Chemotaxonomic clarification of pharmaceutically important species of Cyperus L .

The evaluation of the crude herbal drug which eventually enters the pharmaceutical market is obviously of considerable importance. This operation involves the identification of the material and determination of its quality, purity and of adulterated nature of the adulterants. The present paper is based on the above objectives which confined to chemotaxonomic authentication of Cyprus rotundus L. (Nagar mootha) and its other similar species. Chemotaxonomic techniques including morphology, organoleptography, palynology, anatomy and phyto-chemical analysis were carried out in order to clarify the pharmaceutically important species of Cyperus that is Cyperus rotundus, Cyperus alopecuroides, Cyperus difformis and Cyperus niveus. It is concluded from this study that the genuine source of herbal drug Nagar Mootha is C. rotundus instead of other species. Such type of studies is the need of herbal industry to ensure validation process which applied in the manufacturing of herbal medicines and phytopharmaceuticals. This will provide the credibility in the regulation and grown of pharmaceutically important herbal medicines for the foreseeable.


INTRODUCTION
In several industrialized societies, plant-derived prescription drugs constitute an element in the maintenance of health.Medicinal plants are an integral component of research developments in the pharmaceutical industry.Such research focuses on the isolation and direct use of active medicinal constituents, or on the development of semi synthetic drugs, or still again on the active screening of natural products to yield synthetic pharmacologically-active compounds (Hoareau and Dasilva, 1999).In Germany, for example, over 1500 plant species encountered in some 200 families and 800 genera have *Corresponding author.E-mail: catlacatla@hotmail.com.been processed into medicinal products.In South Africa, likewise, some 500 species are com-mercialized trade products.Today, Bulgaria, Germany and Poland are recognized as major exporters of plant-based medicinal products.The development and com-mercialization of medicinal plant-based bio-industries in the developing countries is dependent upon the availa-bility of facilities and information concerning upstream and downstream bioprocess, extraction, purification, and marketing of the industrial potential of medicinal plants.
Absence of such infrastructure compounded by lack of governmental interest and financial support restricts the evolution of traditional herbal extracts into authenticated market products.Furthermore the absence of modernized socio-economic and public healthcare systems reinforces reliance of rural and lower-income urban populations on the use of traditional medicinal herbs and plants as complementary aids to routine pharmaceutical market products.The prophylactic and therapeutic effects of plant foods and extracts in reducing cardiovascular disease has been reviewed (Walker, 1996).
At the same time, accurate plant identification is the foundation of the safe use of plant based natural health products in pharmaceutical sciences.Without proper identification as a starting point, the safe use of quality products cannot be guaranteed.There is recognition within pharmaceutical industry and 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;Zafar et al., 2010).Authentication of pharmaceutical products originated from traditional herbal medicines would facilities accurate documentation of taxa traded, medicinal usage and assists in identifying material implicated in poisoning cases.Dried products sold in the medicinal plant trade are generally difficult to identify, as many useful diagnostic characters are lost through desiccation.Many herbs are sold through brokers where the material can change hands several times.The originality of herbal drugs in terms of raw material extraction, preparation and marketability requires proper guide lines according to WHO standards (WHO, 2003).
Lack of authentic sources of herbal drug is one of the common example and important issue in pharmaceutical industry.It is well known that in course of time, drug materials get changed to or substituted with other plant species.Nagar mootha is recent day examples in this regard.On discussion with herbal drug suppliers, herbalists and local medicinal plant collectors, it came to be known that in the past, roots of this herbal drug was obtained from different species of Cyperus.But now a day due to lesser availability in field these drugs are sold in market with quite similar roots from different species.Due to lack of proper documentation of genuine drug and research is this field the situation to know authentic drug is complex.
The overall objectives of the present project is to use classical and modern techniques of chemotaxonomy to authenticate the original raw material of correct species marketed at herbal shops.This quality assessment mode is effective for their standardization, modernization and acceptance by the world market.

Morphological characters
Four medicinal plant species that is, Cyperus rotundus, Cyperus alopecuroides, Cyperus difformis and Cyperus niveus 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 andK.C.S.I., 1885 ab, 1894;Nasir and Ali, 1974;1975).

Palynological features
The pollen study was carried out under the Meiji light microscope (MX 5200H, Japan).Qualitative characters includes type of pollen, shape of polar view, shape in equatorial view, sculpturing whereas 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 to 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-MODEL TM 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 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 g 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) and modified method of Ahmad et al. (2010).

