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:

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)

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

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.

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.

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)

• the adequate processing of the fresh material, including drying, transportation and storage

• the use of appropriate and reproducible extraction techniques

• storage under appropriate conditions (generally dry, cool, in the dark)

• use of the material only within the generally accepted shelf-life of the botanical drug.

Specifically, quality control needs to assure:

• the correct botanical identity of the drug (i.e. the correct species and plant part) in an appropriate quality (time of collection, age)

• the purity of the material used (i.e. that other botanical drugs are only present in minimal amounts)

• contaminants such as insects, mites, bacteria, fungi, heavy metals, herbicides, fungicides, pesticides, insecticides and any other toxins are below the legal (e.g. Eur. Ph.) threshold

• that the required level of active compounds (only if a minimum level of these natural products is defined) or a defined level of biological activity (if the drug is characterized biologically or pharmacologically) is reached.

The best level of quality control can be achieved if the above requirements are defined in a monograph in a legally binding pharmacopoeia. In Europe, the relevant one is the European Pharmacopoeia (Eur. Ph.). Typically, such a monograph includes the following:

• Title (English name, Latin name used in international trade)

• Definition of the drug (plant part to be used; whether it is fresh, dried, cut or powdered, and possibly also specifying constituents typical for the drug, with minimal amounts required)

• Characteristics: i.e. organoleptic or other properties of the drug (smell, colour, other similar characteristics; taste is rarely included in the Eur. Ph. for reasons of safety)

• Identification (macroscopic and microscopic description, and in some cases thin-layer chromatographic (TLC) characteristics; see Table 9.3)

• Tests for purity (providing data on maximum amounts of foreign matter, i.e. non-acceptable substances, loss on drying, ash)

• Required level of biologically active or lead compounds

• Storage (general information about required forms of storage).

Pharmaceutical drugs have to comply with all the characteristics as they are defined in such a monograph, and material that does not comply must be rejected. In many cases, TLC methods for quality control are included, such as those given in Table 9.3.

Table 9.3 Examples of botanical drugs characterized with the help of TLC

There are several other methods in current use which help to assure a reproducible quality of the botanical material, whether it is used as a drug as such or whether it is used for preparing an (standardized) extract.

Botanical (classical pharmacognostical) methods

One of the main tools for analysing botanical material is the microscope. Since botanical drugs have characteristic features, these can easily be used to establish the botanical identity and quality of a drug (see also Chapter 3).

A typical example is the various types of crystals formed by calcium oxalate. Several species of the family Solanaceae are used for obtaining atropine, which can be used as a spasmolytic in cases of gastrointestinal cramps and asthma, and as a diagnostic aid in ophthalmology for widening the pupil. Species with high concentrations of atropine include Atropa belladona (deadly nightshade or belladonna), Datura stramonium [thorn apple or Jimson (Jamestown) weed] and Hyoscyamus niger (henbane). Each is characterized by typical crystal structures of oxalate: sand, cluster crystals and microspheroidal crystals, respectively. These are subcellular crystal structures, which can easily be detected using polarized light and are thus a very useful diagnostic means, even though they are not involved in the medically relevant effects described above.

Another typical example is that of the glandular hairs, which in many species contain the essential oil. They are a very useful diagnostic feature because they have a characteristic structure. Fig. 9.2 shows glandular hairs that are typical of the Lamiaceae and Asteraceae.

Fig. 9.2 Diagnostic features of botanical drugs that are revealed upon microscopic examination include typical glandular hair as found in the Lamiaceae and the Asteraceae. The top drawings show the lateral view; the bottom drawings show the view from above.

A third example is the structure of the cells that form the surface of leaves and which contain the stomata (pores for the exchange of respiratory and photosynthetic gases). Form, size, number of stomata and many other features can be used to identify a certain species, or to detect untypical contaminating plant material in a drug.

Other methods rely on typical properties of botanical drugs. The bitterness value, for example, is used for solutions of drugs that are used for their bitter(appetite stimulating) effect (Eur. Ph. 2002, Chapter 2.8.15). It is determined organoleptically (i.e. by taste) by comparison with quinine as standard. The bitterness value is important for centaury herb (Centaurii herba, Centaurium erythraea Rafn.), gentian root (Gentianae radix, Gentianalutea L.) and wormwood herb (Absinthii herba, Artemisia absinthium L.).

