. A defined and especially constant composition of the drug is therefore one of the most important prerequisites for any kind of clinical experiment. Otherwise, a statistical analysis of the results is almost impossible. Given the nature of products of plant origin, which by definition are never constant, and may furthermore vary with climatic and soil conditions, this is wheree quality control comes in (Bauer & Tittel, 1996; Bauer, 1998).
A practical approach to quality control
Natural products in medicine form a vast array of "raw materials", making a clear definition important. The term herbal drugs denotes plants or plant parts (roots, bark, leaves, flowers, seeds) which have been converted into herbal drugs by means of simple processes such as drying (i.e. they are in an unprocessed state)(EMEA, 1998). A practical addition to the definition is also to include some more crude products derived from plants, which no longer show any organic structure, such as essential oils, fatty oils, resins and gums. Derived products in the processed state, e.g. extracts or even isolated purified compounds (strychnine from Strychnos nux-vomica L.) or mixtures of compounds (abrin from Abrus precatorius L.) are as a rule not included in the definition. Their production is already based on adequate quality control of the respective starting materials.
The following paragraphs will focus on quality control of herbal drugs in compliance with the above definition.
Identity, purity and content
In general, quality control is based on 3 important pharmacopoeial definitions:
- Identity: "is the herb the one it should be?"
- Purity : "are there contaminants e.g. in the form of other herbs which should not be there?"
- Content (assay): "is the content of active constituents within the defined limits?"
It is obvious that the content (assay) is the most difficult part, since in most herbal drugs the active constituents are unknown. Sometimes markers can be used which are by definition chemically defined constituents that are of interest for control purposes, independent of whether they have any therapeutic activity or not (EMEA, 1998). In all other cases, a simple determination of extractable matter is an approach often seen in Pharmacopoeias.
For a practical approach to control the quality of herbal drugs there are 2 possibilities: either a monograph has been published in a Pharmacopoeia of the material, or not.
Monograph available in a Pharmacopoeia
Several of the principal Pharmacopoeias contain monographs of herbal drugs, for instance the European Pharmacopoeia (Council of Europe, 1997-2001a) and the United States Pharmacopoeia – The National Formulary (USPC, 1994-2001). Pharmacopoeias which follow the guidelines stated by these globally recognized Pharmacopoeias and publish additional monographs on herbals include the German Pharmacopoeia (DAB 1997/2000) (Anonymous, 2000a) and regional Pharmacopoeias like the Asian Herbal Pharmacopoeia (ASEAN, 1993) and the Thai Herbal Pharmacopoeia (DMS, 1995). Other valuable sources of monographs include the World Health Organization (WHO, 1999), the journal Pharmeuropa (finalised drafts, which will be published in the European Pharmacopoeia) (Council of Europe, 1989-2001) and the German Homeopathic Pharmacopoeia (HAB 2000, HAB 1) (Anonymous, 2000b; Anonymous, 1978-1991); the latter is available in English translation as well (known as GHP)(British Homeopathic Association, 1990-1993).
The major advantage of an official monograph published in a Pharmacopoeia is that standards are defined and available, and that the analytical procedures used are fully validated. Especially the latter is of major importance since it can be a rather time-consuming process. By definition, validation is the process of proving that an analytical method is acceptable for its intended purpose (Green, 1996). For pharmaceutical methods, guidelines from the United States Pharmacopoeia (USPC, 1994-2001), the International Conference on Harmonization (ICH) (Anonymous, 1995), and the Food and Drug Administration (FDA) (FDA, 1987, 1994) provide a framework for performing such validations. In general, validation investigations must include studies on specificity, linearity, accuracy, precision, range, detection limit, quantitation limit and robustness, depending on whether the analytical method used is qualitative or quantitative (Green, 1996).
Monograph not available
When no pharmacopoeial monograph is available for the herbal drug, development and validation of the analytical procedure has to be done by the manufacturer himself. The best strategy is to follow closely the pharmacopoeial definitions of identity, purity and content (assay). Valuable sources for general analytical procedures include the methods sections of the Pharmacopoeias mentioned above, the guidelines published by the World Health Organization (1998), and Pharmeuropa (Council of Europe, 1989-2001). Additional information, especially on chromatographic and/or spectroscopic methods can be found in the general scientific literature.
Also of utmost importance is the availability of standards. For macroscopic and microscopic procedures in general, this means that reliable reference samples of the plant must be available. A defined botanical source e.g. voucher specimens, will normally solve this problem. Standards for chromatographic procedures might be less easy to obtain. Characteristic plant constituents, either active or markers, are seldomly commercially available. Sometimes a liquid chromatography - mass spectroscopy approach can be referred to as a characterization. Going one step further, after isolating such a compound, elucidations to prove its definite structure will not be easy. The method often employed is to use readily available compounds which behave similarly in the chosen chromatographic systems, and to calculate retention values and/or times towards these compounds as a standard. Following the Pharmacopoeial guidelines of identity, purity and content (assay), the next paragraphs will elaborate on factors involved in a quality control approach for herbal drugs.
