Journal of Medicinal Plants Research Vol. 4(15), pp. 1584-1593, 4 August, 2010 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2010 Academic Journals Full Length Research Paper

Authentication of misidentified crude herbal drugs marketed in Pakistan

Muhammad Zafar1, Mir Ajab Khan1, Mushtaq Ahmad1*, Shazia Sultana1, Rahmatullah Qureshi2

and Rasool Bakhsh Tareen3

1Department of Plant Sciences, Quaid-i-Azam University Islamabad Pakistan. 2Department of Botany, PMAS Arid Agriculture University, Murree Road Rawalpindi, Pakistan

3Department of Botany, University of Balochistan, Pakistan

Accepted 22 June, 2010

The main aim of this paper is to use taxonomic and chemical method for authentication of a traditional herbal drug locally known as “Kiandari”. This herbal drug is commonly traded at herbal shops of Indo-Pak subcontinent region. The genuine botanical source of this drug is controversial throughout the region. Four different species; Argemone mexicana L., Cnicus benedictus L., Fagonia indica Burm. and Solanum suratense Burm. f. are found in herbal shops under the same trade name Kiandari. In this study morpho-palynological scanning electron microscopy (SEM) analysis and chemotaxonomic thin layer chromatographic (TLC), ultra-violet (UV) and IR analysis markers were used to differentiate herbal raw material with resolution of botanical identity and origin of correct species. Compared with other characters, chemotaxonomic markers are more robust, accurate, reproducible and reliable source for authentication of genuine herbal drugs and their adulteration. Key words: Pakistan, authentication, misidentified, crude herbs, market.

INTRODUCTION Plants have been used for medicinal purposes for many centuries. Today, herbal medicines are being employed worldwide in a variety of health care systems and as home remedies. In developing countries, communities rely heavily on traditional herbal medicines in order to meet their primary health care needs. In many industri- alized countries herbal medicines are gaining popularity as alternative and complementary therapies (Ekinic et al., 2004).

In the indigenous system of medicine, the plants in crude form, either fresh or dried are utilized for their cura-tive effects against a variety of mankind’s ailments. Due to some morphological similarities and lack of correct identification, the crude drugs are often adulterated or substituted in commerce which obviously results in the loss of drug efficacy. Correct identification of herbal drug is the foundation of the safe use of plant based natural health products. Without proper identification as a starting point, the safe use of quality products cannot be guaranteed. There is recognition within industry and *Corresponding author. E-mail: catlacatla@hotmail.com, mushtaq@qau.edu.pk

government that there is a need to protect access and choice by consumers when it comes to natural health products. At the same time, consumers have a right to expect that these products can be used with confidence regarding their safety and quality (Ahmad et al., 2009).

Dried products sold in the market are generally difficult to identify, as many useful diagnostic characters are lost during drying. At the same time other numerous problems are confronted to taxonomists in the identification of traded herbal drugs. The existence of several common names for the same plant species in different areas may confuse end users for selection and utilization of genuine drug. Another problem is superficial resemblance of plant species within the same tribe or family (Khan et al., 1996). Problem of adulteration in medicinal plants arose due to the potential use of different species for similar ailments (Shinwari et al., 2002).

Due to the nature of their utilization, in the production of medicinal plants either cultivated or wild crafted origin, quality has always been a focal issue. In this regard, histological and morphological inspections have been the usual methods for authentication, but they are not applicable to most forms of modern herbal drugs, e.g. herbal raw material, extracts and pills. Chemical and

