INTRODUCTION:

Herbal drugs have been used since ancient times as medicines for the treatment of a range of diseases. Medicinal plants have played a key role in world health. In spite of the great advances observed in modern medicine in recent decades, plants still make an important contribution to health care.¹ In recent era, there has been great demand for plant derived products in developed countries. These products are increasingly being sought out as medicinal products, nutraceuticals and cosmetics.²

At least 120 distinct chemical substances derived from plants that are considered as important drugs currently in use in the world, while several other drugs are simple synthetic modifications of the natural products.³ World Health Organization (WHO) encourages, recommends and promotes traditional/herbal remedies in natural health care programmed because these drugs are easily available at low cost, safe and people have faith in them. The WHO assembly in number of resolutions has emphasized the need to ensure quality control of medicinal plant products by using modern techniques and applying suitable standards.⁴WHO has provided some terms related to herbal drugs, according to their definitions? Herbal medicines include herbs, herbal materials, herbal preparations and finished herbal products. In some countries herbal medicines may contain, by tradition, natural organic or inorganic active ingredients that are not of plant origin (e.g. animal and mineral materials). Herbs include crude plant material, such as leaves, flowers, fruit, seeds, stems, wood, bark, roots, rhizomes or other plant parts, which may be entire, fragmented or powdered. Herbal materials include, in addition to herbs, fresh juices, gums, fixed oils, essential oils, resins and dry powders of herbs. In some countries, these materials may be processed by various local procedures, such as steaming, roasting or stir-baking with honey, alcoholic beverages or other materials. Herbal preparations are the basis for finished herbal products and may include comminuted or powdered herbal materials, or extracts, tinctures and fatty oils of herbal materials. They are produced by extraction, fractionation, purification, concentration, or other physical or biological processes. They also include preparations made by steeping or heating herbal materials in alcoholic beverages and/or honey, or in other materials. Finished herbal products consist of herbal preparations made from one or more herbs. If more than one herb is used, the term “mixture herbal product” can also be used. Finished herbal products and mixture herbal products may contain excipients in addition to the active ingredients. However, finished products or mixture herbal products to which chemically defined active substances have been added, including synthetic compounds and/or isolated constituents from herbal materials, are not considered to be herbal (WHO guideline, 2000). Standardization of herbal formulations is essential in order to assess of quality drugs, based on the concentration of their active principles, physical, chemical, phytochemical, standardization, In-vitro and In-vivo parameters. The quality assessment of herbal formulations is of paramount importance in order to justify their acceptability in modern system of medicine.⁵ So that it is necessary to maintain reproducible efficacy and safety of phytopharmaceuticals therefore if phytopharmaceuticals have to regarded as rational drug they should be standardized and pharmaceutical quality must be approved.⁶ One of the major problems faced by the herbal industry is the unavailability of rigid quality control profiles for herbal materials and their formulations. In India, the department of Ayush, Government of India, launched a central scheme to develop a standard operating procedures for the manufacturing process to develop pharmacopeial standards for ayurvedic preparations. The subject of herbal drug standardization is massively wide and deep. There is so much to know and so many seemingly contradictory theories on the subject of herbal medicines and their relationship with human physiology and mental function. India needs to explore the medicinally important plants. This can be achieved only if the herbal products are evaluated and analyzed using sophisticated modern techniques of standardization.⁴ Also World health Organization (WHO) gives guidelines for the herbal standardization and evaluation.

STANDARDIZATION:

Standardization as defined by American Herbal Product association: “Standardization refers to the body of information and control necessary to product material of reasonable consistency. This achieved through minimizing the inherent variation of natural product composition through quality assurance practices applied to agricultural and manufacturing processes.⁷ Standardization expression is used to describe all measures which are taken during the manufacturing process and quality control leading to a reproducible quality. It’s also involving the study from birth of plant to its clinical application. It’s also include the herbal drugs preparation to a define content of a constituent or a group of substance with known therapeutic activity respectively by addition of excipients or by mixing herbal drugs preparation.⁸ Standardization should take into consideration all aspects that contribute to the quality of the herbal drugs, namely correct identity of the sample, organoleptic evaluation, pharmacognostic evaluation, volatile matter, quantitative evaluation (ash values, extractive values), phytochemical evaluation, test for the presence of xenobiotics, microbial load testing, toxicity testing, and biological activity. Of these, the phytochemical profile is of special significance since it has a direct bearing on the activity of the herbal drugs. The fingerprint profiles serve as guideline to the phytochemical profile of the drug in ensuring the quality, while quantification of the marker compound/s would serve as an additional parameter in assessing the quality of the sample. Phytochemical standardization encompasses all possible information generated with regard to the chemical constituents present in an herbal drug. Hence, the phytochemical evaluation for standardization purpose includes the following:

1. Preliminary testing for the presence of different chemical groups.

2. Quantification of chemical groups of interest (e.g., total alkaloids, total phenolics, total triterpenic acids, total tannins).

3. Establishment of finger print profiles.

4. Multiple marker-based fingerprint profiles.

5. Quantification of important chemical constituents.¹

METHODS OF STANDARDIZATION:

Standardization of herbal raw drugs:

Standardization of herbal raw drugs include passport data of raw plant drugs, botanical authentification, microscopic & molecular examination, identification of chemical composition by various chromatographic techniques and biological activity of the whole plant.⁹ Macroscopic and microscopic evaluation and chemical profiling of the herbal materials for quality control and standardization have been in use for standardization. Macroscopic identity of medicinal plant materials is based on sensory evaluation parameters like shape, size, colour, texture, odour and taste while microscopy involves comparative microscopic inspection of powdered herbal drug. Further, advances in microscope technology have increased the accuracy and capabilities of microscopy as a mean of herbal crude material identification due to the implication of light and scanning electron microscopes (SEM) in herbal drug standardization.¹⁰

1. Authentication:

In India, two governments organizations first Central council for research in Unani medicine (CCRUM) and central council for research in ayurvedic medicine are working for quality control in authentication the plant material collect from an appropriate region of the country at an appropriate stage of its growth is well authentication by details taxonomical study and the correct botanical identity is established.¹¹ To ensure and enhance the quality of herbal medicines, the Government of India has notified Good Manufacturing Practices (GMP) under Schedule ‘T’ of the Drugs and Cosmetics Act 1940 which also ensures raw materials used in the manufacture of drugs are authentic, of prescribed quality and are free from contamination. The guidelines for Good Agricultural Practices (GAP) seek to lay down a cultivation programmed designed to ensure optimal yield in terms of both quality and quantity of any crop intended for health purposes.

Ø Name of Institutes

Central Council for Research in Ayurveda and Siddha (CCRAS),

Central Council for Research in Unani medicine (CCRUM),

Central Council for Research in Homoeopathy (CCRH),

Central Council for Research in Yoga and Naturopathy (CCRYN)

Central Council for Indian Medicine (CCIM),

Central Council for Homoeopathy (CCH)

Ø Laboratories

Pharmacopoeial Laboratory for Indian Medicine (PLIM),

Homoeopathy Pharmacopoeia Laboratory (HPL)

Ø National Institutes

National institute of Homoeopathy (NIH),

National Institute of Ayurveda (NIA),

National Institute of Unani Medicine (NIUM),

National Institute of Naturopathy (NIN),

National Institute of Siddha (NIS),

Institute of Post-Graduate Training and Research in Ayurveda (IPGTRA),

Rashtriya Ayurved Vidyapeeth (RAV),

Morarji Desai National Institute of Yoga (MDNIY)

2. A. Macroscopic evaluation:

In this methods, description, general condition of the drug size, shape outer surface inner surface are referred. A sensory or organoleptic character describes colour, odour taste and consistency.⁹

The fractured surface in cinchona, quillia and cascara barks and quassia wood are important characteristics. Aromatic odour of umbellifrous fruits and sweet taste of liquric are the example of this evaluation. The ovoid tears of gum acacia ribbon shaped characterizes of tragacanth disc shaped structure of nux vomica concial shape of aconite quills of cinchona.

