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Sayyada Khatoon*.
Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, India.

Volume 3, Issue 1, Page 58-64, January-April 2015.

Article history
Received: 1 April 2015
Revised: 15 April 2015
Accepted: 18 April 2015
Early view: 20 April 2015

*Author for correspondence


Unani drugs of plant origin are herbaceous whole plant or their parts. The ever increasing demands of these drugs are leading to the adulteration and substitution of genuine drugs and the poor quality of Unani products. In India, the crude drug supplies are usually obtained through various trade channels and are generally lacking in uniform quality. It is very difficult to authenticate the commercial crude drugs because these are available as dried whole plant or some part of it. The Macro-microscopy & Planar chromatography (TLC/HPTLC) are the basic and important tools for proper identification of adulterants/substitutes of Unani drugs and their quality control. The macroscopy includes organoleptic characters while the microscopy encompasses the detection of the cell type and cell contents as well as the arrangement of cells in tissues. Banafshan, Bhuamla, Pershiaoashan, Rehan, Resha Khatmi and Zarnab can easily be differentiated from their adulterants/substitutes by observing the surface characters or comparing leaf surface microscopy. Similarly, the arrangement pattern and size of fibres, stone cells, crystals, secretary canals etc in phelloderm and phloem region are valuable parameters for the identification of most of the bark drugs. However, the quality assurance of Unani drugs still remains a challenge because of the high variability of chemical components viz. alkaloids, phenolics, terpenoids, steroids, glycosides etc. The variation may be diurnal, dioecy, chemotypic, genotypic, ecotypic, seasonal etc. Fingerprint analysis using TLC/HPTLC has become the most potent tools for quality control of Unani medicines because of its simplicity, reliability, rapidity and economy. Not only the general finger print profile but also chemical reference markers can be identified and estimated for quality evaluation and authentication of adulterants/substitutes of Unani medicine viz. asarone in Acorus, phyllanthin & hypophyllanthin in Phyllanthus species, berberine & tinosporaside in Tinospora, glycyrrhizine in Glycyrrhiza, gallic and ellagic acids in Terminalia species etc.