An example of a simple biophysical method is the swelling index (Eur. Ph. 2002, Chapter 2.8.4). This index is an indicator for the amount of polysaccharides present in a certain drug. It is defined as the volume (in ml) occupied by 1 g of a drug, including any adhering mucilage, after it has swollen in an aqueous liquid for 4 h. The drug is treated with 1.0 ml of ethanol (96%) and 25 ml water in a graduated cylinder, shaken every 10 min for 1 h and allowed to stand as specified. Some of the drugs are tested without pretreatment (e.g. fenugreek, ispaghula, linseed); others have to be powdered to a defined particle size prior to measuring the swelling index (e.g. marshmallow root). The required minimal swelling indices for a variety of botanical drugs are given in Table 9.4. If these values are not reached, it may be an indication that the botanical drug is contaminated with other drugs or that it is not of adequate quality (e.g. because it was not stored properly).

Table 9.4 Swelling indices for a variety of botanical drugs

Drug	Swelling index^a
Agar (from several genera of marine algae, especially Gelidium and Gracilaria)	> 10
Cetraria (Lichen islandicus, from Cetraria islandica (L.) Ach. s.l.)	> 4.5
Fenugreek (Foenugraeci semen, from Trigonella foenum-graecum L.)	> 6
Ispaghula husk (Plantaginis ovatae testae, from Plantago ovata Forssk.)	> 40 (determined on 0.1 g of powder)
Ispaghula seed (Plantaginis ovatae semen)	> 9
Linseed (Lini semen, whole drug from Linum usitatissimum L.)	> 4
Linseed (Lini semen, powdered drug)	> 4.5

a Measured as a multiple of the original volume.

The key to modern industrial quality control is the phytochemical methods for the identification of active ingredients and their quantification. Phytochemical analysis indicates whether a sample contains the correct drug in the specified quality, whether it has been extracted in an appropriate manner and stored under the right conditions. The most commonly used analytical techniques are:

• HPLC (high-performance liquid chromatography), used especially in the quantification of compounds and fingerprinting of extracts

• GC (gas chromatography), used mostly for essential oils, and sometimes combined with mass spectrometry (MS)

• TLC (thin-layer chromatography), which is simple and cheap and provides a good analytical tool for establishing the identity of a drug and for detecting contaminants containing similar types of compounds. The method is widely used, but not detailed, and it is not possible to quantify substances fully using TLC

• NIR (near-infrared spectroscopy), used to assess the identity and quality of a botanical sample, and also used for crude drug material. This technique is gaining in popularity

DNA-barcoding, that is the identification of a specific DNA sequence of selected genes in a species, is currently being developed to clearly identify from which species a botanical drug is derived. This method allows for the authentication of a species, but does not provide information on the purity of a botanical drug (i.e. if other plant parts were included, too) nor does it provide information on the quality of the material (e.g. its level of active constituents).

For most widely used botanical drugs, the relevant protocols can be found in the respective pharmacopoeias (see Box 9.1) or in protocols provided by the manufacturer. In the future, we may see the use of DNA-fingerprinting techniques as a novel and very sensitive tool for analysing the quality of all sorts of botanical material, including medicinal drugs.

Box 9.1 Sample monograph from the European Pharmacopoeia

Devil’s claw root

Harpagophyti radix

Definition

Devil’s claw root consists of the cut and dried tuberous, secondary roots of Harpagophytum procumbens DC. [and/or H. zeyheri Decne]. It contains not less than 1.2% harpagoside (C₂₄H₃₀O₁₁; M_r 494.5), calculated with reference to the dried drug.

Characters

Devil’s claw root is greyish-brown to dark brown and it has a bitter taste. It has the macroscopic and microscopic characters described in identification tests A and B.

Identification

A. It consists of thick, fan-shaped or rounded slices or of roughly crushed discs. The darker outer surface is traversed by tortuous longitudinal wrinkles. The paler cut surface shows a dark cambial zone and xylem bundles distinctly aligned in radial rows. The central cylinder shows fine concentric striations. Seen under a lens, the cut surface presents yellow to brownish-red granules.

B. Reduce to a powder (355). The powder is brownish-yellow. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of cork layer consisting of yellowish-brown, thin-walled cells; fragments of cortical parenchyma consisting of large, thin-walled cells, sometimes containing reddish-brown granular inclusions and isolated yellow droplets; fragments of reticulately thickened vessels and tracheidal vessels with associated lignified parenchyma from the central cylinder; small needles and crystals of calcium oxalate are present in the parenchyma. The powder may show rectangular or polygonal pitted sclereids with dark reddish-brown contents. With a solution of phloroglucinol in hydrochloric acid, the parenchyma turns green.

C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance.

