Production, standardization and quality control

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Chapter 9 Production, standardization and quality control

Most people look at herbal medicines and the products derived from them from the perspective of the benefits they expect to receive. However, in pharmaceutical terms, aspects of production (agricultural production or wild-crafting, extraction, fractionation, formulation, quality assurance), the legal framework of their use, and clinical aspects (safety, pharmacovigilance) are of at least equal importance. Consequently, plant-derived medicines are diverse and include, for example:

• pure compounds, which are often isolated from botanical drugs (and which are not considered to be herbal medicines)

• traditionally used medicinal plants, loose or in teabags to form infusions, including ‘instant teas’, and tinctures, ethanolic extracts, essential oils, fatty acids and dried extracts

• cut or powdered botanical crude drugs (generally simply called crude drug throughout the text), used as such (i.e. unprocessed)

• non-standardized extracts, with varying information about quality and, consequently, sometimes uncertain information about clinical efficacy and pharmacological effects

• standardized extracts, generally with relatively well-established clinical and pharmacological profiles.

Here a very short overview is given of the whole process, from the agricultural production of materials collected from the wild, to the processing and production of the pharmaceutical product or health-food supplement. A more detailed discussion is beyond the scope of this introduction, but can be found, for example, in Evans (2009).

In all cases, the basis for drug production is the botanical drug (see Chapter 2), which can be defined as:

image dried parts of entire plants, plants organs, or parts of plant organs for use as medicines, aromatics and spices, or as excipients used in the production of pharmaceuticals. A typical example is the flower of camomile (Matricaria recutita)

image Isolated products obtained directly from plants but which no longer have an organ structure, such as essential and fatty oils, balsams, etc. An example is the exudate of the leaves of aloe [Aloe vera (L.) Burm. f., or Aloe barbadensis Mill.], obtained by cutting the fleshy leaves and collecting the resulting liquid, which is called ‘aloes’ when dried.

Biological resources and conservation

At least 250,000 species of higher plants are known. Of these, a large number have important uses for humans, including foods, building materials, dyes, spices and as medicinal plants. It is impossible to say how many of these are ‘medicinal’, since a plant may be used only locally or on a worldwide level. Fewer than 300 are truly universal or widely used and researched in detail for their pharmacological and toxicological effects. Even fewer have been tested for clinical efficacy. In Europe, at least 2000 medicinal and aromatic plant species are used on a commercial basis. About two-thirds of these are native to Europe and a large proportion of these are still collected from the wild. This fact is not detrimental in itself, but it may pose risks, including, for example:

• Problems in obtaining a consistent quality of product, including the risk of adulteration

• Over-exploitation of native populations of certain plant species.

More than half of all medicinally used species are still collected from the wild, including the less frequently used species. An example of an over-exploited resource collected from the wild has been discussed in detail by Lange (2000). Pheasant’s eye (false hellebore or Adonis vernalis L.) is a native of Southern and Central Europe and is used there for cases of minor cardiac arrhythmias. The plant is threatened not only by its pharmaceutical use (as a phytomedicine as well as a homoeopathic remedy), but also by its use as an ornamental plant and a dye source. Exploitation of Adonis vernalis affects many south-eastern European countries, including Hungary, Romania and the Ukraine. Importantly, detrimental (unsustainable) harvesting techniques are still used and there is a constant risk that the exploited biomass exceeds the sustainable levels and that techniques are used which harm the population severely (Lange 2000).

Coptis teeta Wallich [Mishmi (gold thread), Ranunculaceae] is an example of a species that is under threat of extinction due to over-exploitation. It is found in the eastern Himalayan regions, particularly a small mountainous region of Arunachal Pradesh in north-eastern India. The rhizome is a prized medicinal commodity and is used for gastrointestinal complaints and malaria. However, it has been brought close to extinction by deforestation and over-exploitation. Conservation schemes have been proposed, but it is too early to be certain whether the species can be saved from extinction.

Agricultural and biotechnological production

Most important medicinal plants are now produced under controlled agricultural conditions (Franz 1999). Such production systems require certain conditions for each species with respect to:

• temperature and annual course of temperature

• rainfall (if it is not possible to irrigate the fields)

• soil characteristics and quality (edaphic factors)

• day length and sun characteristics

• altitude.