In traditional systems of medicine, drugs of plant, animal and mineral origin are used in their natural or so called „Crude? forms singly or in their mixture or in combination, to make a formulation (Khatoon et al., 2006a). Nearly 90 % of the Crude Drugs are obtained from the plant sources which may be whole plant especially of herbaceous plants; otherwise their parts such as Root, Rhizome, Stem, Wood, Bark, Leaf, Flower, Anther, Pollen, Seed, Fruit and their Exudates or Gums etc constitute single drugs in all Traditional Systems of Medicine even in Unani. The ever increasing demand of herbal drugs led to a spurt of large-scale commercial production with multicrores Rs/$ investments in many countries including India (Aneesh et al., 2009). With the ever-increasing demand of medicinal plants the supply line is adversely affected causing adulteration and substitution for genuine drugs. Such adulteration and substitution lead to the poor quality of herbal products (Khatoon et al., 2006a; Mehrotra et al., 2001). It is very difficult to authenticate the commercial crude drugs as these are available in the form of dried whole plant or some part of it. WHO has emphasized on the need to ensure the quality of medicinal plants/products (Anonymous, 2003). Countries like China have taken various measures to check the adulteration by raw drug standardization, domestication and cultivation of medicinal plants to ensure sustained supply of quality plant material to production centers (Li et al., 2011; Zhong et al., 2014). In India, the supplies are usually obtained through various trade channels and are generally lacking in uniform quality. Further, due to the enormous demand of herbal drugs resulted in depletion of natural resources is also one of the main causes for substitutions/adulterations (Khatoon et al., 2006a; Mehrotra et al., 2001).
Adulteration may broadly be defined as admixture or substitution of genuine article with spurious inferior, defective or otherwise useless or harmful substances. Adulterants are those herbs/materials having resemblance with that of genuine herbs/materials but have no biological or very non significant medicinal activity while substitutes are those herb/material having more or less similar medicinal properties of that of genuine one viz. Shanjaar/Ratanjot – Arnebia nobilis is genuine drug and A. euchroma, Onosma hispidum can be used as substitutes but Jatropha curcus is an adulterant (Khatoon, 1991).
Adulteration/substitution may be deliberate or un-deliberate. The Unani and Ayurvedic nomenclature of drug, traditional and regional names, various vernacular names of single plant species, are important factors of un-deliberate adulteration (Khatoon et al., 2006a). There are several controversial drugs where botanical identity of plant sources has not been established (Sagar, 2014; Kunle et al., 2012, Mehrotra et al., 2001). Sometimes not only the various species of a particular genus but entirely different plant taxa are being sold or used under the local names (Nizami and Jafri, 2006, Mehrotra et al., 2001). Another un-deliberate aspect of lack of quality of plant based drugs is „Natural variation? in therapeutically active constituents which are either primary or secondary metabolites (Pavarini et al., 2012; Kroymann, 2011; Kale, 2010). The expression of many of these compounds particularly those of the secondary metabolites are controlled and conditioned by a variety of factors such as habitat of the plant, agro climatic conditions, diurnal, seasonal and also the age and stage of plant/ part, dioecy, genetic predisposition, etc. Some of the examples are:
Ø Asgand (Withania somnifera)- Two varieties of Asgand have been mentioned in classical Unani literatue i. e. Asgand Nagori and Asgand Dakani. Asgand Nagori is preferred for its more potential medicinal properties (Anonymous, 1982, Ghani, 1920). However, researches showed various chemotypes with phenotypic variations and distinct pharmacological activities (Tuli and Sangwan, 2009; Kumar et al., 2007).
Ø Rewand-chini (Rheum emodi) should be collected in summer because anthraquinones (therapeutically active constituents) were present in this season but in winter anthranols (inactive) were present (Anonymous 1999).
Ø Degitelis (Digitalis purpurea) showed diurnal variation in therapeutic active constituent in leaves which contain more active glycoside at day time.These glycosides breakup into aglycone and sugar, a less active substance at night (Rowson, 1961).
Ø Dirmanah (Artemisia maritima) contains maximum therapeutic active constituent – santonin in unexpanded flower buds but it is absent in open flowers (Nadkarni, 1996).
The deliberate adulteration of therapeutically important plants with a cheaper material is very common to gain more profit. Various species of a particular genus or similar looking entirely different plant taxa are being sold under the same vernacular name or partially mixed with the genuine drugs. Substances like cloves, fennel, caraway, which are used to obtain volatile oils by steam distillation are mixed with genuine articles after removal of their volatile oils contents. In case of plant based drugs it is not limited to substitution of one plant to another or exhausted products, but various traditional medicines have been found to contain undeclared synthetic materials or even clay (Khatoon et al., 2006a; Mehrotra et al., 2001).
The identification of crude herbal drugs differ from that of living plants since the herbal drugs are usually in a dried form and may also have been subjected to other processing procedure. It is also a well known fact that the therapeutic activity of a medicinal plant is due to the presence of certain biologically active chemical constituents. The old literature of Traditional Indian System of Medicines like Unani and Ayurveda etc. provided specific instructions for collection by indicating location/edaphic conditions, habitat, seasonal and even the stage of the plant/part, growth and developmental stage of herb (Ibn-Al-Bitar, 1985; Sharma, 2001; Tewari et al., 2014).
The Macro-microscopy & Planar chromatography (TLC/HPTLC) are the basic and important tools for proper identification of adulterants/substitutes of Unani drugs and their quality control. Some of the important drugs are discussed in this paper under the heads macroscopy, microscopy and planar chromatography.

