This article throws light upon the top three approaches for herbal drugs standardization. The approaches are: 1. Chromatographic Techniques 2. Biotechnological Approaches to Herbal Drugs Standardization 3. Biopharmaceutical Equivalence: Perspectives and Advantages.
Approach # 1. Chromatographic Techniques:
a. High Performance Liquid Chromatography: One of the most versatile and flexible technique with high throughput, a variation of column system enables a scientist to use one equipment for multifaceted and reasonably faster standardization (Bournot et al., 1992). The only general problem is in case of herbal drugs without and authenticated marker or complex herbal medicinal products.
Preparative HPLC and use of sophisticated Ultra Pressure Liquid Chromatographic apparatuses UPLC’s have started to revolutionize the concept of standardization using liquid chromatography. A vast array of detector modules also simplifies the extent as well as degree of detection and sensitivity.
b. High Performance Thin Layer Chromatography: HPTLC with the modern scanners for TLC plates have also improved the way this vastly used technique could be utilized in herbal medicinal research. It is now an acceptable form of standardization for most single as well as complex herbal mixtures
c. Gas Chromatography with Flame Ionization or mass detection (GC-FID or GC- MS) is a well-documented techniques for volatile herbal components as well as sugar complexes in standardization.
Apart from these vastly used techniques a lot of day to day modifications for individual herbal drugs are being experimented in various research laboratories globally, providing a growing evidence towards herbal drug standardization.
Approach # 2. Biotechnological Approaches to Herbal Drugs Standardization:
Biotechnology and use of biomarkers has facilitated the herbal drug standardization in more than one way, a brief look at the techniques available can shed better light on the future that these concepts may hold:
DNA Fingerprinting:
Some of the newly emerging techniques for ensuring correct botanical identity and quality include Herboprint, which in addition to chemo- profile also considers ayurvedic properties. Various types of DNA-based molecular techniques can be utilized to evaluate DNA polymorphism.
Hybridization-Based Methods:
Hybridization-based methods include restriction fragment length polymorphism (RFLP) and variable number tandem repeats. Labelled probes such as random genomic clones, cDNA clones, probes for microsatellite and mini-satellite sequences are hybridized to filters containing DNA, which has been digested with restriction enzymes.
Polymorphisms are detected by presence or absence of bands upon hybridization.
a. PCR-Based Methods:
PCR-based markers involve in vitro amplification of particular DNA sequences or loci, with the help of specific or arbitrary oligonucleotide primers and the thermo-stable DNA polymerase enzyme.
b. Sequencing-Based Markers:
DNA sequencing can also be used as a definitive means for identifying species. Variations due to trans version, insertion or deletion can be assessed directly and information on a defined locus can be obtained.
Genetic Variation/Genotyping:
This technique is useful for identifying geographical variations in different plant drugs due to geographical variation.
Thus in a nutshell the usefulness of DNA based techniques can for herbal drug research can be identified as follows:
a. Authentication of medicinal plants
b. Detection of adulteration/substitution
C. Marker assisted selection of desirable chemo- types
d. Genetic variation, cultivar variation, cross breeding studies and disease resistant gene identification in foods and nutraceuticals.
Approach # 3. Biopharmaceutical Equivalence: Perspectives and Advantages:
Achievement of a known therapeutic benefit is an important aim from a pharmacological point of view. Since herbal medicinal products may also fall under the category of phytogenerics, therefore, it is important to draw a detailed information about their pharmacological potential as well as pharmaceutical equivalence.
An approach in this direction is not only important from standardization point of view, but also from the lookout towards rationalization of herbal medicine practice.
According to an excellent review on the perspective of bioequivalence of herbal medicinal products by Loew and Kaszkin, 2002, ‘Herbal medicinal products (HMP) are medicinal products containing as active substances exclusively herbal drugs or herbal drug preparations (HDP), precisely defined by the botanical scientific name according to the binominal system (genus, species, variety and author).
In contrast to chemically defined drugs, HMP contain complex mixtures of different compounds with active, synergistic, complementary, antagonistic or toxic substances. In many cases, the active constituents responsible for efficacy are at present unknown. This often makes a confirmation of pharmaceutical and biopharmaceutical quality difficult.
Due to the insufficient definition of the active ingredient, evidence of the therapeutic equivalence of different HDP in bioavailability studies is not possible. Therefore herbal drug preparations are considered as being active ingredients in their total form and do not relate to the active principle, thus underlining the problem of comparability of the results of clinical studies’.
This clearly denominates the probable shortcomings as well as voids in the role of biopharmaceutical equivalence in herbal drug standardization.