Quality control of herbal drugs has traditionally been based on appearance. A primary visual evaluation, which seldom needs more equipment than a magnifying lens, is to ensure that the plant is of the required species, and that the right part of the plant is used. Although the latter may seem obvious, it is of key importance when for instance different parts of the same plant are to be used for different treatments. Stinging nettle (Urtica urens L.) is a classical example: the aboveground parts are used to treat rheumatism, whereas the roots are applied for benign prostrate hyperplasia.
Microscopical analysis is a powerful tool to determine the correct species and/or the right part of that species. For instance in the case of flowers, pollen morphology may be used to identify the species. The presence of certain microscopical structures may be used to identify the plant part used, as illustrated by the presence of fragments with leaf stomata in case of a leaf preparation.
Thin layer chromatography (TLC)
A simple chromatographic technique like thin layer chromatography (TLC) may provide valuable additional information to establish the identity of the material. This is especially important for those species which consist of types accumulating different (active) constituents, i.e. chemical races. TLC-fingerprinting provides a powerful and relatively fast solution to distinguish between chemical races, where macroscopy and microscopy will fail.
Furthermore, TLC-fingerprinting is of key importance for those herbal drugs that no longer have any organic structure (essential oils, fatty oils, resins etc.). Chromatograms of e.g. essential oils are widely published in the scientific literature, which can be of invaluable help in identification.
Classically, purity is in general closely linked with the safe use of herbal drugs. It therefore deals with factors such as foreign matter, ash values and heavy metals. However, due to the application of improved analytical methods, a modern purity evaluation generally also includes microbial contamination, aflatoxins, radioactivity and pesticide residues as well.
Determination of foreign matter
Herbal drugs should be entirely free from visible signs of contamination by moulds or insects, including animal excreta. In general, no poisonous, dangerous or otherwise harmful foreign matter or residue should be allowed. Special care should also be taken to avoid the development of a strong microbial contamination, since micro-organisms might produce toxins (WHO, 1998). Herbal drugs should be made from the plant part stated, should not be contaminated with another part of that plant, or even other plants, and should be free of sand, stones etc. Macroscopic examination can conveniently be employed for determining the presence of foreign matter in herbal drugs (sand, other parts), although microscopy is indispensible in certain special cases (e.g. starch deliberately added to “dilute" the plant material). Furthermore, when foreign matter consists e.g. of a chemical residue, thin layer chromatography will often prove valuable (WHO, 1998).
Determination of ash
The ash that remains after herbal drug material has been burnt is generally determined by 2 subsequent procedures, which measure total ash and acid-insoluble ash, respectively. The total ash method is designed to measure the total amount of material remaining after burning. It includes both ash derived from the plant tissue itself, as well as acid-insoluble ash. The latter is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This second method measures the amount of silica present, especially in the form of sand and siliceous earth (WHO, 1998).
Determination of heavy metals
Contamination of herbal drugs with arsenic and heavy metals like mercury, cadmium and lead can be attributed to many causes, including environmental pollution. They represent a possible danger for the health of the user and should therefore be limited (WHO, 1998). A simple, straightforward determination of heavy metals can be found in many Pharmacopoeias, and is based on colour reaction with reagents such as thioacetamide or diethyldithio-carbamate (Council of Europe, 1997-2001a). The amount of arsenic in herbal drugs can be estimated by matching the depth of colour with that of a standard stain, as published by the WHO.
In those cases where the exact amounts of the different heavy metals and arsenic have to be known, instrumental methods have to be employed. The highly specific Atomic Absorption Spectrophotometry (AAS) is an appropriate method. In this technique, atoms of a metal are volutized in a flame, and their absorption of a narrow band of radiation is accurately measured (Watson, 1999). A draft method is available in Pharmeuropa (Anonymous, 2001).
Determination of microbial contamination including aflatoxins
Herbal drugs normally carry a great number of bacteria and moulds often originating in the soil. While a large range of bacteria and fungi are from the naturally occurring microflora, aerobic spore-forming bacteria frequently predominate. Also poor methods of harvesting, cleaning, drying, handling and storage may cause additonal contamination, as may be the case with Escherichia coli or Salmonella spp. (WHO, 1998).
