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S. G. Uttarwar*et al. /International Journal Of Pharmacy&TechnologyISSN: 0975-766XCODEN: IJPTFIAvailable through OnlineReview Articlewww.ijptonline.comCOMPENDIAL TESTING METHODS FOR HERBAL CRUDE DRUGS: AN OVERVIEWS. G. Uttarwar *, R. S. Wanare † and P. A. Pangarkar ††*S. N. Institute of Pharmacy, Pusad-445204, Dist-Yavatmal, Maharashtra, India.Email: shtl.uttarwar@rediffmail.comReceived on 15-04-2013 Accepted on 05-05-2013AbstractIn the last few decades there has been an exponential growth in the field of herbal medicine and tremendous budgetowards the use of medicine of herbal origin. As the risks and the deficiencies of modern medicine have started gettingmore apparent, majority of formulation are prepared from herbs. In the global perspective herbal medicines and productshas been enjoying renascence among the clients owing to its natural origin and lesser side effects for healthy living.Recently there is a squirt in the interest regarding survival of ayurvedic formulations. Nevertheless, one of thehindrances in the acceptance of the herbal formulation is the lack of standard quality control profile. The profile ofconstituents in the final product has implication in its efficacy and safety. Herbal remedies derived from botanicalsrepresent substantial portion of global market and in this respect internationally recognized guidelines for their qualityassessment and quality control are necessary. WHO has punctuated the need to ensure quality control of botanicals byusing modern technique and by applying suitable parameters and standards. In order to get-over certain unavoidableshortcoming of the Pharmacopoeial monograph other quality control measures must be explored. Quality controlmeasures covers; many aspects of drug manufacture, distribution and, sale is not restricted to final product analysiseither regulatory or otherwise, while engaging in this chore, it must be realized that some of the Quality control practicesthat work excellently either modern drug may not be appropriate with Herbal drugs.Keywords: Crude drug, Herbal medicines, Quality control testing, Standardization.Introduction: Generally, all medicines, whether they are synthetic or of plant origin, should fulfill the basicrequirements of being safe and effective 1 . World Health Organization (WHO) has defined herbal medicines as finishedIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2509

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technologylabeled medicinal product that contain active ingredients, aerial or underground parts of the plant or other plant materialor combinations 2 .In all traditional systems of medicine, the quality control aspect has been considered from its inspection itself byPractitioners for effective and safe use. However, modern concept require necessary changes in their access by that wayconcrete method of quality control in terms development of modern methodologies. Hence quality assurance is hurl areafor the evaluation of botanicals and herbal formulations so utmost chore is to evolve such parameter by which thepresence of the entire ingredients can be identified. For standardization of herbal drugs various chromatographicprofiles, spectrophotometric methods and evaluation of physicochemical properties can be tried to evolve pattern foridentifying the presence of different ingredient 3 .Stream Regulations for Standardization of Crude DrugsInternationally various pharmacopoeias have provided monographs stating parameters and standards of many herbs andherbal products to maintain their quality in their respective nations. British Herbal Pharmacopoeia British Herbal Compendium Chinese Herbal Pharmacopoeia United States Herbal Pharmacopoeia Japanese Standards for Herbal Medicine The Ayurvedic Pharmacopoeia of India (API)Government of India besides has come out with Ayurvedic Pharmacopoeia, which recommends basic qualityparameters for eighty common Ayurvedic herbal drugs.Standardization and Quality Control Parameters for Evaluation of Herbal Crude Drugs 4Standardization accounting to WHO is the process involving the physicochemical evaluation of crude drug covering theaspects, as selection and handling of crude material, safety, efficacy and stability assessment of finished product,documentation of safety and risk based on experience, provision of product information to consumer and productpromotion.IJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2510