1. Macroscopy
The macroscopic study includes organoleptic characters i.e. the occurrence, size, shape, colour, surface markings, margins (leaf), texture, fracture, internal appearance, cut surfaces, odour and taste of the crude drug. These external characters also play an important role for the identification of adulterants/substitutes of herbal drugs (Mehrotra, 2003; Khatoon et al., 2006a; Prakash et al., 2013).

i. ‘Banafsha’
Flowers of Viola odorata L. are the official drug of „Banafsha? but other species of Viola viz. V. pilosa and V. betonicifolia are also commonly being sold as „Banafsha?. All three species of Viola can easily be identified by their macroscopical characters especially by observing the style and stigma. Style is inflated, broad and smooth in V. odorata; subclavate, subtruncate and shortly beaked at the apex in V. pilosa; geniculate at base and clavate above in V. betonicifolia. However, stigma is decurved & hairy in V. odorata; truncate with sub-lateral opening in V. pilosa while convex, forming hump like structure with lateral side opening in V. betonicifolia (Mehrotra et al., 1998).

ii. ‘Bhuamla’
Previously Phyllanthus niruri was reported as „Bhuamla? in India but after 1985 it was distributed in three species P. amarus, P. fraternus and P. debilis (Mitra and Jain, 1985). During market surveillance of herbal drugs, it was observed that almost all the commercial samples, either comprise of Phyllanthus amarus Schum & Thonn. or P. maderaspatensis Linn. or mixture of P. amarus, P. fraternus Webster. and P. maderaspatensis (Khatoon et al., 2006b). These species can be identified on the basis of following morphological characters:

·P. amarus- Branchlet 2-6 cm long; leaves 10-20, elliptic, oblong to ovate, obtuse or minutely apiculate at apex; flowers axillary, proximal 2-3 axils with unisexual 1-3 male flowers and all succeeding axils with bisexual cymules, sepals 5 (Fig. 1A).
? P. fraternus- Branchlet 2-11 cm long; leaves 10-30, elliptic oblong rounded at the apex; flowers in axillary, unisexual cymules, proximal 3-4 male flowers, succeeding solitary female flowers, sepals 6 (Fig. 1B).
? P. maderaspatensis – Branchlet absent; leaves, rounded truncate or somewhat obcordate at the apex, mucronate, much tapering into a very short petiole; flowers axillary, male flowers minute in small clusters, sub-sessile; female flowers solitary and larger with short stalk, sepals 6 (Fig. 1C).

Figure 1. Macroscopy of Phyllanthus species.
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iii. ‘Resha Khatmi’- Drug consists of the roots of Althaea officinalis L. (Fam. Malvaceae). Due to the limited distribution of this species the roots of another species Alcea rosea L. syn. Althaea rosea is also being sold in Indian market. Both the species can be identified by external characters. The roots of Althaea officinalis are strongly longitudinally furrowed, often spirally twisted with short-medium fracture but in Alcea rosea these are finely longitudinally furrowed, straight
with medium-hard fracture (Khatoon et al., 2008a).

2. Microscopy
The microscopical character encompasses the detection of the type of cells and cell contents as well as the arrangement of cells in tissues.
i. Bark drugs
During the drying process the bark drugs develop some sort of markings, cracks on the surfaces and sometimes occupy a different shape e.g. flat, curved, recurved, quilled etc. The arrangement pattern and size of fibres, stone cells and crystals in phelloderm and phloem region are valuable parameters for the identification of most of the bark drugs (Khatoon and Mehrotra, 2009). For example- „Asok?- Saraca asoca L. is official drug but Indian herbal drug markets are full of several adulterants/substitutes. These can easily be identified under the microscope- TS S. asoca shows phloem with small scattered groups of fibres and stone cells, uni-biseriate medullary rays while in S. declinata phloem with solitary or small groups of fibres, presence of fibre sclereids, uni-triseriate medullary rays; in Polyalthia longifolia phloem with broad concentric bands of fibre patches, interrupted by broad radiating multiseriate medullary rays embedded with mucilage canals; in Bauhinia variegata with scattered solitary fibres and calcium oxalate crystals and Shorea robusta with large scattered stone cells and interlocking medullary rays (Figure 2) (Khatoon and Mehrotra, 2009)

