Pharmacognostic Methods for Analysis of Herbal Drugs, According to European Pharmacopoeia

At the present time, according with the WHO reports, about 80 % of the world’s population use herbal medicines for some aspects of their primary health care. This phenomena is in relationship with a rapidly expanding of the phytopharmaceutical industry and market, especialy for dietary supplements. Unfortunately, these supplements are insufficiently studied and have a low quality. For this reason, today, the tendency is to militate for to decrease the number of supplements and to increase the number of herbal medicinal products, which are more rigorously analyzed before marketing authorization and after that, according to European Medicines Agency guidelines.


Introduction
Plants had been used for medical purpose long before recorded history.
At the present time, according with the WHO reports, about 80 % of the world's population use herbal medicines for some aspects of their primary health care.This phenomena is in relationship with a rapidly expanding of the phytopharmaceutical industry and market, especialy for dietary supplements.Unfortunately, these supplements are insufficiently studied and have a low quality.For this reason, today, the tendency is to militate for to decrease the number of supplements and to increase the number of herbal medicinal products, which are more rigorously analyzed before marketing authorization and after that, according to European Medicines Agency guidelines.

Herbal drug
Herbal It comprises qualitative and quantitative tests in order to verify or to establish the identity, purity and quality of a herbal drug.
Purity parameters: foreign matter.
Quality parameters: loss on drying; soluble-substances; total ash and ash insoluble in hydrochloric acid; heavy metals; swelling index; bitter value; assay of active principles; microbiological examination (bacteria, yeasts and moulds, specified microorganisms); pesticide residues; aflatoxines; ochratoxines.

Macroscopic examination
This test have in view to determ the morphological characteristics.It gives details concerning the drug aspect, size, colour, odour and taste.

Methodology
Morphological characters and the colour may be examination with the naked eye or by using a magnifyed glass.
The size can be determ by using a ruler or a caliper.
The odour can be determ by shattering the drug between two fingers and smell, or using an extractive solution.
The taste can be determ by putting a piece of drug or an extractive solution in the mouth.

Evaluation of results
There are not general recomandations in EP in order to the macroscopical examination.
The main characteristics which are frequently analyse are included in table 3.

Limits
The morphologic characteristics vary in large limits, due to the vegetal drug.
Organoleptic characteristics may inform about chemical composition: coloured in yellow conduct to flavones, xanthones, carotenoids; coloured in red for anthocyanins; bitter taste for antracenic-derivatives, alkaloids, cardiotonic glycosides.

Microscopic examination
This test have in view to determ the anatomic characteristics.

Methodology
According to EP chapter 2.8.23., the microscopic examination of herbal drugs is carried out on the powdered drug (355).Chloral hydrate is the most commonly prescribed reagent.Other reagents are: lactic reagent, alcoholic solution of phloroglucinol and hydrochloric acid, ruthenium red solution, glycerol.

Evaluation of results
Phloroglucinol is used to identify the presence of lignin, ruthenium red solution is used to show the presence of mucilage, glycerol is used to show the presence of starch and inulin.
In the case of Plantaginis ovatae semen and Plantaginis ovatae semen tegmentum, lactic reagent makes it possible to visualize lignified cells, cutinized membranes and starch.The Stomatal index is the ratio (expressed as ,,%") of the number of stomata in a given area of leaf and the number of total epidermal cells (including stomata, trichomes) in the same area of leaf.

Qualitative chemical analysis
For unknown vegetal product, this test may establish the chemical composition, and for known herbal drugs this test may confirm the presence of a chemical compound (which may be not the main active principle).

Methodology
Because EP is a reference used for the control of herbal drugs, only reactions in an extractive solution apply.

Evaluation of results
EP examples are included in table 5.

Qualitative chromatography
Chromatography is a method of separation based on adsorbtion, repartition, ion exchange.It brings supplementary informations about chemical composition.

Methodology
Experimental conditions differ depending of chemical compound have to identify.Examples of mobile phases, references substances and reagents used for some active principles and some herbal drugs are included in tabel 6.