These factors are assessed in detailed studies for each species that is cultivated. Generally, it is essential that the production is based on the principles of GMP (good manufacturing practice) and/or ISO (International Organization for Standardization) certification, in this case GAP (good agricultural practice) and the subsequent processing steps (drying, cutting, grinding, storage, packaging, transport, etc., which are covered by GMP) are essential for high-quality and reproducible batches. Medicinal plants are a delicate product and in many cases inadequate storage of transport can ruin a whole year’s work. For example, essential oil containing drugs will easily lose their active ingredients if the botanical drug is exposed to heat or humidity.

Moisture levels

All drugs are at risk of decaying if the humidity in the drug material exceeds 15%. Improperly stored botanical drugs have a musty smell and often change colour (green material turning yellow or brown). However, different levels of moisture are acceptable for each drug. For example, the moisture contents given in Table 9.1 are considered to be within the normal range and do not pose any problems for these drugs.

Table 9.1 Acceptable moisture content for storage of some botanical drugs (after Franz 1999)

Botanical drug Moisture content (%)
Chamomile flower (Matricariae flos, from Matricaria recutita L.) 8–10
Linseed (Lini semen from Linum usitatissimum L.) 5–9
Digitalis leaf (Digitalis lanatae folium, from Digitalis lanata Ehrh.) 8–12
Frangula bark (Frangulae cortex, from Rhamnus frangula L, syn. Frangula alnus Mill.) 5–8
Thyme herb (Thymi herba, from Thymus vulgaris L.) 8–11
Gentian rootstock (Gentianae radix, from Gentiana lutea L.) 8–15
Fennel fruit (Foeniculi fructus, from Foeniculum vulgare Mill. subsp. vulgare) 6–12

Microbiological contamination

Other specific requirements for storage and transportation of the drug have to be met and, in many cases, this is now controlled via GAP and GMP standards. A similar requirement relates to microbial contamination of botanical drugs. Each natural material naturally harbours a large number of spores and other microorganisms. The maximum number of microorganisms allowed is regulated in the European Pharmacopoeia (for details see Eur. Ph. 2002, Chapter 2.6.12):

• Up to 105 aerobic microorganisms per g or ml, including:

– up to 103 yeast and fungi per g or ml
– up to 103 enterobacteria per g or ml

• No detectable Escherichia coli (in 1 g or ml)

• No detectable Salmonella sp. (in 10 g or ml).

Pesticides

Pesticides are commonly used to prevent the infestation of the botanical material with large amounts of unwanted species of plants, insects or animals. These may cause harm or otherwise interfere with the production, storage, processing, transport and marketing of botanical drugs. Acceptable limits have been defined in the Eur. Ph. (Chapter 2.8.13). There is also a detailed description of the sampling methods and the relevant qualitative and quantitative methods. Importation of herbs from countries with less restrictive legislation on pesticides means that this material should be checked very carefully. Examples of relevant limits for some important pesticides are given in Table 9.2.

Table 9.2 Limits of some important pesticides allowed in botanical material

Pesticide Concentration limit (mg/kg)
Aldrin and dieldrin (sum of) 0.05
Chlordane (sum of cis-, trans– and oxychlordane) 0.05
DDT (sum of various related compounds) 1.0
Endrin 0.05
Fonofos 0.05
Malathion 1.0
Parathion 0.5
Permethrin 1.0
Pyrethrins (sum of) 3.0

Source: Eur. Ph. 2002.

Plant breeding

Cultivated species are normally optimized to relatively high and constant levels of active ingredients. Other important goals for breeding relate to the reproducibility of the drug. Matricaria recutita (the common camomile), for example, should have large and homogenous flower heads. Simple, mechanized harvesting and good storage characteristics are normally required for a botanical drug. Other desired features include high yield, high resistance to pathogens (insects, mites, fungi, bacteria and viruses), reproducibility of the yield, good adaptation to the location, low demands regarding ecological requirements, low content of water (for easier drying) and stability of the plant’s organs. The degree to which a certain trait is inherited from one generation to the next is important; if this cannot be assured there is a constant risk of losing high-yielding strains. This risk can be reduced by in vitro culture or cloning, or by propagating through cuttings, but this is not ideal for high-production systems as it is time-consuming and therefore costly.