S. G. Uttarwar*et al. /International Journal Of Pharmacy&TechnologyThe quality and purity required in a drug are achieved by standards given in the official work of reference. To establishthe identity and quality of the drug, several methods may be considered 5,6-8 .These are enumerated below,• Macroscopic Examination: In case of whole drug, the macroscopic and sensory characters are sufficient foridentification of right variety and search of adulterants.• Microscopic Examination: These are valuable both for powders and ungrounded drugs for identification ofright variety and search of adulterants.• Solubility: The solubility, especially exceptional behavior towards solvent, is useful in examination of many oilsand oleo-resins.• Physical constants: Physical constants such as specific gravity, optical rotation, viscosity and refractive indexare especially valuable for the evaluation of fats, oleoresins, balsams and similar substances.• Foreign Organic Matter: Remove of matter other than source plant to get the drug in pure form.• Ash Values: It is criteria to judge the identity and purity of crude drug – Total ash, sulfated ash, water solubleash and acid insoluble ash etc.• Extractive Values: These are indicating the approximate measure of chemical constituents of crude drug.• Moisture Content: To check moisture content helps prevent degradation of product.• Crude Fiber: To determine excessive woody material Criteria for judging purity.• Chromatographic Examination: Include identification of crude drug based on use of major chemicalconstituent as marker.• Qualitative Chemical Evaluation: Covers criteria for identification and characterization of crude drug withrespect to phytochemical Constituents.• Quantitative Chemical Evaluation: Covers criteria to estimate the amount of active constituents.• Volatile oils: its cover the measurement of the volatile content of the plant.• Bitterness value: The bitter properties of plant material are determined by comparing the threshold bitterconcentration of an extract of the materials with that of a dilute solution of quinine hydrochloride R.IJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2511

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technology• Hemolytic activity: The hemolytic activity of plant materials, or a preparation containing saponins, isdetermined by comparison with that of a reference material, saponins R.• Foaming index: The foaming ability of an aqueous decoction of plant materials and their extracts is measuredin terms of a foaming index.• Pesticide residues: it measures the pesticide residues in the plant.• Swelling index: it measures the swelling property of the medicinal plant.• Arsenic and heavy metals: Contamination of medicinal plant materials with arsenic and heavy metals can beattributed to many causes including environmental pollution and traces of pesticides.• Microorganisms: Current practices of harvesting, handling and production may cause additional contaminationand microbial growth.• Alfatoxins: Minute presence in crude drug can be toxic and hence their presence is being tested.• Chemical Fingerprinting: To allow the detection of all the components in extracts.• DNA Fingerprinting: Technique is useful for identification of phytochemically indistinguishable genuine drugfrom substituted or adulterate drug.• Biological profiling: It identifies the biologically active plants allowing highly sophisticated standardization andquality control.• Radioactive contamination: A certain amount of exposure to ionizing radiation cannot be avoided since thereare many sources, including radio nuclides occurring naturally in the ground and the atmosphere.• Toxicological Studies: To help to determine pesticide residue, potentially toxic elements, safety studies inanimals like LD50 and Microbial count approach to ascertain their presence or absence.The processes mentioned above involves wide array of scientific investigations, which include physical, chemicaland biological evaluation employing various analytical methods and tools. The specific aims of such investigation inassuring herbal quality are as varied as the processes employed 9 .IJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2512