Figure 2. TS of stem bark of Asok .
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ii. ‘Pershiaoashan’
It is a drug from Pteridophytes or fern group of plant kingdom. Adiantum capillus-veneris L. is the official drug but other abundantly distributed and similar looking Adiantum species viz. A. lunulatum Burm., A. peruvianum Klotzsch., and A. venustum D. Don are commonly found as substitute/adulterant. The salient distinctive characters under the microscope are the presence of slightly wavy elongated epidermal cells in A. capillus-veneris; epidermal cells strongly wavy in A. lunulatum; star shaped epidermal cells in A. peruvianum; stomata on lower surface of pinnule but on both the surfaces only in A. venustum. In addition, rachis anatomy showed different cellular and stellar characteristics as identifying characters of aforesaid four Adiantum species (Singh et al., 2013).

iii. ‘Rehan’
Ocimum sanctum L. of family – Lamiaceae is the official drug „Rehan? but dried leaves of other Ocimum species viz. O. basilicum L. , O. canum Sims, O. gratissimum L. are also being used or sold as drug „Rehan?. All the aforesaid Ocimum species can easily be differentiated by visualizing the surface after removal of chlorophyll (boiled the leaves with saturated chloral hydrate solution, stained with safranin and mounted in glycerol). Ocimum sanctum can be identified by angular epidermal cells and its peculiar glandular and simple trichomes – glandular trichomes with 8 celled head and simple trichomes 3-5 celled with pointed apex (Figure 3a) while in other species epidermal cells are wavy and different type of trichomes. For example, two types of glandular trichomes – unicellular, 18 – 28 ?m and 72 – 88 ?m with globular head in O. basilicum (Figure 3b); two types of trichomes – glandular trichomes with single basal cell but simple and larger trichomes with multicellular bases in O. canum (Figure 3c); single type of large sized glandular trichome in O. gratissimum (Figure 3d).

Figure 3. Leaf structure anatomy of Rehan.
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iv. ‘Zarnab’
The official drug consists of leaves of Abies spectabilis Spach of the family – Pinaceae but leaves of Taxus wallichiana Zucc. of the family – Taxaceae are being used or sold as drug „Zarnab?. The leaves of A. spectabilis are linear, flattened with a prominent midrib and bifid apex while in T. wallichiana these are linear lanceolate with pointed apex. In A. spectabilis upper epidermis has elongated rectangular epidermal cells, sinuous, thick walled while lower surface has 7-9 rows of stomata in the middle of the lamina. On the contrary, In T. wallichiana upper epidermal cells are angular, hexagonal and thin walled interrupted with trichomes and lower epidermis has peculiar sunken stomata arranged in 6-7 rows in the middle of the lamina (Rawat et al., 1996).

3. Planar chromatography
The official IUPAC term “planar chromatography”, covers all chromatographic techniques using a planar open stationary phase: thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), ultrathin-layer chromatography (UTLC), overpressure layer chromatography (OPLC) and preparative layer chromatography (PLC) (Anonymous 2014). However, TLC/HPTLC is the most powerful analytical version of this form of chromatography due to its advantages which include simple, cost-effective, versatile, precise sample application, wide choice of stationary phases, multiple sample handling, visual chromatogram, simultaneous processing of standards, simultaneous scanning in different light sources, disposable layer and usable in all laboratories etc. The uses of this analytical technique in quality evaluation of plant materials include fingerprint profiling for the assessment of chemical constituents, identification of adulterants/substitutes and quantitative estimation of bio-markers in plant drugs.