2. B. Microscopic Evalution:⁸

The inner pseudoparenchyma cells are oval or rounded, the contain fixed oil & protein the whole tissue is devoid of cellulose and lignin.

Various parameters includes in microscopy

A. Leaf content

B. Trichome

C. Stomata

3. Physical Evaluation:

Determination of foreign matter, total ash, extractive value, moisture content, specific optical rotation, pH, Solubility, Refractive index, Volatile oil content, Pesticide residue, Microbial contamination, Radioactive contamination.²

4. Chromatographic Fingerprinting and Marker Compound Analysis:

A chromatographic fingerprint of an Herbal Medicine (HM) is a chromatographic pattern of the extract of some common chemical components of pharmacologically active and or chemical characteristics. This chromatographic profile should be featured by the fundamental attributions of “integrity” and “fuzziness” or “sameness” and “differences” so as to chemically represent the HM investigated. It is suggested that with the help of chromatographic fingerprints obtained, the authentication and identification of herbal medicines can be accurately conducted (integrity) even if the amount and/or concentration of the chemically characteristic constituents are not exactly the same for different samples of this HM (hence, “fuzziness”) or, the chromatographic fingerprints could demonstrate both the “sameness” and “differences” between various samples successfully. Thus, we should globally consider multiple constituents in the HM extracts, and not individually consider only one and/or two marker components for evaluating the quality of the HM products. However, in any HM and its extract, there are hundreds of unknown components and many of them are in low amount. Moreover, there usually exists variability within the same herbal materials. Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the HM.

Thin layer chromatography (TLC)

Thin layer chromatography is simply known as TLC. It is one of the most popular and simple chromatographic technique used of separation of compounds. In the phytochemical evaluation of herbal drugs, TLC is being employed extensively for the following reasons:

1. It enables rapid analysis of herbal extracts with minimum sample clean-up requirement,

2. It provides qualitative and semi quantitative information of the resolved compounds.

3. It enables the quantification of chemical constituents.

Fingerprinting using HPLC and GLC is also carried out in specific cases In TLC fingerprinting, the data that can be recorded using a high-performance TLC (HPTLC) scanner includes the chromatogram, retardation factor (R_f) values, the color of the separated bands, their absorption spectra, λ_maxand shoulder inflection/s of all the resolved bands. All of these, together with the profiles on derivatization with different reagents, represent the TLC fingerprint profile of the sample. The information so generated has a potential application in the identification of an authentic drug, in excluding the adulterants and in maintaining the quality and consistency of the drug. HPLC fingerprinting includes recording of the chromatograms, retention time of individual peaks and the absorption spectra (recorded with a photodiode array detector) with different mobile phases. Similarly, GLC is used for generating the fingerprint profiles of volatile oils and fixed oils of herbal drugs. Furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as High- Performance Liquid Chromatography–Diode Array Detection (HPLC–DAD), Gas Chromatography–Mass Spectroscopy (GC–MS), Capillary Electrophoresis- Diode Array Detection (CE DAD), High-Performance Liquid Chromatography–Mass Spectroscopy (HPLC–MS) and High-Performance Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy (HPLC–NMR) could provide the additional spectral information, which will be very helpful for the qualitative analysis and even for the on-line structural elucidation.^13,14

High Performance Thin Layer Chromatography (HPTLC)

HPTLC technique is widely employed in pharmaceutical industry in process development, identification and detection of adulterants in herbal product and helps in identification of pesticide content, mycotoxins and in quality control of herbs and health foods.¹⁵ It has been well reported that several samples can be run simultaneously by use of a smaller quantity of mobile phase than in HPLC.¹⁶ It has also been reported that mobile phases of pH 8 and above can be used for HPTLC. Another advantage of HPTLC is the repeated detection (scanning) of the chromatogram with the same or different conditions. Consequently, HPTLC has been investigated for simultaneous assay of several components in a multi-component formulation.¹⁷ With this technique, authentication of various species of plant possible, as well as the evaluation of stability and consistency of their preparations from different manufactures. Various workers have developed HPTLC method for phytoconstituents in crude drugs or herbal formulations such as bergenin, catechine and gallic acid in Bergenia cilliata and Bergenia lingulata.¹⁸