chromatographic techniques are currently used for iden-tification of herbal drugs marketed among various countries and regions. Chromatographic fingerprints (TLC and HPLC, GC etc.) are used as reference standards which indicate purity, identity and quality of herbal drug. A standard and effective quality assurance program for herbal drugs is necessary for their standardization and acceptance by world market. In this paper an effort was made to authenticate the genuine source of herbal drug “Kiandari” by using morpho-palynological and chromato- graphic fingerprints thin layer chromatographic (TLC). MATERIALS AND METHODS Morphological characters Four medicinal plant species were collected during field visits. Crude raw material of herbal drugs was procured from herbal markets of Akbari Mandi Lahore and Peshawar herbal shops. The morphological and organoleptography of plant species and herbal parts were carried out by using light microscope (Koywa SZF 0.75 x -3.4x). The morphological characters were reconfirmed by using various Floras (Hooker, 1875; Tutin and Heywood, 1972; Hooker and K.C.S.I., 1885ab; 1894; Nasir and Ali, 1974, 1975). Palynological features Pollen features were studies by using light microscope Meiji (MX 5200H, Japan). Qualitative characters includes type of pollen, shape of polar view, shape in equatorial view, sculpturing as quantitative character including polar diameter, equatorial diameter, P/E ratio, length and width of colpi, exine thickness, fertile and sterile pollen. Each value was taken five times to ensure the accuracy. Scanning electron microscopy (SEM) analysis were carried out by the dissection of anther and then placement in the center of clean glass slide with 1 - 2 drops of acetic acid for one minute, crushed to release pollen. Then the pollen were transferred to already marked specimen stub and allow them to air dried and then coated with gold with a SPI-MODELTM sputter coater. After coating, stubs were placed in Jeol Vacuum evaporator. It takes about 15 min to produce the vacuum and then observations were made by using 30 KV scanning electron microscope (JSM5910, JEOL Japan). Pollen terminology was determined by with Ronald (2000). Leaf epidermal anatomy The fresh leaves were taken in a test tube covered with 4 ml of concentrated nitric acid, to which 0.2 gm of potassium chloride and 0.1 ml of distilled water was added then mixture was carefully boil and after a few second as soon as the epidermis of leaves were separated in the form of thin pellicle, the contents were emptied into a Petri dish partly filled with water. Macerated leaves were washed with water for 2 - 3 times then placed in Petri dish containing fresh water. To prepare adaxial surface, the leaf was placed in such a way that it is adaxial side of the leaf with the help of fine forceps. Same procedure was followed for the preparation of adaxial surface of leaf. Leaf epidermal samples were prepared according to the methods of Cotton (1974) and Clark (1960). Phytochemical study The plant materials (4 species) were dried under shade and were

Zafar et al. 1585 made into small pieces. Thin layer chromatographic (TLC) studies were performed for flavonoids (Chemotaxonomic markers) finger printing. Commercially available pre-coated polyamide-6 TLC plates (Sorbent Technologies USA) were used. The modified method was applied for flavonoids extraction. TLC plates were observed and photographed under ultra-violet (UV) lamp (Model UVL-56 Black Ray USA) (Ahmad et al., 2010). RESULTS The result of this work illustrated in Tables 1 to 4 and Plates 1 to 4. DISCUSSION The need for quality control of Unani (Greek) medicines has attracted attention in recent times but most people seems to forget that without standards in plant sources of drugs, we can not evaluate standard in Unani medicines. This long standing problem of accurate determination of drug sources is yet to receive adequate attention of Unani practioners and researchers (Ahmad et al., 2008). The Unani medicinal plants are generally collected by professionals who may not be botanists or taxonomists. Similarly the identity of crude drug purchased from the market which is based on trade or vernacular name is taken for granted, without subjecting the plant material for stringent method of botanical identification (Ahmed et al., 2005).

It is equally possible that one may not get the desired plant as the trader may supply other plant having trade or vernacular name. In addition to nomenclatural ambiguity, the traditional and Unani drugs sold in the Pakistani market are adulterated, sophisticated or substituted by quite unrelated plant materials (Ahmad et al., 2009). Similarly herbal drug with vernacular name “Kandiari” is marketed in Pakistan with many but quite different botanical materials. This vernacular name is applied to more than one plant species. In this study under the name of Kandiari four different plant species belonging to different families were procured from market. After appli-cation of chemotaxonomic markers, it was found very interesting that all 4 species were taxonomically quite distinct. Phytochemical constitutents and their phytothe-rapeutic uses were found to be varies from area to area. In case of Argemone mexicana the pollen are tri-tetracolporate, leaf epidermal cells are with undulating wall, amphianisocytic stomata and presence of trichomes and mircrohairs. Phy- totherapeutic uses are diuretic, purgative and destroy worms. TLC of aerial parts shows the presence of two major amounts of phenolic acids and one chalcones (Plate 1). In case of Cnicus benedictus the pollen are echinate, tri-tetracolporate. Leaf epidermal characteristics are undulating wall, amphianisocytic stomata and presence of trichomes and mircrohairs. Phytotherapeutic uses are purgative, ophthalmic and anti intestinal worms. TLC shows the presence of three