Quality Evaluation & Identification of adulterants/substitutes using TLC/HPTLC
In pharmacopoeias (i.e. Indian Pharmacopoeia; Unani Pharmacopoeia of India & Ayurvedic Pharmacopoeia of India) the Rf values of general TLC profiles have been included as minimum requirement of quality standards. However, TLC/HPTLC is being used extensively in the recent years for fingerprinting of medicinal plants as quality control markers, identification of adulterants/substitutes and batch to batch consistency of the products (Khatoon et al., 2005, 2010, 2011, 2014; Singh et al., 2009; Gupta et al., 2011). Examples:

i. Bach
Bach consists of dried rhizome of Acorus calamus L.; Fam. Araceae. Major constituent is asarone. Samples were collected from Lucknow, Dehradun and also procured from Delhi market. Applied the test and reference solutions (10?l) at 4 different tracks on a precoated silica gel G plate 60 F254 of uniform thickness (0.2mm). The plate was developed in the solvent system- toluene : ethyl acetate (9 : 1), to a distance of 9 cm and scanned densitometrically at 254 nm. Asaron (at Rf 0.56) ranges from 0.54 – 1.16 % (Govindarajan et al., 2003).

ii. ‘Khulanjan’
Khulanjan consists of dried rhizome of Alpinia galanga Willd. (Fam. Zingiberaceae). Another species A. officinarum is also known as „Khulanjan?. The samples of A. galanga were collected from Dehradun, Jammu & Tirunelveli. TLC of the methanolic extract on precoated silica gel G plates using toluene : ethyl acetate : methanol (80 : 20 : 0.4) shows under UV (366 nm) blue fluorescent zones of yellow, green and blue at Rf. 0.15, 0.25, 0.69 respectively. After derivatization with anisaldehyde- sulphuric acid reagent 10 minutes at 1200C, spots appear at Rf. 0.15 (greyish green), 0.35 (violet), 0.48 (greyish green), 0.63 (greyish green), 0.69 (green) and 0.91 (violet). The TLC profile of A. officinarum showed additional spot at Rf 0.31 (yellow) under UV 366 nm and at Rf 0.31 (dark blue), 0.51 (orange), 0.57, 0.59 (both dark blue), 0.63, 0.78 (both pink) and 0.90 (brown) after derivatization (Figure 4).

Figure 4. TLC fingerprint of Khulanjan.Samples- AO- A. officinarum 1-3. A. galanga. 1.Dehradun; 2. Jammu; 3. Tirunelveli .
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iii. Resha Khatmi
Roots of Althaea officinalis are official drug „Resha Khatmi? but the roots of Alcea rosea syn. Althaea rosea are being mixed with this drug. Sample of Althaea officinalis was collected from Srinagar while roots of Alcea rosea were collected from Lucknow. TLC of the methanolic extract on precoated silica gel G plates (0.2 mm thick) using toluene : ethyl acetate : methanol (80 : 20 : 0.05) shows common as well as differentiating bands i.e. at Rf 0.24 and 0.73 (both blue) under UV 366 nm and blackish blue at Rf 0.59 and 0.69 under visible light after derivatization in both the species. Additional orange bands at Rf 0.11, 0.13, 0.37 and 0.41 were only visible in Althaea officinalis (Khatoon et al., 2008a).

iv. Phyllanthus sps.
P. amarus, P. fraternus Webster. and P. maderaspatensis can be identified by their TLC profile even in extract form. TLC fingerprints were developed in a solvent system – toluene : ethyl acetate (8.5 : 1.5) and visualized the plate after derivatization with anisaldehyde sulphuric acid reagent. TLC finger print profiles of P. amarus and P. fraternus were almost similar. However, an additional band of bright blue colour at Rf 0.90 was observed only in P. fraternus and the bio-markers- phyllanthin and hypophyllanthin were detected only in P. amarus. In P. maderaspatensis only two bands at Rf 0.28 and 0.38 were observed (Khatoon et al., 2006b).