High Performance Liquid Chromatography (HPLC)

Preparative and analytical HPLC are widely used in pharmaceutical industry for isolating and purification of herbal compounds. There are basically two types of preparative HPLC: low pressure HPLC (typically under 5 bar) and high pressure HPLC (pressure >20 bar). The important parameters to be considered are resolution, sensitivity and fast analysis time in analytical HPLC whereas both the degree of solute purity as well as the amount of compound that can be produced per unit time i.e. throughput or recovery in preparative HPLC.¹⁹ In preparative HPLC (pressure >20 bar), larger stainless steel columns and packing materials (particle size 10-30 μm are needed. The examples of normal phase silica columns are Kromasil 10 μm, Kromasil 16 μm, Chiralcel AS 20 μm whereas for reverse phase are Chromasil C18, Chromasil C8, YMC C18. The aim is to isolate or purify compounds, whereas in analytical work the goal is to get information about the sample. This is very important in pharmaceutical industry of today because new products (Natural, Synthetic) have to be introduced to the market as quickly as possible. Having available such a powerful purification technique makes it possible to spend less time on the synthesis conditions. ^{10, 21- 22}

Liquid Chromatography- Mass Spectroscopy (LC-MS)

LC-MS has become method of choice in many stages of drug development.²³ Recent advances includes electrospray, thermospray, and ionspray ionization techniques which offer unique advantages of high detection sensitivity and specificity, liquid secondary ion mass spectroscopy, later laser mass spectroscopy with 600 MHz offers accurate determination of molecular weight proteins, peptides. Isotopes pattern can be detected by this technique.²⁰

Liquid Chromatography- Nuclear Magnetic Resonance (LC-NMR)

LC-NMR improves speed and sensitivity of detection and found useful in the areas of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process. The combination of chromatographic separation technique with NMR spectroscopy is one of the most powerful and time saving method for the separation and structural elucidation of unknown compound and mixtures, especially for the structure elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC-NMR improves speed and sensitivity of detection. The recent introduction of pulsed field gradient technique in high resolution NMR as well as three-dimensional technique improves application in structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process.²⁴

Gas Chromatography-Mass Spectroscopy (GC-MS)

Gas Chromatography equipment can be directly interfaced with rapid scan mass spectrometer of various types. GC and GC-MS are unanimously accepted methods for the analysis of volatile constituents of herbal medicines, due to their sensitivity, stability and high efficiency. Especially, the hyphenation with MS provides reliable information for the qualitative analysis of the complex constituents.^25-26The flow rate from capillary column is generally low enough that the column output can be fed directly into ionization chamber of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD). In this instrument, ions are created from the eluted sample by electron impact or chemical ionization and stored in a radio frequency field; the trapped ions are then ejected from the storage area to an electron multiplier detector. The ejection is controlled so that scanning on the basis of mass-to-charge ratio is possible. The ions trap detector is remarkably compact and less expensive than quadra pole instruments. GC-MS instruments have been used for identification of hundreds of components that are present in natural and biological system.²⁷

Gas Chromatography- Flame Ionization Detector (GC-FID)

A number of detectors are used in Gas Chromatography. The most common are the Flame ionization detector (FID) and the thermal conductivity detector (TCD). Coupling capillary column gas chromatographs with Fourier Transform Infrared Spectrometer provides a potent means for separating and identifying the components of different mixtures.²⁷ Both are sensitive to a wide range of components, and both work over a wide range of concentrations. While TCDs are essentially universal and can be used to detect any component other than the carrier gas (as long as their thermal conductivities are different from that of the carrier gas, at detector temperature), FIDs are sensitive primarily to hydrocarbons, and are more sensitive to them than TCD. However, an FID cannot detect water. Both detectors are also quite robust. Since TCD is non-destructive, it can be operated in-series before an FID (destructive), thus providing complementary detection of the same analytes.