Test solution. Heat on a water-bath at 60°C for 10 min 1.0 g of the powdered drug (355) with 10 ml of methanol R. Filter and reduce the filtrate to about 2 ml under reduced pressure at a temperature not exceeding 40°C.

Reference solution. Dissolve 1 mg of harpagoside R in 1 ml of methanol R.

Apply to the plate as bands 20 μl of each solution. Develop over a path of 10 cm using a mixture of 8 volumes of water R, 15 volumes of methanol R and 77 volumes of ethyl acetate R. Dry the plate in a current of warm air. Examine in ultraviolet light at 254 nm. The chromatograms obtained with the test solution and the reference solution both show in the middle a quenching zone corresponding to harpagoside. The chromatogram obtained with the test solution shows other distinct bands, mainly above the zone corresponding to harpagoside. Spray with a 10 g/l solution of phloroglucinol R in alcohol R and then with hydrochloric acid R. Dry the plate at 80°C for 5–10 min. In the chromatograms obtained with the reference solution and the test solution the zone corresponding to harpagoside is green. The chromatogram obtained with the test solution also shows several yellow to brown zones below and above the zone corresponding to harpagoside.

Tests

Starch. Examine the powdered drug (355) under a microscope using water R. Add iodine solution R1. No blue colour develops.

Foreign matter (2.8.2). It complies with the test for foreign matter.

Loss on drying (2.2.32). Not more than 12.0%, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100–105°C.

Total ash (2.4.16). Not more than 10.0%.

Assay

Examine by liquid chromatography (2.2.29) using methyl cinnamate R as the internal standard.

Internal standard solution. Dissolve 0.130 g of methyl cinnamate R in 50 ml of methanol R and dilute to 100.0 ml with the same solvent.

Test solution. To 0.500 g of the powdered drug (355) add 50 ml of methanol R. Shake for 1 h and filter. Transfer the filter with the residue to a 100 ml flask, add 50 ml of methanol R and heat under a reflux condenser for 1 h. Cool and filter. Rinse the flask and the filter with 2 quantities, each of 5 ml, of methanol R. Combine the filtrate and the rinsing solution and evaporate to dryness under reduced pressure at a temperature not exceeding 40°C. Take up the residue with 3 quantities, each of 5 ml, of methanol R and filter the extracts into a 25 ml volumetric flask. Whilst washing the filter, dilute to 25.0 ml with methanol R. To 10.0 ml of this solution add 1.0 ml of the internal standard solution and dilute to 25.0 ml with methanol R.

Reference solution. Dilute 0.5 ml of the reference solution described in identification test C to 2.0 ml with methanol R. The chromatographic procedure may be carried out using:

– a stainless steel column 0.10 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 μm)

– as mobile phase at a flow rate of 1.5 ml/min a mixture of equal volumes of methanol R and water R

– as detector a spectrophotometer set at 278 nm

– a 10 μl loop injector.

Inject the test solution. Adjust the sensitivity of the detector so that the height of the peak due to methyl cinnamate is about 50% of the full scale of the recorder. Determine the retention time of harpagoside using 10 μl of the reference solution examined under the same conditions as the test solution.

Calculate the percentage content of harpagoside from the expression:

m₁ = mass of the drug, in grams

m₂ = mass of methyl cinnamate R, in grams in the internal standard solution

F₁ = area of the peak corresponding to harpagoside in the chromatogram obtained with the test solution

F₂ = area of the peak corresponding to methyl cinnamate, in the chromatogram of the test solution.

Storage

Store protected from light.

(reprinted, with modifications, from European Pharmacopoeia 2002, p 1013–1014, with permission)

Pulverized and other crude drug material may be sold as a medicine or health supplement, in which case there is no (chemical) quality control. In the pharmacopoeias of some EU countries a minimum amount of an ingredient, which is considered to be the active one, may be required for some crude drugs (see above). Some alternative and complementary forms of phytotherapy (e.g. Bach flower remedies) have no (or very limited) quality control. Exceptions are homoeopathic medicines (which are based on a completely different philosophical principle) and some high-quality but unlicensed herbal medicines.

Drug: solvent ratio and drug extract ratio

Several indicators are used to describe the extract characteristics as it relates to the production process. In essence one can provide quality parameters by defining an extract in relation to the amount of a specific solvent used or by defining it in relation to the amount of starting material (plus type of solvent(s) used and the mode of extraction).