1586 J. Med. Plant. Res. Table 1. Kandiari case, A. mexicana L. English name Mexican prickly poppy Local name (s) Kandiari, Peeli Bindiari Drug name Satia Nasi Family Papaveraceae Distribution in Pakistan and World In Pakistan; Malir, Amir Laki, Hyderabad, Rawalpindi, Kalar Syedian, Thatta, Gujranwalam,

Pasrur, Multan and Azad Jammu Kashmir. In world; It is native of Tropical America, North America, India and Pakistan.

Occurrence Rarely distributed Morphology It is a prickly, glabrous, branching herb with yellow juice and showy yellow flowers. The height

of this plant varies between 0.3 to 0.12 meters, Leaves are thistle like measurement. Stem clasping, Oblong, sinuately pinnatifid, spinous and viens are white. Flowers are terminal, yellow and of 2.5–5.0 cm diameter. Fruits are capsule. Prickly and oblong ovoid. Seeds numerous, globose, netted and brownish black (Plate 1 A).

Flowering period May to September (Yellow Flower) Voucher No. 15 Pollen description Pollen is tri-tetracolporate. Shape in polar view is square to rectangular. Polar diameter is 29.7

µm (27.5 - 32.5 µm). Length and width of colpi is 5.35 µm (2.5 - 7.5 µm) and 3.9 µm (2.5 - 5 µm) respectively. Exine thickness is 2.5 µm (1.2 - 5 µm) (Plate 1 C and D)

Leaf epidermal anatomy Types of epidermal cells are long with less undulating walls. Diameter of epidermal cell is 95.5 µm (62.5 - 129.17 µm) x 34.75 µm (41.76 - 45.58 µm). Length of stomatal complex is 50.84 µm (45 - 62.5 µm). Type of stomata is amphianisocytic. Diameter of stomata is 21.45 µm (20 - 22.9 µm) x 8.75 µm (6.67 - 8.34 µm). Trichomes and microhairs are also present. The diameter of trichome is 150 x 25 µm (Plate 1 E).

Part used Roots, leaves, seeds and yellow juice Phytotherapeutic uses Diuretic, purgative and destroys worms. It cures lepsory, skin-diseases, inflammations and

bilious fevers. Roots are anthelmintic. Juice is used to cure ophthalmia and opacity of cornea. Seeds are purgative and sedative. Seeds resemble mustard seeds and it is used to adulterate with mustard seed.

Toxicity Seed yield non edible toxic oil and causes lethal dropsy when used with mustard oil for cooking. The plant is toxic to animals and cattle avoid grazing this plant.

Organoleptography (Aerial parts) Branches, leaves and flowers are mixed. Branches ranges from 0-11 cm. Spines are present, light green in color, smooth. Ligule is present. Leaves light green, spines are present on edges/margin. Flowers are yellow (Plate 1 B).

TLC fingerprinting TLC of aerial parts shows the presence of two major amounts of phenolic acids and one chalcones (Plate 1 F).

Table 2.Cnicus benedictus L., Syn: Carduus benedictus Steud., Carbenia benedicta Adans. English name Blessed thistle or Holy thistle Local name (s) Kindiari, Tarili Booti Drug name Unknown Family Asteraceae Distribution in Pakistan and World

In Pakistan; Director, Chitral, Drosh, Swat, Khawaja Khela and Shahderi, Attock, Campbellpur. In world; Native to the Mediterranean region, from Portugal north to southern France and east to Iran. S. Europe to W. Asia. An infrequent casual in Britain.

Occurrence Common in the field of wheat Morphology Annuals, to 60 cm. Stems often spreading or prostrate, usually branched throughout, usually

reddish, loosely tomentose. Leaves mostly cauline, sessile and often short-decurrent or proximal tapering to winged petioles, blades lanceolate to oblanceolate, 6 - 25 cm, margins coarsely dentate or pinnately lobed, lobes and teeth armed with short, weak spines, faces sparsely to densely hairy with jointed multicellular hairs and slender cobwebby hairs, resin-gland-dotted. Heads disciform, borne singly, sessile, each subtended by involucre-like cluster of leaf-like bracts. Involucres spheric, 20–40 mm. Phyllaries in several series, tightly overlapping, outer ovate with tightly appressed bases and spreading spine tips, inner lanceolate, tipped by pinnately divided spines more than 5 mm. Florets many; corollas yellow, those of sterile florets linear, 3-lobed, not exceeding disc corollas, very slender, those of disc florets 19 - 24 mm (Plate 2 A)

Zafar et al. 1587 Table 2. Cont’d.