Supercritical Fluid Chromatography (SFC)

Supercritical fluid chromatography is a hybrid of gas and liquid chromatography that combines some of the best features of each. SFC permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. SFC has been applied to a wide variety of materials including natural products, drugs, food and pesticide.²⁸ These compounds are either nonvolatile or thermally labile so that GC procedures are inapplicable or contain no functional group that makes possible detection by the spectroscopic or electrochemical technique employed in LC.²⁴

DNA Fingerprinting

DNA analysis has been proved as an important tool in herbal drug standardization. This technique is useful for the identification of phytochemically indistinguishable genuine drug from substituted or adulterated drug. It has been reported that DNA fingerprint genome remain the same irrespective of the plant part used while the phytochemical content will vary with the plant part used, physiology and environment.²⁹ Deoxyribonucleic acid (DNA) is the fundamental building component of all living cells. Our characteristics, traits and physical features are determined by the 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 the Central Dogma Theory. Central Dogma theory can be defined as the fundamental theory of molecular biology that genetic information flows from DNA to RNA to proteins.³⁰ This concept of fingerprinting has been increasingly applied in the past few decades to determine the ancestry of plants, animals and other microorganisms. Genotypic characterization 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 commercial herbal drugs is the availability of intact genomic DNA from plant samples after they are processed. Adulterants can be distinguished even in processed samples, enabling the authentication of the drug.³¹ The other useful application of DNA fingerprinting is the availability of intact genomic DNA specificity in commercial herbal drugs which helps in distinguishing adulterants even in processed samples.³²

Genetic Marker

A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism SNP), or a long one, like mini satellites.

Some commonly used types of genetic markers are

RFLP (or Restriction fragment length polymorphism)

AFLP (or Amplified fragment length polymorphism)

RAPD (or Random amplification of polymorphic DNA)

VNTR (or Variable number tandem repeat)

Micro satellite polymorphism

SNP (or Single nucleotide polymorphism)

STR (or Short tandem repeat)

SFP (or Single feature polymorphism)

They can be further categorized as dominant or co-dominant. Dominant markers allow for analyzing many loci at one time, e.g. RAPD. A primer amplifying a dominant marker could amplify at many loci in one sample of DNA with one PCR reaction. Co-dominant markers analyze one locus at a time. A primer amplifying a co-dominant marker would yield one targeted product.³³

5. Other techniques:

Capillary electrophoresis (CE)

Researchers evaluated the importance of CE for quality control of herbal medicinal products. Several CE studies dealing with herbal medicines have been reported and two kinds of medicinal compound i.e. alkaloids and flavonoids have been studied extensively. The methodology of CE was established to evaluate one herb drug in terms of specificity, sensitivity and precision, and the results were in agreement with those obtained by the HPLC method. Furthermore, the analysis time of the CE method was two times shorter than that in HPLC and solvent consumption was more than 100-fold less.^34-36

Thermal analysis of herbal drugs

Thermo gravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC) have been employed to study any physical or chemical changes in various products including herbal drugs and also used to study pre formulation or drug excipients compatibility. TGA may be operated under sub ambient conditions to analyze ethanol in herbal formulations such as asavas and arista. TGA and DTA analysis of mercury based Indian traditional metallic herbal drug Ras-sindoor indicated the presence of mercury sulphide based on a sharp peak at 354^o C which corresponded to melting temperature of mercury sulphide. The optimized extraction obtained by distillation showed the presence of volatile oil in dry ginger as a component of volatile oil-beta cyclodextrin inclusion compound using DTA. DSC thermo grams data confirmed the formation of phospholipids complex with emodin (an anthraxquinone) and naringen.^37-42

Differential Pulse Polarography (DPP)