The drug:solvent ratio is calculated by dividing the amount of (dried) plant material by the amount of solvent used for extracting it. If the following information is provided ‘DSR = 1:4 (EtOH, 70%) by maceration’, the botanical drug was macerated with a four-fold amount of 70% ethanol. Importantly, the ratio does not provide information on the quality of the plant material used or other parameters influencing the result of the extraction. In addition to this ratio, the solvent used, and the form of extraction (e.g. percolation – an extraction by moving fluids through powdered materials; maceration soaked in a liquid in order to produce an extract) needs to be included.

Drug:extract ratio (DER): The ratio of a botanical drug to the amount of extract obtained, for example, 4 to 1 (4:1) – four units of a dried starting material (the botanical drug) yield one unit of extract (e.g. kg)

Drug:solvent ratio (DSR): The ratio of a botanical drug to the amount of solvent used in the extraction, for example, 1 to 8 (1:8) – eight units are used to extract one unit of a botanical drug. In general m/v (mass/volume) or m/m (mass/mass) are used as units and very often a range is given (e.g. 1 to 6–10). In addition, the solvent and the type of extraction must also be stated.

The drug:extract ratio, on the other hand, gives information on the amount of extract obtained from a botanical drug: 4:1 (maceration, 70% ethanol). In this case 4 units (e.g. kg) of a drug yield 1 unit of dried extract. This DER is often given as a range (e.g. 3–5) and always in whole numbers. It varies considerably depending on the type of botanical drug which is extracted and the solvents used. If chamomile flowers, for example, are extracted in water, the DER is in the range 6–8:1; if, on the other hand turmeric is extracted with 96% EtOH, the DER typically is in the range of 20–50:1. In other words, in the first case a large amount of extract can be obtained from a botanical drug (12–18%), in the second case only a very small amount (2–5%) can be obtained.

Standardization

The concept of standardization is relatively recent for phytomedicines, but it is rapidly becoming essential to ensure that patients are provided with high-quality botanical products. Standardization can be defined as a requirement to have a minimum amount of one or several compounds or groups of compounds in the extract. Often a range from a minimum to a maximum amount is given. In the field of phytomedicines standardization only applies to extracts. For example, if an extract has to contain at least 8% of compound class X expressed as compound X1, this would indicate that the extract has to contain at least 8% of a class of compounds (e.g. flavonoids) as expressed in one specific compound of this group (as for example rutin). This quantification is normally done using HPLC or another appropriate analytical tool. An essential basis (but not a substitute) for this is the establishment of reproducible pharmaceutical quality (see previous chapter).

Why is standardization necessary and important? There are many reasons for using well-defined extracts, including, for example:

• reproducible composition and generally higher quality of the product. Standardization may require that the quantity of unwanted material in the extract should not exceed a certain limit, while active ingredients will have to be above a minimum concentration

• provided that the product is registered, it thus becomes a medicine that should comply with the basic standards required for all drugs

• standardization allows comparison of the clinical effectiveness, pharmacological effects and side effects of a series of products (e.g. against placebo). If a product is not standardized to active compounds, the comparison with other products derived from the same botanical drug is more problematic, since the composition may vary greatly

• such products give patients greater (objective and subjective) security and thus increase the level of trust people have in herbal products

• ensuring the quality of products sold has always been, and continues to be, a key responsibility of the pharmacist.

An example of a standardization in which a range (a maximum and minimum amount) of a certain compound or of several compounds is defined, is the extract of the leaves of Ginkgo biloba L. (Ginkgo folium). In this case, a standardized extract with a content of 2.8–3.4% ginkgolides, 2.6–3.2% bilobalide, 22–27% flavonoids and less than 5 ppm ginkgolic acid is commonly used. Such an extract is often called a ‘special extract’ and is obtained by further processing an extract in order to enrich desired compounds and reduce the amount of undesired ones. The production of ginkgo special extract is not a standardization in the strict sense, but a process of defining a range for certain compounds or classes of compounds.

Types of extract

In dealing with the standardization of phytomedicines, there is a further problem. Extracts contain a mixture of active and inactive ingredients, but often it is not known which compounds contribute to the activity or the pharmacological effects of the extract. Generally, the whole herbal drug preparation (e.g. the extract) is regarded as the active pharmaceutical ingredient. For quality control, several systems of classification have been proposed. A particularly useful one is now widely used on an EU-wide level; it has been described in a monograph of the Eur. Ph. (2003) and is thus accepted as a binding quality standard. Three classes of extracts are distinguished:

• (Truly) standardized extracts (type A): extracts standardized to active constituents

• Quantified extracts (type B1): extracts standardized to constituents that contribute to the activity

• Other extracts (type B2): extracts standardized to lead compounds of unknown pharmacological relevance, which serve as quality markers.