Pharmaceutical and Biopharmaceutical Equivalents:
According to the Note for Guidance on the investigation of bioavailability and bioequivalence medicinal products are pharmaceutically equivalent if they contain the same amount of the same active substance(s) in the same dosage forms that meet the same or comparable standards.
These principles of pharmaceutical similarity applied to chemically defined drugs should also be applied to HDP as far as possible.
The quality and quantity of a HMP depends on different factors:
The starting material, effect of geographic origin on the composition of plant material, free as possible from pesticides and diseases in order to guarantee healthy plant growth, natural fluctuations, collection and/ or cultivation, harvesting, primary processing, washing, cutting, fumigation, freezing, distillation, drying etc.
GAP (good agricultural practice), in particular the solvents used for extraction, the manufacturing process, control of the finished products, stability and GMP (good manufacturing practice). In this context the quality of herbal medicinal products is characterized (Quality of Herbal Remedies, 1998) as follows.
Since a herbal drug product is comparable to an active pharmaceutical ingredient (API):
A. Extracts containing constituents (single or groups) that are solely responsible for the known and acknowledged/well documented therapeutic activity. Adjustment (standardisation) to a defined content is acceptable.
B1. Extracts containing chemically defined constituents (single or groups) possessing relevant pharmacological properties (active markers). These substances are likely to contribute to the clinical efficacy; however, evidence that they are solely responsible for the clinical efficacy is not yet available.
The characterisation of these extracts should take into consideration as far as possible the particular state of knowledge concerning the documented efficacy, quality and safety of an extract. Standardisation by blending different lots of a herbal drug before extraction, or by mixing different lots of herbal drug preparations is appropriate. Adjustment using excipients is not acceptable.
B2. Extracts containing no constituents documented as being determinant or relevant for efficacy, or as having pharmacological or clinical relevance. In these cases, chemically defined constituents (markers) without known therapeutic activity may be used for control purposes. These markers may be used to monitor good manufacturing practice or as an indication for the assay/ content of the drug product.
Therefore the category from 3(B2) to 1 (A1) can be achieved if a standardization approach is appropriately adopted, depending on the recommended techniques adopted by the manufacturer of herbal API’s.
Although, there are only a very few plant drugs that can be placed in the category A and therefore unless most of the therapeutically used drugs are tested and brought under the level A, it would be a big question to answer for their complete bioequivalence determination.
Bringing these herbal drugs into the category of API also involve the following contentions:
a. Pharmaceutical equivalence, i.e. raw material quality, extraction, manufacturing process, standardization.
b. Biopharmaceutical equivalence, i.e. the same extract/extract fractions, the same presentation form, the same dose, in vitro qualitative and quantitative conformity.
c. Equivalence in bioassays with regard to the pharmacological profile, i.e. the same profile in cell cultures, isolated receptor systems, enzymes, isolated organs and in the whole animal.
d. Bioequivalence in pharmacokinetics with regard to the rate and extent of the active substance.
e. Bioequivalence in effect kinetics, i.e. in clinical and pharmacological models.
f. If none of these conditions are fulfilled then it is a requisite to conduct controlled clinical studies according to the accepted guidelines on clinical efficacy and safety, as per the Pharmacopoeia guidelines.
Limitations for a Bioequivalence determination in HDPS:
The search for an indirect evidence via bioequivalence studies with herbal drugs is a logical step specially in cases where such evidence is a prerequisite for registration of a product or an inclusion in monograph. Although, it is worth understanding that it is one of the prime requirements that the product under investigation is in it-self well understood and defined.
In general, a bioequivalence study in herbal drugs encompasses the measurement of a surrogate end point, especially when the substance under investigation is a marker isolated from the herbal drug mixture or extract.
As a burgeoning question in the modern scientific scenario is the lack of herbal drugs that have been studied systematically, a few well studied examples include Silybium marianum, Piper longum, Aesculus hippocastanum etc.
However it is very clear in the studies conducted on these herbs as well as there are numerous other constituents having biological activity in comparison to their original markers.
As an example for the study conducted on horse chest nut, the extract was standardized to 16-20% if aescin content. In a single dose and four multiple dose immunological assay a variation based on steady state concentrations, clearly demarcated the fat that bioequivalence parameters especially from a immunological perspective holds serious limitations.
A careful and well-conceived use of bioavailability or bioequivalence data is very necessary, especially when regulatory considerations have to be kept in mind. In this context there has been a misleading concept about using the chemical markers towards standardizing the pharmacological activity and especially drawing out the bioequivalence.
Markers are by definition chemically defined substances that are useful in calculating the quantity of herbal drug in a preparation or a finished herbal product. An example to this can be illustrated by discussing the case of Hypericum perforatum.