DPP can be used to study trace amounts of chemicals with detection limits on the order of 10-8 M. Some heavy metals, including Pb, Cd, Zn, Cu and Fe were successfully identified and determined in chamomile and calendulea flowers by DPP. Accumulation of heavy metals, namely Pb, Cd, Cu and Zn was estimated in market as well as genuine samples of important herbal drugs of India viz., Alpinia galanga, Artemesia parviflora, Butea monosperrma, Coleus forskohlii, Curcuma amada, Euphorbia prostrate, Leucas aspera, Malaxis accuminata and Pueraria tuberose. The concentration of Pb and Cd was found beyond the WHO permissible limits in most samples. Trace a mounts of selenium in Chinese herbal medicines and flavonoids in small amount of medicinal herb samples were determined by DPP. A DPP method has been for the determination of total hypericin in phytotherapeutic preparations (drops, tablets and capsules) in various buffer systems over the pH range 3.5–10.0.^43-47

Metabolomics technique

This technique has been used for identification of active phyto constituents from herbal medicine. Metabolomic approach was employed to identify the chemical constituents in Sophora flavescens, which were further analyzed for their effect on Pregane X receptor activation and Cytochrome P3A regulation. The greater potential of metabolomics has been reported in the development of active secondary metabolites from medicinal plants as novel or improved phyto therapeutic agents. The recent studies showed that NMR-based metabolomics approach combined with orthogonal projections to latent structure-discriminant analysis identified the purity of an herbal medicine.

X-ray Powder Diffractometry (X-RPD)

This technique is used to identify minerals, crystalline materials and metallic based herbal formulations. The tin based herbal drug Vanga Parpam was estimated by XRD and the intense sharp diffraction peaks clearly confirmed the presence of high crystallinity in Vanga Parpam. XRD analysis of metallic based Indian traditionally medicine Ras-sindoor indicated the presence of mercury sulphide which is represented by sharp peak. X-ray powder diffractometry data confirmed the formation of phospholipid complex with emodin, naringenin, quercetin, gallic acid.

Infrared Spectroscopy (IR)

FTIR along with the statistical method principal component analysis (PCA) was applied to identify and discriminate herbal medicines for quality control in the fingerprint region 400-2000 cm^-1. The ratio of the areas of any two marked characteristic peaks was found to be nearly consistent for the same plant from different regions, thereby, an additional discrimination method for herbal medicines. PCA clusters herbal medicines into different groups, clearly showing that IR method can adequately discriminate different herbal medicines using FTIR data. Near-infrared spectroscopy technique has been used for rapid determination of active components, species, geographic origin, special medicinal formula, on-line quality control, identification of counterfeit and discrimination of geographical origins of Chinese herbal medicines. Two-dimensional near-infrared (NIR) correlation spectroscopy was applied to the discrimination of Fructus lycii (a traditional Chinese medicinal herb) of four different geographic regions.

Standardization of herbal formulation:

Standardization of herbal formulation requires implementation of Good Manufacturing Practices (GMP) (WHO guideline, 1996) In addition, study of various parameters such as pharmacodynamics, pharmacokinetics, dosage, stability, self-life, toxicity evaluation, chemical profiling of the herbal formulations is considered essential.⁴⁸ Heavy metals contamination, Good Agricultural Practices (GAP) in herbal drug standardization is equally important.⁴⁹

Standardization of polyherbal formulations:

Standardization is an important aspect for maintaining and assessing the quality and safety of the polyherbal formulation as these are combinations of more than one herb to attain the desire therapeutic effect.²⁷ Standardization minimizes batch to batch variation; assure safety, efficacy, quality and acceptability of the polyherbal formulations.⁵⁰

CONCLUSION:

Standardization of the herbal drug the physical, chemical, biological, analytical parameters are required to carried out. It assures the quality, purity and safety of herbal drug. Studies involve the safe and accurately selection and handling of crude materials, ensure efficacy and stability of finished product. Implication of standardization of herbal drug is essential because of increasing demand for developing an herbal dosage form which improve patient convince and compliance. Implications are done by making strong rules and act for standardization of herbal drug & its herbal formulation.

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Received on 22.03.2016 Modified on 08.04.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(2): 93-100

DOI: 10.5958/0975-4385.2016.00018.2