iv. Terminallia sps.
Barks of Terminalia species are being used as cardiac tonic, diuretic. These are widely used as important timber yielding plants and are rich source of tannins and dyes. The stem bark (waste of timber industry) can be exploited for important polyphenols- ellagic acid and gallic acid, which have potent antioxidant potentials. HPTLC markers and a method for quantification of ellagic acid and gallic acid in seven Terminalia species viz T. arjuna (Roxb.) Wight & Arn, T. bellirica Roxb. , T. bialata Steud., T. catappa Linn., T. chebula Retz.., T. manni King and T. tomentosa W. & A. Prodr., have been developed. The study showed that common as well as distinguishing bands were observed for all seven Terminalia species in UV light at ?= 254 nm and 366 nm and after derivatization with anisaldehyde sulphuric acid reagent. The percentage of ellagic acid and gallic acid varies from species to species. The maximum concentration of ellagic acid was found in T. chebula and that of gallic acid in T. catappa i e. 2.69% and 1.04% respectively (Khatoon et al., 2008b).

vi. Effect of microbial load on glycyrrhizin of Glycyrrhiza glabra L.
Glycyrrhiza glabra, commonly known as „Asl-us-soos?, is an age old plant used to cure varieties of ailments from simple cough to hepatitis more complex like Severe Acute Respiratory Syndrome (SARS) and cancer. Microbial contamination in the crude drug may occur through handling by personnel. The presence of E. coli, Salmonella spp. and moulds indicate poor quality. It was observed that the concentration of glycyrrhizin (active constituent) decreases by microbial contamination. The TLC plate was developed in the solvent system chloroform: acetic acid: methanol: water (6 : 3.2 : 1.2 : 0.8). The band of glycyrrhizin was observed at Rf 0.72 under UV 254 nm which converted purplish blue colour after derivatization with anisaldehyde-sulphuric acid reagent (Agarwal et al., 2014).

vii. Effect of seasons & dioecy on bio-markers of Tinospora cordifolia (Thunb.) Miers
T. cordifolia (family-Menispermaceae) is a dioecious creeper, commonly known as „Gilo?. Male and female plants of T. cordifolia were collected in the months of January, April, June, August and October for studying seasonal as well as dioecy variation. The HPTLC was developed with chloroform: methanol: water (8: 2: 0.2) as mobile phase. Densitometric scanning at 220 nm for tinosporaside and at 320 nm for berberine was evaluated as shown in Figure 5. The biomarkers- tinosporaside and berberine reached in their highest concentration in monsoon season and female samples showed significantly higher concentration of aforesaid bio-markers. (Choudhry et al. 2014).

Figure 5. HPTLC Densitometric fingerprint profile of methanolic extracts of Tinospora cordifolia stem collected in different seasons along with biomarkers. 1, 3, 5, 7, 9: male samples; 2, 4, 6, 8, 10: female samples (1-2: January; 3-4: April; 5-6: June; 7-8: August; 9-10: October); Ber: Berberine; Tin: Tinosporaside .
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WHO has developed a series of technical guidelines and documents relating to the safety and quality assurance of medicinal plants and herbal drugs. These include, Guidelines on good agricultural and collection practices (GACP) for medicinal plants (Anonymous 2003), Quality control methods for medicinal plant (Anonymous 1998) and Guidelines on good manufacturing practices (GMP) for herbal medicines (Anonymous 2007). From the ongoing studies it can be concluded that the macro-microscopy and planar chromatography are the primary and important tools for quality evaluation and identification of adulterants/substitutes of Unani drugs. The concentration of therapeutically active phytoconstituents can easily be assessed by using TLC/HPTLC. These parameters not only are utilized by students and academicians but also by pharmaceutical companies for proper quality of Unani drugs and batch to batch consistency of their compound formulations.


Author is thankful to the Director, CSIR-National Botanical Research Institute, Lucknow, India for providing the facilities and Dr. Shanta Mehrotra (Ex-Head) & Dr. AKS Rawat, Head Pharmacognosy and Ethnopharmacology Division for encouragement throughout the work.


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