In vitro cultivation

In vitro culture involves the use of non-differentiated cell aggregates (submersed or callus cultures). These are most commonly used in the production of ornamental and food plants (e.g. orchids, strawberries). In the case of medicinal plants, cell cultures are used:

• as a starting point for investigating the biochemical basis of the biosynthesis of natural products and for breeding new varieties/strains

• as a basis for vegetative propagation of plants to be used as a phytomedicine or for isolating a pure natural product, especially if a consistent quality of the strains or a fungi- or virus-free culture is required. For example, high-yielding strains of Catharanthus and Dioscorea may be required, or the development and maintenance of pyrrolizidine-free strains of common comfrey (Symphytum officinale L.) and coltsfoot (Tussilago farfara L.), or virus-free Digitalis lanata Ehrh. cultures

• for the semi-synthetic production of some natural products (e.g. production of digoxin from digitoxin)

• for the direct synthesis of a medicinal natural product, although this is only rarely achieved by this method. Today, this process is restricted to the production of paclitaxel and its precursors (first isolated from the Pacific yew, Taxus brevifolia Nutt.) and shikonin (a dye and medicinal product used in Asian medicine from Lithospermum erythrorhizon Sieb. & Zucc.).

The Pacific yew as an example

The Pacific yew (Taxus brevifolia Nutt., Taxaceae) is a botanical drug which exemplifies all the various approaches for producing a medicinally used natural compound. In 1962 several samples of Taxus brevifolia Nutt. were collected at random for the National Cancer Institute (NCI) and the US Department of Agriculture. These samples were included in a large screening programme at the NCI. A potent cytotoxic effect was documented in one in vitro system. After a lengthy development process, clinical studies started 13 years later in 1984. It took a further 10 years before paclitaxel was approved for the treatment of anthracycline-resistant metastasizing mammary carcinomas. In the meantime the compound had been licensed for a variety of other cancers and semi-synthetic derivatives produced such as docetxel, which are also now employed (see also Chapter 8).

The strategy for obtaining the pure active ingredient thus moved from collection from the wild during (1962)/1975–1990 to the commercial silvicultural production of a biosynthetic precursor of paclitaxel in another species of Taxus (European yew, T. baccata L.) during 1990–2002, to the current (2003) commercial in vitro production using fermentation technology:

• Taxus brevifolia is a very slow-growing species, which produces the active ingredients only in very small amounts. Since paclitaxel was for many years isolated from the bark, trees had to be felled in order to obtain it. The requirement for paclitaxel increased dramatically with progress in the clinical development of the drug in the mid-1970s (the amount required to meet the annual therapeutic requirements of patients with ovarian cancer in the USA was estimated to be 15–20 kg). If other cancers common in the USA were to be treated with this compound, around 200–300 kg of the pure compound would have been required per year. This amount can be isolated from approximately 145,000 tons of bark. Collecting such amounts would have been completely unsustainable and would have resulted in the extinction of the species within a few years.

• In the 1990s the semi-synthetic production of paclitaxel from natural products in other Taxus spp. (10-desacetylbaccatin II isolated from the European yew, T. baccata) allowed for the production of large amounts of paclitaxel. Up to this point, a conflict of interest between conservation and medicinal use was unavoidable.

• Large-scale production using cell-culture techniques is now feasible and has been approved by the FDA. Today, most of the paclitaxel required is produced using fermentation technology (Goodman & Walsh 2001).

Drug preparation and extraction

A number of diverse overall approaches and specific techniques are available for processing crude plant (or rarely animal) material. For phytomedicines, the general framework is relatively well circumscribed, based on European and national legislation.

Extracts

According to the Eur. Ph. (2002, Chapter 01/2002-765), an extract is a concentrated preparation of liquid (fluid extract or tinctures) or intermediate (semi-liquid) or solid (dry extract) consistency normally produced from dried botanical or zoological material by a technique involving the use of adequate solvents for obtaining a mixture of compounds. For some preparations, the material to be extracted may undergo a preliminary treatment prior to extraction. Examples for the latter include defatting, inactivation of enzymes or most commonly simply grinding.

Extracts are prepared by maceration, percolation or other suitable, validated methods using ethanol or another suitable solvent. After extraction, unwanted material may be removed if this is deemed appropriate.

Preparation and extraction is the core process of the industrial production of phytopharmaceuticals, and require a detailed analysis of the best conditions for each plant-derived drug. An important difference is whether a plant is going to be used as a phytomedicine or an individual biologically active compound is to be isolated from the material. For the former purpose, the botanical drug must conform to Pharmacopoeial requirements or another process that assures reproducible quality; for the latter, optimization to obtain large yields of the relevant compound(s) is essential.