Evaluation of results
Each compound has a characteristic spot, with a definit Rf-value, colour and / or fluorescence.
In some cases, using this technique it may distinguish vegetal sources / herbal drugs, like Panax sp.; Panax ginseng C. A. Meyer is the vegetal source for Ginseng radix, and Panax pseudoginseng Wall.var.notoginseng (Burk.)Hoo et Tseng is the source for Notoginseng radix.
TLC may be used as a purity test, too (see section 2.5.Foreign matter).

High Pressure Liquid Chromatography (HPLC)
This technique is used both for identification and for assay.
For example, HPLC technique is used as an identification test in the case of the following herbal drugs: Echinaceae angustifoliae radix, E. pallidae radix, E. purpureae folium, E. purpureae herba.

Gas-chromatography (GC)
This technique is used both for identification and for assay.
Identification by using GC technique is mentioned for the following herbal drugs: Thymi herba, Lavandulae flos, and Sabalis serrulatae fructus.

Foreign matter
According to EP chapter 2.8.2., foreign matter is material consisting of foreign organs (matter coming from the source plant but not defined as the drug) and / or foreign elements (matter not coming from the source plant and either of vegetable or mineral origin).

Methodology
A macroscopic examination, microscopic examination, reactions or chromatography are used for to identify foreign matters.
A quantitative evaluation may be applied, too.

Evaluation of results
Organoleptic, morphologic, anatomic and chemical characteristics for the sample are compared with the ones are known for ,,pure" herbal drug.
The content of foreign matter is expressed as ,,%, m/m".

Limits
The EP recommendation (monograph ,,Herbal drugs") is that the content of foreign matter is not more than 2%, unless otherwise prescribed or justified and authorized.

Loss on drying
This parameter is stricken by the humidity of the environment.On the other hand, it may affect the quality of the herbal drugs among the storage.A high content of water may favor the growth of microorganisms (fungi which produce mycotoxins), or may activate enzymatic systems which will generate compounds with a less activity (specific hydrolases may degrade primary cardenolic glycosides to secondary cardenolic glycosides, which have less activity).

Methodology
Usually, the powdered drug is dried in an oven at 105 0 C for 2 h.

Evaluation of results
The result is expressed as ,,%, m/m" or ,,mL/kg".
In the case of Lini semen, mucilages may favor the growth of microorganisms (fungi which produce mycotoxins), if the content of water is higher.
In Digitalis purpureae folium, water may activate enzymatic systems (specific hydrolases) and so, primary cardenolic glycosides degrade to secondary cardenolic glycosides, which have less activity.
If the vegetal drug with a high content of lipids is stored in a light, hot and wet place, unsaturated fatty acids degrade (peroxidation, polymerization); the lipids are ranciding.

Soluble substances
This parameter refers to all vegetal compounds which can be extracted with a certain solvent, in certain experimental conditions.When the solvent is water, the parameter calls ,,water-soluble extractive".When another solvent is used, it calls ,,extractable matter".

Methodology
Any general EP method exists.Generally, the powdered drug is extracted with the solvent (definite quantity) under the conditions specified in monograph, the solvent is evaporated, the residue is dried up to fixed mass and finally weighed.
Experimental protocols are included in table 8.

Evaluation of results
The result is expressed as ,,%, m/m". www.intechopen.com

Limits
The limits vary, due to the vegetal drug (table 8).For a medicinal product development, the results obtained by using a vegetal raw material and different solvents and different experimental parameters help to evaluate the efficiency of the extraction, to establish the proper solvent and the optimum working technology.

Total ash and ash insoluble in hydrochloric acid
These parameters express the content of metallic ions (mineral compounds) of a vegetal drug, and they are in relationship with the pedoclimatic conditions.

Methodology
Essentially, according to EP general monograph (chapter 2.4.16), for to determ the total ash, the vegetal drug is ignited to constant mass in a muffle furnace at about 600 o C.
According to EP general monograph (chapter 2.8.1.),for to determ the ash insoluble in hydrochloric acid, the following method apply: the residue from the determination of total ash is boiled with dilute hydrochloric acid, and the solution is filtered through a ashless filter; the filter is dried, is ignited, allow to cool in a desiccator and finally is weighed.

Evaluation of results
The result is expressed as ,,%, m/m".