Phytochemical study
The plant materials (4 species) were dried under shade and were made into small pieces.Thin layer chromatographic (TLC) studies were performed for flavonoids (Chemotaxonomic markers) finger printing by adopting the method of Ahmad et al. (2010) and Hassan et al. (2007).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 UV lamp (Model UVL-56 Black Ray USA).

RESULTS AND DISCUSSION
The English names, local names, drug names and families of different species and their distribution, occurrence, morphology, etc were given in Tables 1 to 4, while the species parts and characteristics are given in Figures 1 to 4.
Cyperus L. (Sedge) belongs to the family Cyperaceae

Flowering period April to October
Voucher No. 261

Leaf epidermal anatomy
Abaxial and adaxial surfaces with costal and intercostals zonation.Long-cells in costal and intercostals zones rectangular, with markedly sinuous walls.Whole plant including roots (rhizomes) is dried in shade and crushed to obtain powder.Equal quality of water is mixed in powder to make paste.The paste is externally applied on infected skin to cure eczema, scabies and chronic skin sculpturing.The paste is also applied on skin for softness and cooling effects.

Toxicity
It has no toxic effect when consumed in normal dosage.

Organoleptography (Roots)
Root sample contain irregular pieces of length 0.5 to 10 cm and diameter of 0.3 cm to 2.8 cm.root surface has woody dark brown color with vertical and horizontal ridges on surface.Root surface also have tiny hairy projections.Root is branched with some nodded regions.Root is slightly soft than above three root samples.Internally root is light brown (skin) in color.Root is bitter in taste (Figure 1B).
TLC Fingerprinting TLC under UV shows the presence to three minor flavanoles (Figure 1F).

Morphology
Perennial, tufted with short rhizome.Aerial stem up to 140 cm long and 3 to 7 mm diameter, trigonous, smooth.Leaves up to as long as stem; sheaths up to 35 cm, leaf blade up to 60 cm long and up to 10 mm broad, keeled, apex trigonous, scabrous.Inflorescence a compound umbel; involucral bracts up to 5-7, leaf like, up to as long and as wide as leaf, primary branches 8-12; secondary branches up to 5-7, leaf like, up to as long and as wide as leaf, primary branches 8-12; secondary branches up to more than 10, with several foliose bracts; some primary and most secondary branches ending with cluster of spikes, sometimes with small teritry anthelodia; cluster of spikes 2-6 x 1-2 cm, each cluster with 70 spirally arranged spikes; spikes 5-15 x 1.5 mm, compressed; glumes 2 mm, keeled, midnerve strong, greenish, side yellowish or grey, with reddish brown stripes, margins narrowly scarious, stamens 2, stigmas 2 or 3. Nut elliptic to obovate-oval, biconvex, slightly flattened (or obcompressed-trigonous in tricapellary pistil), yellowish brown, finely reticulate or almost smooth (Figure 2A).

Flowering period April to September
Voucher No. 299

Indigenous recipes
Roots are collected, washed and dried in shade.Roots are cut into small pieces and ground to obtain powder.5-10 gm of powder is taken with amla juice (Phyllanthus emblica) in order to cure menstrual cycle, digestive problems and pain in body.½ teaspoon of root powder is taken twice a day for 3-4 days to cure diarrhea.

Organoleptography (roots)
Root length varies from 5 cm to 14 cm and irregular pieces of 0.5 to 3 cm diameter.Externally root is hard and dark brown in color with irregular vertical ridges.Root is branched with swollen nodes.Internally root is comparatively light brown as compared to outer surface.Root is slightly bitter.Root have woody smell (Figure 2B).

Occurrence
Very common in moist and shady places
Flowering period July to December Voucher No. 296
Exine with striate elements.The sculpturing elements less than 1 µm.The granules are uniformly distributed with narrow spaces over the tectum (Figures 3D and E).

Indigenous Recipes
Fresh aerial parts are crushed to obtain paste which externally applies on eyes to reduce redness, pain and for conjunctivitis.The paste is applied for 15-20 days on skin to cure eczema and scabies.The aerial parts are dried in shade and ground to obtain powder.Half teaspoon at morning before breakfast and at night with glass of water is taken for 20-25 days to reduce obesity.

Organoleptography (Roots)
Root length is 4-8 cm and 1 to 3.5 cm in diameter.Root texture is hard; color of surface is dark brown.Ridges and furrows on the root surface.Small prickles on root surface.Internally root is light brown in color.Root has smell like wet soil.Taste of root is slightly bitter (Figure 3B).
TLC fingerprinting TLC under UV shows the presence of two minor flavonls (Figure 3F).