Very often a botanical drug is gathered during the flowering period of the plant (aerial parts, leaves, flowers), during spring (bark) and at the end of the vegetative season (root and rootstock). However, there are many exceptions; for example, cloves [Caryophylli flos, Syzygium aromaticum (L.) Merr. & L. M. Perry] are collected prior to the opening of the flowers and the flower buds are used pharmaceutically. For digitalis, the leaves are collected rather late during the vegetative process (October).

Extraction (e.g. percolation, maceration, pressing of fresh plant material for expressed juice) is again specific for each drug and depends on the phytotherapeutic product required.

There are many different types of drug preparations, including:

• fresh plant material, used popularly as an infusion or decoction

• dried and cut drug material, often used in industrial production

• dried and powdered drug material, commonly used as an infusion or decoction. If such material is to be used pharmaceutically it must comply with standards as defined in the monograph for the specific botanical drug. If no such monograph exists the material has to comply with the general monograph for herbal drugs (Eur. Ph. 2002, 01/2002-1433)

• extraction and subsequent bulk production of pure natural products (e.g. morphine, digoxin, digitoxin, camptothecin) or a mixture of closely related ones (e.g. sennosides from Senna, aescin from horse chestnut, quillaia saponins from soapbark) using validated, standard phytochemical techniques (chromatography, partitioning between solvents of differing polarity, precipitation, etc.)

• unstandardized tinctures are hydroalcoholic (or alcoholic) extracts of crude drug material used as a liquid botanical drug

• an extract prepared from dried drug material using defined solvent systems is processed into a variety of pharmaceutical products (e.g. tablets for crude extracts). Such extracts are often characterized by the drug:solvent ratio, which gives the relationship of the volume of solvent to the amount of drug extracted (e.g. 1:10) (see p. 154). In many high-quality products, these extracts are ‘standardized’ by mixing high- and low-yield material. By ‘spiking’ the extract with an enriched extract, a ‘modified’ extract with a defined range of active natural products is obtained (e.g. dry aloe extracts standardized to 19.0–21.0% of hydroxyanthracene derivatives calculated as barbaloin)

• a particularly interesting case is that of the so-called ‘special extracts’. A special extract is prepared by first extracting the drug with a defined solvent system and then processing the extract so that a well-defined extract with specific ranges of ingredients is obtained. These extracts have a significantly reduced percentage of unwanted compounds, and an increased percentage of compounds that contribute significantly to the pharmacological activity and clinical effectiveness. In the case of ginkgo leaves (Ginkgo folium, Ginkgo biloba L.), for example, the desired natural products include the flavone glycosides (16–26%) and the terpene lactones (5–7%); whereas the polyphenols, polysaccharides, and especially the ginkgolic acids, are less desirable constituents (for details see p. 158–159 and 249)

• there are several special methods of extraction; for example, the cold pressed extract of the rootstock of Echinacea species is developed into an immunostimulant product. The fresh rootstock is used for this and the sap is processed into a commercial botanical pharmaceutical. For material to be used pharmaceutically, the process must be validated.

Effect of preparation methods on content

Different methods of preparing botanical material and subsequent extraction result in extracts with differing composition and different concentrations of active (as well as undesired) ingredients. A wide range of factors both in relation to the production of the botanical starting material (the botanical drug) and its processing, extraction and formulation have an impact on the chemical composition and thus the pharmacological activity of a phytotherapeutic preparation (Fig. 9.1). Strictly speaking, for an assessment of the pharmacological effect and clinical effectiveness of a botanical drug, precise data on the composition of the extract are needed. Just as importantly, pharmacological or clinical data on two products can only be compared meaningfully if the composition of the extracts is known. This implies, for example, that a meta-analysis of clinical studies is only feasible if the botanical drug materials used are similar and the resulting extracts have a comparable composition, a consideration often omitted by authors of such studies.

image

Fig. 9.1 The extraction process and selected factors that impact on the composition (and quality) of an extract and the final product.

Quality control and standardization

Quality control: general procedures

Quality-control measures vary considerably. The most relevant apply to plant extracts and unprocessed plants (the quality control of pure compounds is covered by standard pharmaceutical procedures). Quality control is a multistep process that covers all stages from the growing of the botanical material to the final control of the finished product and the evaluation of its stability and quality over time. It is essential at all stages of the production of the botanical material, including transportation, extraction and processing, storage and the elaboration of the finished pharmaceutical product. Many factors can influence the quality of the finished product, for example:

• the quality of the botanical material used, which in turn is influenced by a multitude of biogenic (e.g. infections with fungi) and climatic factors, and also includes the risk of contamination with heavy metals, pesticides, herbicides and the like (see above)

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