Limits
Parameter ,,Total ash" is included in all monographs.
Parameter ,,Ash insoluble in hydrochloric acid" is included in most of monographs; exceptions are the following: Agni casti fructus, Agrimoniae herba, Alchemillae herba, www.intechopen.com The limits vary, due to the vegetal drug (for examples see table 9).

Heavy metals
This parameter espress the pollution.

Methodology
Atomic absorption spectrometry is used.This is described in EP general chapter 2.4.27. www.intechopen.com

Evaluation of results
The limits of suitability are given as a maximum value expressed as units ppm.

Swelling index
The swelling index is the volume (expressed in milliliters) occupied by 1 gram of an herbal drug and the adhering mucilage, after it has swollen in an aqueous liquid.
It expressed a high content of mucilage in an herbal drug.

Methodology
A general EP method is described in chapter 2.8.4.Generally, 1 gram of herbal drug (the degree of comminution prescribed in the monograph) is placed in a 25 mL ground-glass stoppered cylinder graduated and then is moistened with alcohol.Add 25 mL water, close the cylinder and shake it for 1 h, with a standard frequency.Allow to stand 3 h.Finally, note the volume occupied by the drug and the adhering mucilage.

Evaluation of results
The result is given by the mean of the 3 tests.

Limits
The limits vary, due to the vegetal drug (table 10).

Bitterness value
The bitterness value is the reciprocal of the maximum dilution of a vegetal drug that still has a bitter taste.It is determined by comparison with quinine hydrochloride, the bitterness value of which is set at 200000.
It expressed a high content of bitter-compounds in an herbal drug, active principles which are used to stimulate the appetite.But, attention: not all vegetal substances with bitter taste are used to stimulate the appetite (e.g.cardenolic glycosides, antracenic derivatives and alkaloids).

Evaluation of results
Depending to the dilutions of the reference substance (quinine hydrochloride) and of the test solution, expressions for correction factor and for bitterness value are described in the monograph.
The result is given by the mean of all 6 tests.

Limits
The acceptance limits vary, due to the vegetal drug (table 11).

Colouring intensity
This parameter expresses the content of pigments (yellow and /or red pigments).

Methodology
Spectral methods apply.These methods consist of the measuring the absorbance at a specific wavelength for a solution having a definit concentration.

Evaluation of results
The absorbance is recorded using a suitable spectrophotometer.

Limits
Specific limits of admisibility are mentioned in monographs.The details are mentioned in tabel 12.

Assay
In the case of herbal drugs with constituents of known therapeutic activity or with active markers, assays of their content are applied.When the constituents responsible for the therapeutic activity are unknown assays of analytical markers are required.
determined by following the variation of the potential difference between two electrodes immersed in a sample solution as function of the quantity of the titrant solution added.
Other times, visual indicators may be used, too.

Evaluation of results
The content of active or analitycal marker is calculated by having in view the stoechiometry of the titration reaction.

Limits
Specific limits of admisibility are mentioned in monographs.The details are mentioned in tabel 13.  13. Assay -titrimetric methods

Spectrophotometric methods
Spectrophotometric analysis is based on the measurement of radiation intensity as a function of wavelength.
These methods may be used because the active / analytical marker or its derivatives has a definite UV or VIS spectrum.

Methodology
Specific spectrophotometric methods are described in monographs.Usually, the following parameters vary: the degree of comminution of the herbal drug, solvent for extraction, methodology for obtaining the sample and the reference solutions, coloring reagent, wavelength for detection.Some details are mentioned in tabel 14.
A general method for determination of tannins in herbal drugs is described in EP chapter 2.8.14.This method consists of the following steps: the herbal drug is extracted with water on a water-bath; total polyphenols are determined in this solution, using a spectrophotometric method; shake the solution with hide powder, filter and assay the polyphenols not adsorbed by the hide powder in the filtrate, using the same spectrophotometric method.Table 14.Assay -spectrophotometric methods

Evaluation of results
The content of active or analitycal marker is calculated by using the specific absorbance or by comparing with the reference solution.
In the case of the method described in chapter 2.8.14., the formula for the content of tannins have in view the diference between the value for total polyphenols and the value for polyphenols not adsorbed by the hide powder.

Limits
Specific acceptance limits are mentioned in monographs.