Flowering period March to May Voucher No. 72 Pollen description Pollen is monad, tricolporate and echinate. Shape of pollen in polar and equatorial view is

circular. Polar diameter is 19.7 µm (15 - 20.5 µm). Equatorial diameter is 19 µm (16.5 - 20 µm). P/E ratio is 1.03 µm. The length of spine is 2.3 µm (2 - 2.5 µm). Exine thickness is 1.2 µm (1 - 1.5 µm) (Plate 2 C and D).

Leaf epidermal anatomy Epidermal cells are some what rectangular to hexagonal and polyhedral with smooth walls on adaxial surface. The abaxial surface is irregular with undulating walls. Size of epidermal cells is 60.62 µm (37.5-83.75 µm). Length of stomatal complex is 38.21 µm (30 - 52.5 µm). Types of stomata are amphianisocytic and staurocytic. Diameter of stomata is 13.125 µm (12.5 - 15 µm) x 4.375 µm (3.75 - 5 µm) (Plate 2 E).

Part used Leaves and leafy flowering tops Phytotherapeutic uses Promote lactation, digestive complaints, Astringent; Bitter; Cholagogue; Contraceptive;

Diaphoretic; Diuretic; Emetic; Emmenagogue; Galactogogue; Homeopathy; Stimulant; Stomachic; Tonic. The whole plant was infused overnight in cold water and the liquid drink three times daily in the treatment. Men were required to run after each dose in order to encourage sweating.

Toxicity The treatment often caused nausea and vomiting-excessive doses of the plant cause vomiting. The plant is used externally in the treatment of wounds and ulcers.

Organoleptography (Aerial Parts)

Branches are smooth, hairy with cylindrical lines. Branches are 3 - 5 cm in length. Leaves are hairy and leathery. Spines are present on leaves. Leaves are broad and dissected. Flower is yellow green produced in a dense flower head (Capitulum) surrounded by numerous spiny basal bracts. Leaves are amplexicaul (Plate 2 B).

TLC fingerprinting TLC of aerial parts shows the presence of three phenolic acids and one flavonol and one chalcones (Plate 2 F)

Table 3. Fagonia indica Burm.

English name Southern Cyprus Local name Dhamaya Drug name Dhamasa Family Zygophyllaceae Distribution in Pakistan and World In Pakistan; Found in plain areas of Punjab, Karachi, Sind, Attock, Haripur, Mianwali,

Jhelum, Salt Range, Mirpur and common on dry, bare, stony and sandy soils. In world; Indo-Pakistan subcontinent westwards to North and East tropical Africa in arid and semi-arid regions.

Occurrence Common in arid soils. Morphology Annual to perennial, covered with whitish pruinose or sessile glands or glabrous

shrublet. . Leaves mostly unifoliolate or basal ones trifoliolate and upper unifoliolate, leaflets linear-oblong or lanceolate, 6-35 mm long, 3-4 mm broad, mucronate, short to long petioled or sessile; stipular spines awl shaped, patent to ascending, equal to shorter than leaves, occasionally deficient or minute. Stem basally somewhat woody, branches procumbent or erect, cylindrical, striate, internodes 2.5-5 cm longFlowers mediocre, 1.2 cm across, pinkish-purple; pedicel 4-6 mm long. Sepals ovate, 3-4 mm long, 1.5 mm broad, glandular outside, acute, persistent. Petals spathulate, 6 mm long, 3 mm broad, obtuse. Stamens with 6 mm long filaments. Capsule 3-4 mm long and broad, pubescent, pedicel equal to about twice as long as fruit (Plate 3 A).