This first classification heavily impacts on strategies for pharmaceutical quality control.

Standardized extracts

Truly standardized extracts are extracts for which the active constituents (single or groups) are known. They can thus be standardized to a defined content of the active constituent(s) giving a clearly defined amount of an active natural product. Examples include:

• digitalis leaf (Digitalis folium, foxglove)

• senna dry extracts: standardized to 5.5–8.0% hydroxyanthracene glycosides, calculated as sennoside B with reference to dried extract (Eur. Ph.)

• belladonna leaf dry extract (Belladonnae folium from Atropa belladonna L., deadly nightshade): standardized to 0.95–1.05% of alkaloids calculated as hyoscyamine (Eur. Ph.).

Adjustment (standardization) to a defined content is acceptable using inert excipients or preparations with a higher or lower content of active constituents. This type of extract is sometimes also referred to as ‘normalized’ extract, a term that is a much better description of this process of quality control (see above).

Quantified extracts

These are extracts with constituents having known therapeutic or pharmacological activity. Groups of compounds likely to have the desired pharmacological activity are unknown, but are not solely responsible for the clinical efficacy of the extract. The monograph must define a range of content of the selected constituent(s), some of which are lead compounds. This category includes special extracts, in which a certain compound or groups of compounds have been enriched and unwanted compounds have been excluded. Standardization by blending different batches of a herbal drug before extraction, or by mixing different lots of herbal drug preparation, is acceptable. Adjustment using excipients is not acceptable. Examples:

• Ginkgo biloba L. leaf (Ginkgo folium, ginkgo or maidenhair tree)

• Hypericum perforatum L. aerial parts (Hyperici herba, St John’s wort).

Other extracts

These are extracts generally accepted to be pharmacologically active. However, the constituents responsible for this activity are not known and consequently quality markers have to be defined. These give information on the overall quality of the phytomedicine for control purposes and may be used to monitor good manufacturing practice, but cannot be used as evidence that the relevant active compounds are present in sufficient amounts or not. Examples:

• Echinacea (Echinacea radix and herba)

• Crataegus aerial parts (Crataegi folium cum flore, hawthorn)

• Passiflora incarnata L. aerial parts (Passiflorae herba, passion flower).

Examples of drugs and their quality control and standardization

Several examples of ways to assure the identity of a botanical drug (quality control) and, if applicable, the ways extracts derived from such a drug are standardized have been discussed. Here, examples of how a drug is characterized are given, together with some general background information and a brief discussion of the strengths and weaknesses of the various methods.

Standardized extracts

Digitalis purpurea folium (foxglove leaves)

Note: Digitalis purpurea folium is the botanical drug of the genus Digitalis and is currently monographed in the Eur. Ph. In this discussion, additional data on Digitalis lanatae folium (not monographed in Eur. Ph.) and Digitalis glycosides (monographed) are included.

William Withering’s ‘An account of foxglove and some of its medical uses’ (1785) introduces foxglove as a remedy for dropsy and oedema, later additionally used for heart conditions, especially congestive heart failure. This is largely due to an inhibitory effect on Na⁺/K⁺-ATPase. Today, pure compounds (including semi-synthetic derivatives of digitalis glycosides, rather than standardized extracts) are used. The glycosides digoxin (the most widely used in the UK), digitoxin and lanatoside C all have monographs in the Eur. Ph. and are isolated industrially from the botanical drug using a multistep process. Therapeutically used digitalis glycosides are generally not chemically pure but may contain up to 5% (Eur. Ph.) or even 11% (USP) of other compounds. Digitalis lanata Ehrh. (Scrophulariaceae) is the species cultivated for obtaining the raw pharmaceutical product. Digitoxin is a degradation product of lanatoside A (from Digitalis lanata) [as well as of purpurea glycoside A (from D. purpurea L.)] (Fig. 9.3).

Fig. 9.3

Since the active constituents are known, foxglove extracts clearly fall into the category of (truly) standardized extracts. The extracts are important in the industrial production of the pure compounds and, consequently, numerous methods for quality control have been developed; these show the range of pharmacognostical tools available to assess the quality of the botanical drug and the extract derived from it.

Quality control

Typical microscopical characteristics of the pulverized drug (see Fig. 9.4) include:

• 2–7-celled clothing trichomes (clothing hairs) with cells often collapsed and glandular hairs composed of a characteristic gland and a unicellular stem cell (pedicel)

• characteristic structure of polygonal epidermal cells and stomata

• calcium oxalate and sclerenchyma are absent.