Hypericin, which is a chemical marker for standardization of H. perforatum extract was also considered as biological marker for the purpose of bioequivalence determination as it had potent anti-depressive properties. In later findings another component, hyperforin was found to be active as well, and therefore the whole question of correlating bioequivalence to hypericin content came under serious debate.
Since in herbal drugs the overall effectiveness may be a result of complex self-regulatory components henceforth, in an attempt to rationalize the concept of bioequivalence in herbal drugs the following guidelines were laid out for oral immediate release form drugs not requiring in vivo studies:
a. The active substance does not have a narrow therapeutic range.
b. It has a first pass metabolism > 70% and linear pharmacokinetics within the therapeutic range.
c. It is highly water soluble.
d. It is highly permeable in the intestine.
e. The formulation is not expected to have effects on pharmacokinetic parameters.
It is prominent that an evidence based bioequivalence studies are very difficult if not impossible, and although use of ultramodern analytical apparatuses may have made the estimation easier, there is a lot that needs to be done for a universally acceptable approach to be developed.
Therefore there are in general the following three issues of major concern while evaluating the bioequivalence parameters for herbal drugs (Indian Herbal pharmacopoeia, 1998).
Important parameters for bio/pharmaceutical equivalence of herbal product:
a. Pharmaceutical equivalence, which is dependent on raw material quality, extraction methodology, chemical standardization of extraction procedure, manufacturing processes and machinery involved etc.
b. Biopharmaceutical equivalence: Effect of same dosages and their biological effect replication.
c. Therapeutic equivalence.
Therapeutic Equivalence as a complimenting factor to bio-equivalence:
According to the European as well as WHO guidelines (1998) a medicinal product is therapeutically equivalent with another product if it contains the same active substance or therapeutic moiety and, clinically, shows the same effectiveness and safety as those products, whose effectiveness and safety have been established.
To demonstrate the therapeutic equivalence. Therefore as a complementary evaluation for bioequivalence a therapeutic equivalence determination involves the following determinations: Direct evidence: Clinical studies with primary and secondary end points regarding effectiveness or clinical studies with pharmacodynamics and surrogates as end points.
Indirect Evidence:
Confirmation of bioequivalence in clinical pharmacokinetic studies via surrogates AUC (quantity of absorption), Cmax (maximum concentration) and tmax (rate of absorption) located within the confidence interval. Pharmacological profiles in animal models. In vitro studies in cell systems regarding influence on receptors, enzymes, channels.
In practice indirect evidence, using pharmacokinetic surrogates for bio-equivalence, is generally the most appropriate procedure to substantiate the therapeutic equivalence between medicinal products.
Two medicinal products are bioequivalent if they are pharmaceutical equivalent and their bioavailability (rate and extent) after administration in the same molar dose are similar to such a degree that their effects, with respect to both efficacy and safety, will be essentially the same.
Bioequivalence is based on the measured concentrations of the parent compound or an active metabolite in plasma or urine. Parameters of importance for equivalence are Cmax (maximal concentration), the shape of and the area under the concentration versus time curves (AUC) or the cumulative renal excretion and excretion rate in multiplicative models.
Bioequivalence is assumed, if the 90% confidence interval of the ratio (test/reference) for the parameters AUC and Cmax lies within the range of 0.80-1.25 (log transformed data). In the case of Cmax a broader interval may be acceptable and must be prospectively defined, e.g. 0.70-1.43.
Examples for Bioequivalence Studies from Natural Products:
Gingko biloba is one of the highly studied herbal medicinal plant and a very good example for the application of bioequivalence studies in herbal drug standardization. Gingko extracts are approved by the German Commission E monograph for usefulness in treatment of dementia, vertigo and tinnitus.
In general, the product specifications for this plant include dry extract of the dried leaf, manufactured using acetone/water with subsequent purification Steps, without the addition of concentrates or isolated ingredients and with a drug/extract ratio of 35 to 67:1 an average of around 50:1.
A bioequivalence study carried out by Miiller and Blume compared the bio-availabilities of two gingko extracts, both characterized as containing 24% flavonol glycosides on their labels. While, product A released less than 33% of its triterpene lactone contents in 60 minutes, product B released 99 per cent in 15 minutes. Both products were administered to 12 healthy individuals and using a crossover trial design.
The concentration of gingkolides A, B and bilobalide were measured in blood. A statistically significant maximum plasma concentration, and area under the curves were obtained for product B. Statistical analysis using 90% confidence interval clearly revealed that the two products were not bioequivalent.
Therefore, it is clear that a bioequivalence study could provide some important to the question like a similar claim composition leads to different biological profile and a biological activity as well.