High-Performance Liquid Chromatography (HPLC)
Is a chromatographic technique that is used to separate, identify, quantify and purify the individual components of the mixture, due to a different migration of the compounds through the column (solid stationary phase).
In the case of herbal drugs, the separations are based upon partition mechanisms using chemically modified silica as the stationary phase and polar solvents as the mobile phase.

Methodology
The apparatus consists of a pumping system (which must deliver the mobile phase at a constant flow rate), an injector (which can operate at high pressure and is capable to release an exact volume of solutions), a proper chromatographic column (eventually having a temperature controller), a detector (commonly a ultraviolet / visible spectrophotometer) and a data aquisition system.
Usually, the following parameters vary: column characteristics (type, dimensions, particle size), qualitative and quantitative composition of the mobile phase, method of separation (isocratic flow / gradient elution), the gradient, characteristics of the sample and reference solutions, using an external or an internal standard, flow rate, injection volumes, run time, wavelength for detection.Some details are mentioned in tabel 15.

Evaluation of results
When an extern standard is used, the content of active or analitycal marker is calculated by comparing the response of the sample with the response the reference.When an internal standard is used, the content of this standard must have in view.

Limits
Specific limits of admisibility are mentioned in monographs.

Gas Chromatography (GC)
Is a chromatographic technique that is used to separate, identify, quantify and purify the volatile components (or volatile derivatives of the components) of the mixture, due to a different migration of the species through a solid or a liquid stationary phase.

Methodology
The apparatus consists of an injector, a chromatographic column (which is included in an oven), a detector (commonly a flame-ionisation detector) and a data acquisition system.
Usually, the following parameters vary: the type and the characteristics of the stationary phase, the carrier gas, method of separation (normalisation / derivatisation), temperature (for column, injection port, detector), characteristics of the sample and reference solutions, flow rate, injection volumes, split ratio, run time.Some details are mentioned in tabel 16.

Evaluation of results
The content of active or analitycal marker is calculated by comparing the response of the sample with the response of the reference.The results is expressed as a minim value (%) in the essential oil.

Limits
Specific limits of admisibility are mentioned in monographs.Table 16.Assay -GC methods

Determination of essential oils in herbal drugs
A general method for extraction and assay of essential oils in herbal drugs is described in EP chapter 2.8.12.

Methodology
Essentially, the determination is carried out by steam distilation in a special apparatus in the conditions described in chapter 2.8.12.The distilate is collected in the graduated tube, using (usually) xylene to take up the essential oil, and the aquueous phase is automatically returned to the distillation flask.The mass of herbal drug used for extraction, the type and the volume of solvent, distilation rate and distilation time vary, and so that these parameters are mentioned in specific monographs.

Evaluation of results
The volume of liquid collected in the graduated tube is readed, the volume of xylene is substracted and so the volume of essential oil is obtained.The result is expressed as ,,mL/ kg".

Limits
Specific limits of admisibility are mentioned in monographs.

Pesticide residues
According to EP, chapter 2.8.13, a pesticide is any substance or mixture of substances intended for preventing, destroying or controlling any pest, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of herbal drugs.The item includes substances intended for use as growth-regulators, defoliants or desiccants and any substance applied to crops, either before or after harvest, to protect the commodity from deterioration during storage and transport.
EP chapter refers especially to the organochlorine, organophosphorus and pyrethroid insecticides.

Methodology
A general procedure is described in EP, but this is only for information.
It consists of the following three steps: extraction the pesticides; purification (using sizeexclusion chromatography); quantitative analysis (examine by gas-chromatography, using carbophenothion as the internal standard).

Evaluation of results
The content of an insecticide is calculated from the peak area and the concentrations of the solutions.Lists of relative retention times for main organophosphorus insecticides, and the organochlorine and pyrethroid insecticides respectively, are attached in the monograph.

Limits
The limits are expressed as ,,mg/ kg".A list containing 69 pesticides is presented in the EP chapter.For other substances, the limits are calculated using an expression which have in view acceptable daily intake, body mass and daily dose of the herbal drug.

Microbial contamination
The presence of micro-organisms may reduce or inactivate the therapeutic activity of the herbal drug, and implicitly of the pharmaceutical product.
This parameter refers to the total aerobic microbial count (TAMC), total combined yeasts / moulds count (TYMC) and specific micro-organisms (e.g.Escherichia coli).
According to monograph ,,Herbal drugs", it is a compulsory test.