Fig. 9.4 Microscopic appearance of powdered botanical drug material from digitalis leaf (Digitalis purpurea folium), showing (a) irregularly shaped lower epidermis with stomata, (b) unusual multicellular hairs with some cells having collapsed during the drying process, (c) glandular hairs.

After the identification of the botanical drug using, for example, microscopic methods or methods described below, the material is investigated in order to quantify the active ingredients (phytochemical analysis). HPLC and TLC may be used both to establish the identity of a botanical drug and to quantify the actives.

Although these methods are generally quite reliable and offer a high degree of reproducibility, the core problem of digitalis is that the cardenolides differ widely in their pharmacological potency and that the safe dose range is very narrow. Therefore, biological methods have considerable advantages over chemical analysis. The most widely used is inhibition of the activity of Na⁺/K⁺-ATPase in a solubilized preparation:

If the ATP is labelled radioactively (³²P), the resulting amount of free radiolabelled P_i can easily be determined. Alternatively, the final reaction product of two subsequent reactions can be determined:

In a last step, pyruvate is metabolized by lactate dehydrogenase to yield lactate:

Although the above methods can be used for quantifying compounds in botanical material, the exact determination of digitalis glycosides in blood and plasma is even more important. For this, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) has been shown to be the most reliable method.

• RIA An antigen (digoxin, etc., covalently bound to a hapten) is used to induce antibody production in a rabbit. The quantity of digitoxin is determined by co-incubating the labelled antigen–hapten (defined amount) with the unlabelled antigen (unknown); if more unlabelled antigen is present, the amount of bound antigen is reduced. The analysis of trace amounts is possible, but such a highly specific method is generally only available in specialized laboratories.

• ELISA The antibodies are fixed to the surface of, for example, a microtitre plate. The sample with an unknown amount of antigen (digitoxin) is then added together with hapten-bound enzyme-labelled (e.g. peroxidase) antigen (also digitoxin). Both compete for the binding positions. After washing, the read-out is the rate of production of a coloured compound, which is quantified photometrically. The amount of digoxin in a sample is calculated from the amount of labelled antigen, which is highest when the least unbound antigen is present.

Lastly, in vivo biological determination was used for many years (i.e. the LD₅₀ in guinea pigs). This is determined using an infusion of solution with an unknown amount of digitoxin and measuring the survival time of the animals. The concentration is determined by comparison with a standard solution applied to control animals.

(Truly) standardized extracts

Sennae folium (senna leaf)

This is a commonly used purgative appropriate for short-term use, with well-established effectiveness and significant side effects if used over prolonged periods of time. The species used pharmaceutically are Cassia senna L. (syn. C. acutifolia Delile), also known as Alexandrian senna, and Cassia angustifolia Vahl, or Tinnevelly senna (Caesalpiniaceae). Both names make reference to the former ports of export of the botanical material. The name in international trade for both species is Sennae folium (= senna leaf). The following constituents are important for the pharmacological effects of the drug:

• Sennosides, including sennosides A and B (Fig. 9.5)

• Glucosides of rhein (e.g. rhein-8O-glucoside) and aloe-emodin.

Fig. 9.5

Quality control

Some characteristic microscopic features of the drug include the unique non-lignified warty hairs up to 250 μm long, and small cluster crystals as well as prisms of calcium oxalate. Another characteristic are the stomata with two cells with the long axes parallel to the pore (diacytic stomata), the mid-rib bundle and larger veins of the leaves surrounded by a zone of lignified pericyclic fibres and a sheath of parenchymatous cells containing prisms of calcium oxalate (Fig. 9.6).

Fig. 9.6 Microscopic appearance of the lower surface of senna leaves (Sennae folium) showing (a) the epidermal cells, (b) typical bend hairs with an uneven surface, (c) cross-section of a leaf with an irregular oxalate crystal.

As regards phytochemical methods, according to the Eur. Ph., sennosides and rhein-8-glucoside should be detected using 98% acetic acid/water/ethyl acetate/1-propanol (1:30:40:40). Prior to spraying with Borntraeger reagent (potassium hydroxide), the glycosides are oxidized and hydrolysed by spraying the plate with nitric acid and subsequent heating (120°C, 10 min). In this case an extract is used as reference material. Sennosides show up as red-brown spots at R_F 0.15–0.44; sometimes, rhein-8-glucoside can also be detected as a red zone around R_F 0.40. This methods allows the detection of other anthranoid-containing drugs, but, since the material is grown commercially, adulteration or contamination with other drugs is rare. This is another clear example of a type A extract. The active constituents are well known, they are easily characterized by TLC, and quantification of the active compounds is possible (e.g. with the help of HPLC).