Physical evaluation 2S. G. Uttarwar*et al. /International Journal Of Pharmacy&TechnologyEach monograph contains detailed botanical, macroscopic and microscopic descriptions of the physical characteristics ofeach plant that can be used to ensure both identity and purity. Each description is accompanied by detailed illustrationsand photographic images which provide visual documentation of accurately identified material. Various physicalparameters include viscosity, volatile matter content, density, refractive index, hemolytic activity, foaming index,melting point, solubility. The Q. C. Parameter for evaluation of crude drug Moisture content Specific gravity Opticalrotation Bitterness value Swelling index, Ash value.Microscopic evaluation 3Microscopic analyses of plants are invaluable for assuring the identity of the material and as an initial screening test forimpurities. Most manufacturers of herbal products lack the quality control personnel to accurately assess plant identityand purity microscopically. The Ayurvedic Herbal Pharmacopoeia (AHP) fully characterizes herbal products against theliterature and AHP verified trade mark authenticated materials to assure identity of test materials. Ideally, submittedmaterials should be in their whole or semi-whole (cut) form for microscopic assessment. However, much informationcan be discerned from microscopic evaluation of powders as well. Full and accurate characterization of plant materialrequires a combination of physical and chemical tests.Chemical evaluation 9This covers the screening, isolation, identification and purification of chemical components. Chemical analysis of thedrug is done to assess the potency of vegetable and animal source material in terms of their active principles. Thechemical screening include colour reaction test, which help to determine the identity of the drug substance and possibleadulteration.Biological evaluationPharmacological activity of certain drugs has been applied to evaluate and standardize them. The assays on livinganimal and on their intact or isolated organs can indicate the strength of the drug or their preparations. All livingorganism are used, these assays are known as Biological assays or Bioassay 14 .IJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2513

Analytical Methods 9S. G. Uttarwar*et al. /International Journal Of Pharmacy&TechnologyCritical to compliance with any monograph, standard is the need for appropriate analytical methods for determiningidentity, quality, and relative potency. There are a plethora of analytical methods available. However, it is often difficultto know which is the most appropriate to use. The primary goal of AHP is to provide multiple methods of identificationand testing by which all aspects of the botanical can be appropriately assayed.Chromatographic Characterization 5,9Chromatography is the science which studies the separation of molecules based on differences in their structure and/orcomposition. In general, chromatography involves moving a preparation of the materials to be separated, “the "testpreparation”, over a stationary support. The molecules in the test preparation will have different interactions with thestationary support leading to separation of similar molecules. Test molecules which display tighter interactions with thesupport will tend to move more slowly through the support than those molecules with weaker interactions. In this way,different types of molecules can be separated from each other as they move over the support material. Chromatographicseparations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layerchromatography), very sensitive High Performance Thin Layer Chromatography (HPTLC), volatile gases (gaschromatography), paper (paper chromatography), and liquids which may incorporate hydrophilic, insoluble molecules(liquid chromatography).High performance thin layer chromatography (HPTLC) is a valuable quality assessment tool for the evaluation ofbotanical materials. It allows for the analysis of a broad number of compounds both efficiently and cost effectively.Additionally, numerous samples can be run in a single analysis thereby dramatically reducing analytical time. WithHPTLC, the same analysis can be viewed collectively in different wavelengths of light thereby providing a morecomplete profile of the plant than is typically observed with more specific type of analysis.10, 11-13DNA FingerprintingDNA analysis has been proved as an important tool in herbal drug standardization. This technique is useful for theidentification of phytochemically indistinguishable genuine drug from substituted or adulterated drug. It has beenreported that DNA fingerprint genome remain the same irrespective of the plant part used while the phytochemicalIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2514