Flowering period September to January Voucher No. 124 Pollen description Pollen type is psilate, tricolpate. Shape in paler view is circular and in equatorial is

spheroidal to prolate to apple shaped. Polar diameter is 26.2 µm (25 - 27.5) while equatorial is 24.12 µm (22.5 - 32.5 µm). P\E ratio is 3. Length and width of colpi is 2.5 µm both. Exine thickness is 1.25 µm. Percentage fertility is 80.48. (Plate 38.2 and 38.3) (Plate 3 C and D)

1588 J. Med. Plant. Res.

Table 3. Cont’d. Leaf epidermal anatomy Types of epidermal cells are tetra, penta to polygonal and irregular to less undulating.

Diameter of epidermal cell is 48.335 µm (33.34 - 63.34 µm). Length of stomatal complex is 46.25 µm (35 - 55 µm). Type of stomata is staurocytic. Diameter of stomata is 13.34 µm (12.5 - 15 µm) x 5.417 µm (5 - 6.25 µm). Both sides of the leaf are same. (Plate 38.4 and 38.5). (Plate 3 E)

Part used Aerial parts Phytotherapeutic uses Fever, hepatitis, itching, pimples, monorrhagia, skin diseases, tonic for liver and

stomach, blood purification, cough and diabetes. The decoction of the whole plant (half Kg) mixed with 100 gm sugar is recommended for the control of monorrhagia. One teaspoon thrice a day of the whole plant extract is given for blood purification, diabetes and skin diseases. Half Kg of the whole plant is boiled in 2 liters of water. Patients with hepatitis are advised to take bath with this decoction. This prescription is also used for swelling of body, undiagnosed fever which damages kidney and liver. It is also used for itching and pimples.

Toxicity Non toxic. Organoleptography (Aerial Parts) Herbal drug consist of dried aerial parts. Color of drug is green and light brown.

Branches, leaves and flowers are mixed. Spines on branches are present. Branches are 0.4 - 1.5 cm in length. Braches are with cylindrical lines. Spine length is 0.4 - 0.8 cm in length. Flower is spiny and globular, ligule present. Flower is greenish brown in color (Plate 3 A).

TLC fingerprinting TLC of aerial parts shows the presence of two major amount of flavonols and one phenolic acid (Plate 3 F)

Table 4. Solanum suratense Burm. f., Syn: Solanum xanthocarpum Schrad and Wendl., Solanum virginianum L., Solanum jacquini Willd.

English name Yellow berried nightshade

Local name (s) Mohakari, Kandiari, Jangli Bangan

Drug name Mohakari

Family Solanaceae

Distribution in Pakistan and world Rawalpindi, Attock, Islamabad, Mianwali, Jehelum, Lahore, Talagnag, Bhakkar, Khushab, D.I. Khan, Mardan, Kohat, Sind

Distribution in world

Occurrence Very common in waste places and arid zones

Morphology It is a very prickly perennial herb somewhat with woody base. Stem branched much and younger ones clothed with dense, stellate and tomentose hairs. Prickles are compressed straight, glabrous and shining, often 1 to 3 cm long. Leaves ovate or elliptic, sinuate or subpinnatifid, obtuse or subacute, stellately hairy on both sides, armed on the midrib and often on the nerves with long yellow sharp prickles. Petiole is long, stellately hairy and prickly. Flowers are in cymes or some times reduced as solitary. Calyx tube is short, globose and lobes linear-lanceolate, acute, densely hairy and prickly. Corolla purple, lobes deltoid, acute, and hairy outside. Anther filament is long, glabrous and anthers open by a pore. Ovary is ovoid and glabrous. Berry yellow, green-blotched and sorrounded by enlarged calyx. Seeds are glabrous (Plate 4 A).

Flowering period August to March

Voucher No. 74

Pollen description Pollen type is psilate, tricolpate. Shape in polar view is semi-angular to inter semi-angular and in equatorial is prolate to perprolate to apple shaped. Polar diameter is 18.34 µm (17.5 - 22.5 µm) while equatorial is 17.62 µm (15 - 20 µm). P\E ratio is 20. Length of colpi is 4 µm (2.5 - 5 µm) and its width is 2.5 µm. Exine thickness is 1.15 µm (0.25 - 1.25 µm). Percentage fertility is 90.91. (Plate 4 C and D)

Zafar et al. 1589

Table 4. Cont’d.