Quantified extracts

Ginkgo biloba leaves

Ginkgo biloba leaves are used for improving cerebral and peripheral circulation in the elderly, as well as for vertigo and other complaints that involve reduced cerebral circulation. Unlike many other phytomedicines, most of the pharmaceutically relevant products are hydroalcoholic ‘full’ or ‘special’ extracts, the latter being analytically very well characterized. In this case, a relatively broad range of active ingredients are known, of which two groups of compounds are particularly relevant:

• Flavonoids (0.5–1%): flavone and flavonol glycosides, acetylated flavonol glycosides, biflavonoids

• Terpene lactones (0.03–0.25%).

Special extracts have the largest share of the market in Continental Europe. Although the two groups of compounds are important for understanding the pharmacological effects of the drug, other compounds may also be important. These extracts also have a significantly reduced level of unwanted groups of compounds (polyphenols, polysaccharides and ginkgolic acids) and an increased percentage of flavonoid glycosides (16–26%) and terpene lactones (5–7%). It is, therefore, a typical example of a type B1 extract, in which the constituents have known therapeutic or pharmacological activity. But these compounds are not solely responsible for the clinical efficacy of the extract.

Quality control

Microscopy is less useful in quality control here, so phytochemical methods are predominant.

In the case of the crude drug, TLC is only feasible for the flavonoids. The terpene lactones are only present in a small concentration and they are accompanied by substances which interfere with the analysis. The special extracts can be analysed using the following TLC methods:

• Flavonoids: ethyl acetate/formic acid/glacial acetic acid/water (100:11:11:26), detection with diphenylboryloxyethylamine.

• Biflavones: chloroform/acetone/formic acid (75:16.5:8.5), detection with diphenylboryloxyethylamine.

• Terpene lactones: toluene/acetone (70:30), detection with acetic acid reagent.

Standardization

The above methods are qualitative and, as in the previous examples, do not allow the quantification of the relevant compounds. Consequently, HPLC and GC methods are paramount for the quantification of lead substances:

• HPLC is used for the determination of the three aglycones quercetin, kaempferol and isorhamnetin (after hydrolysis of the glycosides).

• GC is used for the quantification of the terpene lactones.

No single method gives all the results required, thus complex analyses are required.

Other extracts

Passiflorae herba (passion flower)

Passion flower herb (Passiflorae herba, Passiflora incarnata L.) is used as a mild phytomedicine in chronic fatigue syndrome, nervousness and anxiety. Some clinical studies and other data point to the efficacy of the drug, but it is not clear which compounds are responsible.

Quality control

Microscopic analysis gives a very characteristic picture, including prominent pieces of the lower epidermis with cells around the stomata, which cannot be distinguished from the other cells of the epidermis (anomocytic), and cluster crystals below the surface. The material is also characterized by typical hairs (Fig. 9.7).

Fig. 9.7 Microscopic appearance of passion flower herb (Passiflorae herba) showing (a) typical fragment of the lower epidermis of a leaf with some calcium oxalate structures along the small veins, (b) sickle-shaped long unicellular trichomes and (c) the unusual pollen. Contrary to drugs derived from leaves, herb material contains typical elements of the flowers, such as the epidermis of the inner corolla (d) and relatively large amounts of pollen, and/or fruit and also often stem material.

Of the known ingredients, flavone glycosides such as isovitexin are relatively abundant, and traces of essential oil and a cyanogenetic glycoside have been found. The presence of harman-type alkaloids, which was reported in earlier work, has not been corroborated in more recent studies. Clinical data confirm the usefulness of this species for the conditions mentioned above, but the compounds responsible for the activity are not definitively known. Chrysin and other flavonoids have been shown to be biologically active, and, consequently, the flavonoids are used as markers. Thus it is an example of a type B2 extract. In this case, quality control can only assure reproducible quality of the extract. Both qualitative and quantitative methods have been developed:

• Qualitative analysis: TLC analysis of a methanolic extract (Eur. Ph.) using rutin and hyperoside as reference; plate development with acetic acid/water/ethyl methyl ketone/ethyl acetate (10:10:30:50), detection at 365 nm (UV) after spraying with diphenylboryloxyethylamine

• Quantitative analysis: spectrophotometric or fingerprinting with HPLC.