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technologycontent will vary with the plant part used, physiology and environment 10 . Deoxyribonucleic acid (DNA) is thefundamental building component of all living cells. Our characteristics, traits and physical features are determined bythe specific arrangement of DNA base-pair sequences in the cell. It is this distinct arrangement of adenine, guanine,thymine and cytosine (called DNA nucleotides) that regulates the production of specific proteins and enzymes via theCentral Dogma Theory. Central Dogma theory can be defined as the fundamental theory of molecular biology thatgenetic information flows from DNA to RNA to proteins 11 . This concept of fingerprinting has been increasingly appliedin the past few decades to determine the ancestry of plants, animals and other microorganisms. Genotypiccharacterization of plant species and strains is useful as most plants, though belonging to the same genus and species,may show considerable variation between strains. Additional motivation for using DNA fingerprinting on commercialherbal drugs is the availability of intact genomic DNA from plant samples after they are processed. Adulterants can bedistinguished even in processed samples, enabling the authentication of the drug 12 . The other useful application of DNAfingerprinting is the availability of intact genomic DNA specificity in commercial herbal drugs which helps indistinguishing adulterants even in processed samples 13 .Purity DeterminationEach monograph includes standards of purity and other qualitative assessments which include when appropriate: foreignmatter, ash, acid-insoluble ash, moisture content, loss of moisture on drying, and extractives.High performance thin layer chromatography (HPTLC) is valuable quality assessment tool for the evaluation ofbotanical materials. It allows for the analysis of a broad number of compounds both efficiently and cost effectively.Additionally, numerous samples can be run in a single analysis thereby dramatically reducing analytical time. WithHPTLC, the same analysis can be viewed sing different wavelengths of light thereby providing a more complete profileof the plant than is typically observed with more specific types of analyses 15 .Quantitative AnalysisPrimary factors for considering a method as appropriate include accuracy of the findings, speed, basic ruggedness,applicability to a large segment of the manufacturing community, and avoidance of the use of toxic reagents andsolvents. In an attempt to promote harmonization, primary consideration is given to those methods which are alreadyIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2515

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technologyaccepted in official pharmacopoeias or by AOAC International. The validation process minimally includes: standardprecision, linearity, sample precision using replicate samples, sample linearity, selectivity (co-elution, sensitivity toanalyte degradation), retention times, and limits of detection. Other methods which may be of value to the industry maybe included or cited in the monograph but are not required for compliance with the monograph.Who Guidelines for Quality Standardized Herbal FormulationsStandardization and quality control parameters for herbal formulations are based on following fundamental parameters:1. Quality control of crude drugs material, plant preparations and finished products.2. Stability assessment and shelf life.3. Safety assessment; documentation of safety based on experience or toxicological studies.4. Assessment of efficacy by ethno medical information and biological activity evaluations.Quality Control of Crude MaterialAccording to pharmaceutical manufacturers association of U.S. “quality is the sum of all the factors which contributedirectly or indirectly to the safety, effectiveness and acceptability of the product” 4 . Standardization describes allmeasures taken during manufacturing process and quality control leads to reproducible quality of particular product.Standardization of botanicals offers many obstacles because synthetic drugs have well defined structure and otheranalytical parameters as well as reference standard for comparison also established assays and pharmacopoeias.Therefore, quality control is not problematic for synthetic drug 15 . There are several challenges as standardization ofherbal product is considered like controversial identity of various plants, deliberated adulteration of plant material,problems in storage and transport, which should be considered 16 .One of the impediments in the acceptance of the herbalproducts worldwide is the lack of standard quality control profiles. Most of the herbal formulations, especially theclassical formulations of traditional medicine, are polyherbal. In the United States, herbs are used either as dietarysupplements, with minimal standards of safety and efficacy, or as drugs, which require expensive and cumbersometesting procedures.Validation: In order to control quality of herbal drugs in better way, we must amalgamate newer techniques and termsto maximum extent. USFDA defines validation as, “Validation is documented evidence which provides a high degree ofIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2516

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technologyassurance that a specific process will consistently produce a product meeting its predefined specifications and qualityattributes”.Generally, validation investigations must include studies on specificity, linearity, accuracy, precision, range, detection,and quantitative limits, depending on whether the analytical method used is qualitative or quantitative 17 . Also, of utmostimportance is the availability of standards. For macroscopic and microscopic procedures in general this means thatreliable reference samples of the plant must be available. A defined botanical source (e.g. voucher specimens) willnormally solve this problem. This concept of validation is getting well applied to manufacturing of synthetic drugs fromlong time back. But yet not methodically studied and applied for the manufacturing of herbal drugs. Internationalregulations like USFDA, MCC, MHRA, TGA etc. shows the applicability of validation to pharmaceuticalmanufacturing but no one regulation except WHO applies the validation concept to manufacturing of herbal drugs.WHO also emphasize on very little part of validation. Due to aforesaid reason the emphasis on the concept of validationand also the validation model for manufacturing of herbal drugs should be the need of time.Validation Model for Manufacturing of Herbal drugsThere are lots of parameters which has to be considered while certifying the manufacturer the type of herbs as,• Environmental and growing conditions.• Time of harvesting.• Variation in composition.• Drying of herbal drugs.• Packaging of herbal formulations.ConclusionBecause of sobriety of the problem, fundamental knowledge of the important herbs found in India and widely used inAyurvedic formulation along with new standardization techniques is of prime importance and be explored.Incorporation of this will authenticate quality thereby reducing further problems. The development of modern analyticaltools in testing the various quality parameters for an effective quality control herbal product cannot be over emphasized.The assurance of the safety and efficacy of herbal drug requires monitoring of the quality of the product from collectionIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2517