Leaf epidermal anatomy Types of epidermal cells are polygonal. Diameter of epidermal cell is 30.4165 µm (19.167 - 41.67 µm) x 17.9165 µm (14.165 - 19.167 µm). Length of stomatal complex is 30 µm (27.5 - 37.5 µm). Type of stomata is amphianisocytic. Diameter of stomata is 14 µm (12.5-15 µm) x 3.75 µm (2.5 - 5 µm). Type of trichome is unicellular and stellate having arms all around. Lengths of its arms are 196.25 µm (167.5 - 225 µm) (Plate 4 E).

Part used Fruits, whole plant Phytotherapeutic Uses Anticancer (fruit and plant); alleviates spasms, stimulates the cardiovascular system and

antiarrhythmic (fruit); antiviral and spermicidal (plant); expectorant and carminative (plant). Asthma, cough, bronchspasm, sore throat, constipation, an effective expectorant and diuretic. 200 g plant material is boiled in 1 liter of water daily for 20-25 min. 5-6 black pepper and 2-3 teaspoonful (20-25 g) common salt is also added. When ½ liter of water remains, then it is filtered by cloth or filtration pot. This decoction is given to the cattle and patient suffering from indigestion, fever, cough, stomach disorder, asthma and to improve hunger. For Children (5 - 15 years), ½ cup (60 ml) of decoction (at one time) is given with bread (2-3 times) per day for 6 - 7 days. For Adults (16 - 50 years), 1 - 2 cups (150 ml) of decoction (at one time) is given with bread (2 - 3 times) per day for 8-10 days. For Cattle: 1 glass (250 ml) of decoction if given (at one time) two times (Morning – Evening) per day for 4 - 5 days.

Toxicity None Organoleptography (Aerial parts) Crude drug consist of aerial parts. Branches are green or brown in color. Branches are

1.5 - 6 cm in length. Star shaped structures of unequal size on branches. Leaves are dissected and spiny. Spines are yellowish. Branches are hairy. Flower is globular, brown in color. Irregular ridges are present on branches. Epicalyx is present. Branches, leaves and flowers are mixed (Plate 4 A).

TLC Fingerprinting TLC of aerial parts shows the presence of one major flavones and two minor phenolic acids (Plate 4 F).

Plate 1. A- Argemone mexicana, B- Aerial Parts, C- SEM of pollen polar view, D- SEM of pollen sculpturing, E- Stomata in anatomy section, F- TLC finger prints of aerial parts.

1590 J. Med. Plant. Res.

Plate 2. A- Cnicus benedictus, B- Aerial parts, C- SEM of pollen polar view, D- SEM of pollen equatorial View, E- Stomata in anatomy section, F- TLC finger prints of aerial parts.

phenolic acids and one flavonol and one chalcones (Plate 2). In case of Fagonia indica the pollen are psilate and tricolporate. Leaf epidermal characteristics are tetra, penta to polygonal and irregular to less undulating cells, staurocytic stomata are present. Phytotherapeutic uses are anti fever, hepatitis, itching, pimples and monorrha-gia. TLC of aerial parts shows the presence of two major

amounts of flavonols and one phenolic acid (Plate 3). In case of Solanum suratense the pollen are monad, echinate and tricolporate. Leaf epidermal cells are rectangular to hexagonal. Amphianisocytic stomata are present. Phytotherapeutic uses are astringent, diuretic and emetic. TLC of aerial parts shows the presence of one major flavones and two minor phenolic acids (Plate 4).

Zafar et al. 1591

Plate 3. A- Fagonia indica, B- Aerial Parts, C- SEM of pollen polar view, D- SEM of pollen equatorial view, E- Stomata, F- TLC finger prints of aerial parts.

Conclusion In this study, we have therefore attempted to compile data based on chemotaxonomic studies to determine the

authenticity of a particular folk herbal drug. Based on chemotaxonomic features and phototherapeutic uses, the four species are quite distinct but confused in market due to vernacular / trade name “Kiandiar”. In view of globalize

1592 J. Med. Plant. Res.

Plate 4. A- Solanum suratense, B- Aerial parts, C- SEM of pollen polar view, D- SEM of pollen sculpturing, E- Trichomes, F- TLC finger prints of aerial parts.

character of herbal drugs trade and utilization; it should be in the interest of all stallholders to harmonize the

individual approaches and to agree upon internationally acceptable minimum standards.

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