Flowering period April to October
Voucher No. 531

Indigenous recipes
Roots are collected, clean and washed.Roots are dried in shade and ground to obtain powder.5-8 gm of root powder is taken twice a day for a week to cure infection internally.½ teaspoon of powder is taken with water at night time for stimulant, sedative and diuretic.

Toxicity
Excessive use may cause toxicity Organoleptography (roots) Root length varies from 1 cm -5 cm and diameter of root is from 2-5.5 cm.root is globular with both ends slightly pointed.Root surface is dark brown in color.Surface of root is rough and contain small hair.Root is hard in texture.Internally root has comparatively light brown color than external surface.Root is odor less and slightly bitter (Figure 4B).
TLC Fingerprinting TLC under UV shows the presence to two minor flavonols, two minor phenolic acid and one minor aurone (Figure 4F).
which contains medicinally important species used in traditional systems of medicines pharmaceutical industries in Pakistan, India, China and other Asian countries.The herbal drug obtained from Cyperus is commonly called as Nagar Mootha in Urdu, Hindi and Tibb while Nagar Musta in Sanskrit (Bhagwat et al., 2009).Cyperus is known in Chinese as "xiangfu or xiang fuzi" means fragrant and usually applied to strong and pleasant fragrances, such as those occurring in culinary species, perfumes and incenses (Yang, 2002).The character "fu" is the same as that used to describe aconite (Aconitum heterophyllum).
The term was likely to be used because the appearance of the Cyperus rhizomes, the part used, remind herbalists of the aconite roots (Haung and Yuxia, 1993).In much of the rest of the world, the Cyperus is referred as "nut grass" or purple nut sedge (sedge is the term indicating blade like leaves).The nut is the rhizome (or tuber) which forms rounded or elongated roots.

Phytotherapeutic uses
Cyperus is considered by many traditional practitioners to be the best herb used for depression, circulation, skin disorders, digestive complaints etc. (Nadkarni, 1976;Williamsons, 2002).Cyperus is included in dozens of traditional herb formulas.C. rotundus (Nagar mootha) is thought to have originated in Indo-Pak sub continent and these spread the past 2000 years (it first appears in a Chinese medicine book around 500 A.D.).The rhizome is used in Ayurvedic, Unani and Chinese medicines mentioned in the ancient Caraka Samhita (ca. 100 A.D.).Its uses in modern medicines are primarily for treating fevers and digestive system disorders (diarrhea, vomiting and indigestion).It is also known as menagogue to (treat delayed menstruation) and an analgesic use for dysmenorrheal for (painful menstruation) (Nadkarni, 1976).

Problems in authentication
In herbal markets of Indo

Organoleptography and chemotaxonomic differentiation
Morphologically the C. rotundus has slender leaves that are connected to a network of underground rhizomes, roots and tubers (Nishimoto et al., 1998).C. rotundus is  perennial stoloniferous herb, bearing ovoid tubers at the end of stolens (Figure 1A).Rhizomes has dark brown color with vertical and horizontal ridges on surface (Figure 1B).While C. alopecuroides is the perennial, tufted with short rhizome.Externally the rhizome is hard, dark brown color with irregular vertical ridges (Figure 2B).Similarly the C. niveus is perennial herb with woody rhizomes which are globular and dark brown in color (Figure 4B).Gupta et al. (1998) investigated the antiinflammatory activities of oil isolated from the rhizomes of C. scarious and other closely resembling species.Palyno-anatomical features of C. rotundus, C. alopecuroides, C. difformis and C. niveus have quite resemblance to each other and it is difficult to differentiate these species on the basis of microscopic features like pollen and leaf epidermal characters.While it is found that TLC fingerprinting can differentiate the genuine rhizome form its adulterants.TLC under UV shows the presence of three minor flavanoles in the rhizome of C. rotunuds (Figure 1F), while in C. aleopecuroides and C. difformis there are two and three minor flavonoles respectively (Figures 2 and 3F).But C. niveus is quite different which contain two minor flavonoles, two minor phenolic acids and one minor aurone (Figure 3F) indicating that these species of Cyperus can be distinguished due to flavanole pattern.It has been observed during rhizome study that there is a lot of variation in rhizome size which might determine the quality of variant.Further study is required to find the area and soil of the best variant of Nagar Mootha (C.rotundus).It is concluded that the subject of herbal drug standardization is massively wide and deep.There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function.
to apply in skin related ailments; it helps in relieving from itching.It is used in increasing the size of breast.It also improves eyesight and is used in eye related ailments.Powder is used n mental diseases and diseases like psychosis and epilepsy.It improves digestive system.It also helps in maintaining normal body temperature.