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technologythrough processing to the finished packaged product. Our view is to draw the attention of readers to the rapidly evolvingstandardization aspects of herbal drugs with some new trends, so the area could be expanded significantly and minimizequality infract.References1. EMEA (2005). Guidelines on Quality of Herbal Medicinal Products/Traditional Medicinal Products,EMEA/CVMP/814OO Review. European Agency for the Evaluation of Medicinal Products (EMEA), London.2. Agarwal, A., 2005. Critical issues in Quality Control of Herbal Products, Pharma Times, 37(6), pp 09-11.3. Sane, R.T., 2002. Standardization, Quality Control and GMP for Herbal drugs, Indian Drugs, 39 (3), pp 184-190.4. Florey, H. W., Chain, E. and Florey, M. E., 1949. Vol. 1. The antibiotic, Oxford University Press. New York, pp576-628.5. Quality Control of Herbal Drugs, Pulok K Mukherjee , Business Horizons Pharmaceutical publishers, 2002, pp 186-245.6. Hylands P. New approaches for the quality control and standardization of plant delivered pharmaceutical andnutraceutical products. Oxford: Oxford Natural Products Publications. 20027. Indian Pharmacopoeia-2007, Ministry of health and Family Welfare, Controller of Publication, Delhi, Vol -III, pp2015-2073.8. Evans WC.Techniques in microscopy: quantitative microscopy.A Textbook of Pharmacognosy. 14 ed. /London:WB Saunders bCompany Ltd. 1996, pp 568-5789. Kunley, Oluyemisi F. Standardisation of herbal medicines.A review, International journal of biodiversity andconservation vol 4(3), March 2012, pp 101-112.10. Shikha S, Mishra N. Genetic markers - a cutting-edge technology in herbal drug research. J Chem Pharm Res 2009,vol 1, pp 1-18.11. Breithaupt,. H. (2003) Back to the roots EMBO Rep 4(1), pp 10-12.12. Mihalov, J. J., Marderosian, A. D., and Pierce, J. C. DNA identification of commercial ginseng samples, 2000.IJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2518

S. G. Uttarwar*et al. /International Journal Of Pharmacy&Technology13. Lazarowych NJ, Pekos P. The use of fingerprint and marker compounds for identification and standardization ofbotanical drugs. J Drug Inform 1998, 32, pp 497-512.14. Chaudhari, R. D., 1996. Herbal drug industry, 1 st Edn., Eastern Publisher, New Delhi, 498-499.15. Chakravarthy, B. K., 1993. Standardization of Herbal products. Indian. J. Nat. Product,vol 9, pp 23-26.16. Bhanu P. S., Sagar, T. K. and R. Zafer., 2003. Failure and successes of Herbal Medicines, The Indian Pharmacist, pp17-23.17. De Smet PAGM, Keller K, Hansel R, Chandler RF (1997). Adverse Effects of Herbal Drugs, (eds), Springer-Verlag,Heidelberg. vol 129(3), pp 137-145.Corresponding Author:S. G. Uttarwar *,Email: shtl.uttarwar@rediffmail.comIJPT | July-2013 | Vol. 5 | Issue No.2 | 2509-2519 Page 2519