Echinacea root (E. purpurea and E. pallida)

Echinacea purpurea and E. pallida are used as immunostimulants and in the treatment of respiratory infections. Both species are employed in the preparation of pharmaceutical products and some data on efficacy are available for both. Other species are also used, but there is insufficient information available to validate their use. The active constituents are not known. Extracts of these two species on the market (and generally accepted to be pharmacologically active) can therefore be classified as type B2. Prominent constituents include caffeic acid derivatives (about 1%), especially echinacoside (E. pallida), cichoric acid (E. purpurea), alkamides (E. purpurea), minor amounts of essential oil and polysaccharides (both Echinacea spp.).

Quality control

The drug is difficult to identify microscopically, but some botanical characteristics can be used. As a consequence, methods for quality control (identification) focus on phytochemcial techniques.

Drug identification (authentication), using phytochemical methods, is generally performed with TLC (ethyl acetate/formic acid/glacial acetic acid/water, 100:11:11:26). This method detects the caffeic acid derivatives, which thus serve as a marker for the pharmaceutical quality and also identify the plant material used for producing the phytopharmaceutical. Some good HPLC methods are available, but have not yet been systematically employed in quality control. As the active constituents are not known, they cannot, of course, be quantified.

Conclusion

There are many strategies for securing adequate quality. The process of quality assurance covers the whole range from the agricultural production, the extraction and formulation, to dispensing the product. The more commonly used medicinal plants are in the European Pharmacopoeia or other pharmacopoeias and the relevant technical data can be found there. Both botanical/pharmacognostical and phytochemical methods are relevant and which methods are used depends on the type of pharmaceutical product.

Standardization is a method of assuring a minimum level of active ingredients in the extract and it is becoming increasingly important as a means of ensuring a consistent composition of high-quality phytopharmaceutical products and offers opportunities to produce herbal medicines of reproducible composition.

European Pharmacopoeia. Council of Europe (Directorate for the Quality of Medicines), fourth ed. Strasbourg: European Pharmacopoeia; 2002.

European Pharmacopoeia. European Directorate for the Quality of Medicines and HealthCare (EDQM). France: Strasbourg; 2003.

Evans W.C. Trease and Evans’s pharmacognosy, sixteenth ed. London: WB Saunders; 2009.

Franz C. Züchtung und Anbau von Arnzeipflanzen. In: Rimpler H., editor. Biogene Arzneistoffe, 2 Aufl. Stuttgart: Wissenschaftliche Verlagsgesellschaft; 1999:1-19.

Goodman J., Walsh V. The story of taxol. In Nature and politics in the pursuit of an anti-cancer drug. Cambridge: Cambridge University Press; 2001.

Lange D. Conservation and sustainable use of Adonis vernalis, a medicinal plant in international trade. Plant species conservation monographs. Bonn: Federal Agency for Nature Conservation, 2000;vol. 1.

Further reading

Eschrich W. Pulver-Atlas der Drogen der deutschsprachigen Arzneibücher. Stuttgart: Deutscher Apotheker Verlag; 1999.

Heinrich M., Pieroni A., Bremner P. Medicinal plants and phytomedicines. In: Nesbitt M., Prance G., editors. The cultural history of plants. New York: Routledge (Taylor and Francis); 2005:205-238.

Heinrich M., Modarai M., Kortenkamp A. Herbal extracts used for upper respiratory tract infections: Are there clinically relevant interactions with the Cytochrome P450 enzyme system? Planta Med.. 2008;74:657-660.

Hohmann B., Reher G., Stahl-Biskup E. Mikroskopische Drogenmonographien der deutschsprachigen Arzneibücher. Stuttgart: Wissenschaftliche Verlagsgesellschaft; 2001.

Mukherjee P.K., Mukherjee D., Venkatesh M., Rai S., Heinrich M. The sacred lotus (Nelumbo nucifera) – phytochemical and therapeutic profile. J. Pharm. Pharmacol.. 2009;61:407-422.

Rahfeld B. Mikroskopischer Farbatlas pflanzlicher Drogen. Spektrum Akademischer. Heidelberg: Verlag; 2009.

Rimpler H. Biogene Arzneistoffe. Stuttgart: Wissenschaftliche Verlagsgesellschaft; 1999.

Stewart K.M., Cole D. The commercial harvest of devil’s claw (Harpagophytum spp.) in southern Africa: The devil’s in the details. J. Ethnopharmacol.. 2005;100:225-236.

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Fundamentals of Pharmacognosy and Phytotherapy 2e