Methodology
Microbial analysis is performed according to specific microbiologic methods.The following methods are discussed in EP (chapters 2.6.12.) for TAMC and TYMC: membrane filtration method and plate-count methods (including pour-plate method, surface-spread method and most-probable-number method).

Evaluation of results
The limits of suitability are given as a maximum value of units CFU.

Limits
The general chapters 5.1.4,,Microbiological quality of non-sterile pharmaceutical preparations and substances for pharmaceutical use" and 5.1.8,,Microbiological quality of herbal medicinal products for oral use" not include limits for herbal substances.So, acceptance limits should be set in relation to the specific herbal substance and subsequent processing.Reduction of the microbial count at level of herbal substance (e.g.geographical origin, appropriate harvest/collection and drying procedures, treatment with water vapour) should be taken into account when setting the limits.

Determination of aflatoxins
Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus (a fungus).Aflatoxins are toxic and among the most carcinogenic substances known.Aflatoxin-producing members of Aspergillus are common and widespread in nature.They can colonize and contaminate grain before harvest or during storage.Host crops are particularly susceptible to infection by Aspergillus following prolonged exposure to a high humidity environment, or damage from stressful conditions such as drought, a condition which lowers the barrier to entry.The native habitat of Aspergillus is in soil, decaying vegetation, hay and grains undergoing microbiological deterioration and it invades all types of organic substrates whenever conditions are favorable for its growth.Favorable conditions include high moisture content (at least 7%) and high temperature.The toxin can also be found in the milk of animals which are fed contaminated feed.

Methodology
A specific liquid chromatographic method, using an isocratic flow, fluorescence detection and post-column derivatisations apply.An immunoaffinity column containing antibodies against aflatoxin B 1 is used for to obtain the test solution.The method is described in EP general chapter 2.8.18.It is cited as an example of a method that has been shown to be suitable for devil's claw root, ginger and senna pods.

Evaluation of results
The limits of suitability are given as a maximum value expressed as ,,ng/g", or ,, µg/kg" .

Limits
The EP requires a limit of not more than 2 µg/kg of aflatoxin B 1 and a limit of 4 µg/kg for the sum of aflatoxins B 1 , B 2 , G 1 and G 2 .

Determination of ochratoxin A
Ochratoxins are a group of mycotoxins produced by some Aspergillus species and Penicillium species including Aspergillus ochraceus and Penicillium viridicatum.Ochratoxin A is the most prevalent and relevant fungal toxin of this group, while ochratoxins B and C are of lesser importance.Ochratoxin A is known to occur in commodities such as cereals, coffee, dried fruit and red wine.It is nephrotoxic and nephrocarcinogenic.

Methodology
A specific liquid chromatographic method, using a gradient elution and a fluorescence detection apply.An immunoaffinity column containing antibodies against aflatoxin B 1 is used for to obtain the test solution.The method is described in EP general chapter 2.8.22.It is suitable for Liquiritiae radix.The EP recommendation is that the suitability of this method for other herbal drugs must be demonstrated or another validated method used.

Evaluation of results
The limits of suitability are given as a maximum value expressed as ng/g.

Limits
In the case of Liquiritiae radix, the acceptance limit of maximum 20 µg/kg is required.

Conclusion
A herbal drug is a particular and a complex raw material.Its analysis involves specific pharmacognostic methods, which can be undertaken from European Pharmacopoeia or must be developed by the scientist.
Owing to the complexity of all above-mentioned aspects in studying the medicinal plants (herbal drugs), pharmacists, biologists, chemists and biochemists must co-operate.

Table 3 .
Macroscopic characteristics www.intechopen.com Common anatomic elements (which are used for to confirm the organ) and specific elements (which are used for to identify the herbal drug) are analyzed (see table4). www.intechopen.com

Table 5 .
Reactions in an extractive solution.

Table 6 .
TLC experimental conditions

Table 7 .
Some impurities are limited, and others are excluded.Some examples are included in table 7. Foreign matter

Table 9 .
Total ash and ash insoluble in hydrochloric acid (examples)