In addition, the presence of fungi should be carefully investigated and/or monitored, since some common species produce toxins, especially aflatoxins. Aflatoxins in herbal drugs can be dangerous to health even if they are absorbed in minute amounts (WHO, 1998). Laboratory procedures investigating microbial contamination are laid down in the well-known Pharmacopoeias, as well as in the WHO guidelines (WHO, 1998). In general, a complete procedure consists of determining the total aerobic microbial count, the total fungal count, and the total Enterobacteriaceae count, together with tests for the presence of Escherichia coli and Salmonella spp. Limit values can also be found in the sources mentioned; however, they may vary.
Procedures for the determination of aflatoxin contamination in herbal drugs are published by the World Health Organization (1998), and in Pharmeuropa (Anonymous, 1999). After a thorough clean-up procedure, thin layer chromatography is used for confirmation.
A certain degree of exposure to ionizing radiation cannot be avoided since there are many sources, including radionuclides naturally occurring in the environment. Dangerous contamination, however, may be the consequence of a nuclear accident. The World Health Organization, in close cooperation with several other international organizations, has developed guidelines for use in the event of widespread contamination by radionuclides resulting from such a major nuclear accident. These publications emphasize that the health risks from food accidentally contaminated, not only depend on the specific radionuclide and the level of contamination, but also on the quantity of food consumed. In case of herbal drugs, however, taking into account the amounts which are normally used, they are unlikely to be a health risk. Therefore, at present, no limits for radioactive contamination are proposed (WHO, 1998).
Determination of pesticide residues
Herbal drugs are liable to contain pesticide residues which accumulate from agricultural practices, such as spraying, treatment of soils during cultivation, and administering of fumigants during storage (WHO, 1998).
Limits for pesticide residues are published in the European Pharmacopoeia (Anonymous, 1997-2001b) and by the World Health Organization (1998). Samples of herbal material are extracted by a standard procedure, impurities are removed by partition and/or adsorption, and the presence of a moderately broad spectrum of individual pesticides is measured in a single determination by gas chromatography.
Sometimes, however, it may be desirable to test herbal drugs for broad groups in general, rather than for individual pesticides. Many pesticides contain chlorine in the molecule, which for example can be measured by analysis of total organic chlorine. In an analogue way, insecticides containing phosphate can be analyzed for total organic phosphorus. Some simple procedures have been published by the World Health Organization (1998).
Where no active constituent or marker can be defined for the herbal drug, the percentage extractable matter may be used as a form of assay. The choice of solvent depends as such on the nature of the compounds involved, and might be deduced from the traditional use. When, in general, a herbal drug is used to make a tea, the hot water extractable matter, expressed in mg/g air-dried material, may serve this purpose (WHO, 1998). The monograph for ginger (Zingiber officinale Roscoe) of the European Pharmacopoeia is a classic example. A special form of assay is the determination of essential oils by steam distillation. Many limits on content can be found in the literature, and thus serve their purpose.
In all other cases, when active constituents (sennosides in Senna) or markers (alkydamides in Echinacea) are known, the vast array of modern chemical-analytical methods can be employed. These include visible-light and ultraviolet spectroscopy, high pressure liquid chromatography (HPLC), gas chromatography (GC) and quantitative thin layer chromatography (TLC), to name a few (Watson, 1999; lit. ref. #3182). All are more or less instrumental systems, which generally consist of costly equipment. Of the methods mentioned, visible light spectroscopy uses a relatively inexpensive device, but is yet able to perform very precise measurements. This might explain why this method is rather popular, as well as the fact that it can be used to determine many natural substances (e.g. alkaloids, anthraquinones, cardioactive glycosides, flavonoids, tannins). Validation procedures are straightforward, and measurements can be made quickly, although clean-up of the samples may be more time consuming.
When more complex samples have to be analyzed, spectroscopy often performs less well, and chromatographic procedures are preferred because of their vast separating capacity. The most universal method is undoubtedly high pressure liquid chromatography and the literature available on plants, plant fractions, mixtures of isolated compounds and purified components is enormous.
Finally, for separation of essential and fatty oils, gas chromatography is the proven method of choice. Although the equipment for gas chromatography is more demanding in its operation (it uses for instance compressed, extremely purified gases), and although some oils also can be succesfully analyzed by using high pressure liquid chromatography, the latter is not likely to replace gas chromatography for oil analysis in the near future.
In conclusion, quality control for efficacy and safety of herbal products is of utmost importance. It is obvious that for a given plant product its quality will also be determined by the prevailing conditions during the growth cycle of the plant. Therefore for cultivated plants the concept of a system of "good agricultural practices" (GAP) has been introduced in the literature. In such a system, every step has to adhere to a set of requirements, involving e.g. seed selection, growing conditions, use of fertilizers, optimization of harvest time, harvest and drying. In other words such a system will focus on total chain control. It is therefore likely that, GAP-procedures will become an integral part of quality control in the near future.
S.F.A.J. Horsten, N. Bunyapraphatsara & J.L.C.H. van Valkenburg