PHCOG J ORIGINAL ARTICLE Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Sudhakaran M.V Department of Drug Standardisation, Government Ayurveda College, Thiruvananthapuram -695 001, Kerala, India Submission Date: 12-9-2012; Accepted Date: 28-9-2012 ABSTRACT Introduction: Cissampelos pareira Linn. is a dioecious, perennial, twinning and climbing medicinal shrub belongs to the family Menispermaceae. It is being used as a traditional remedy by native peoples of South America for centuries, to treat women’s ailments, such as for menstrual cramps, prevents threatened miscarriage, ease childbirth, to stop uterine hemorrhages after childbirth, postpartum pain and ibroid tumors. Drug is frequently prescribed for treating cough, abdominal pain and fever according to Ayurvedic Pharmacopeia of India. The genus is highly specialized for the richness of a diverse array of biologically active bisbenzylisoquinoline alkaloids. Objectives: The present study was performed with the objectives to elaborate the macroscopic and histo-morpho diagnostic proile of Cissampelos pareira and to analyze the quantitative, luorescent and powder microscopic peculiarities to support its pharmacobotanical characterization. Materials and Methods: microscopic evaluation, quantitative, luorescence standards of the drug and powder microscopy were carried out using the stem, root and leaves of Cissampelos pareira Linn. Results: Distribution of long uniseriate, bicellular clothing trichomes in the epidermal tissues of the aerial parts, presence of actinodromous venation with small, pentagonal shaped areoles, adaxial irregular and wavy epidermal cells, hypostomatic epidermis, abaxial anomocytic stomata, distinctive contour of the midrib, dorsiventral mesophyll with prominent spongy parenchyma zone having lobed and interconnected spongy cells, small palisade ratio, small stomatal index, and the vascular system having a ring of seven free collateral bundles in petiole were features characteristics of the species. Extraxylary ibres, distension and arching over to the vascular strands of the stem. Deposition of simple and compound starch grains, calcium oxalate crystals in the epidermal tissues of the lamina, ground tissues of petiole, stem, secondary xylem vessels of the root. Wagon wheel with spokes like appearance of the cross sectional view of the root, wood with more axial and radial parenchyma and less xylem iber. Apotracheal diffuse axial parenchyma and wide non-ligniied rays are also diagnostic features of the taxon. Conclusion: The present macroscopic and histo-anatomical observations of stem, root and leaves of Cissampelos pareira thus provides useful information for quality control parameters for the crude drugs. Powder, quantitative and luorescence standards put forth could label valuable information as identifying parameters to substantiate and authenticate the phytomedicine. Keywords: Anomocytic stomata, axial parenchyma, calcium oxalate, extraxylary ibers, palisade ratio, vein-islets and stone cells. *Corresponding author. Dr. Sudhakaran, M.V Research Oficer (Botany) Department of Drug Standardisation, Government Ayurveda College Thiruvananthapuram -695 001, Kerala, India. Tel: 0471-2917299 (R): Mob. 9447 500125. E-mail: dr.sudhakaranvasu@gmail.com dr.sudhakaranvasu@yahoo.co.in DOI: 10.5530/pj.2012.34.11 Phcog J | Dec 2012 | Vol 4 | Issue 34 INTRODUCTION The genus Cissampelos is mostly dioecious climbing plant consisting of approximately 30 species belonging to the family Menispermaceae of tribe Cocculeae.[1–2] The genus is distributed throughout tropical and subtropical parts of Asia, East Africa, and America and India. The genus name Cissampelos (ivy-vine) is derived from the Greek words Kissos means Ivy and ampelos=vine, because of the resemblance of the growth of this plant, 57 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. like ivy in the green rambling branches and inlorescence forming fruits having vine or grape like racemes. The speciic epithet pareira is a Portuguse name given in Brazil to the roots of some wild vine, hence the species name so ascribed on account of its supposed resemblance of the roots of wild vine. Cissampelos pareira L. is documented as Patha in classical texts (Charaka and Sushruta). In Ayurveda system of medicine, Stephania japonica (Thunb.) Miers, Cyclea peltala (Lam.) J. Hooker and Thoms and Cissampelos pareira Linn. are being used as Patha. Cissampelos pariera is commonly known as the “Midwife’s herb” in South America. The root is being used for centuries by native peoples of South America to treat most types of women’s ailments, such as for menstrual cramps, prevents threatened miscarriage, to stop uterine hemorrhages after childbirth, ease childbirth and postpartum pain and ibroid tumors.[3] They are frequently prescribed for treating diseases of heart, cough, abdominal pain and fever according to Ayurvedic Pharmacopeia of India and antipoisonus drug according to Dhanwanthiri Nighantu.[4] The root is a promising muscle relaxing agent, used as a substitute for tubocurarine, since the hayatine methiodiodiole of the root has a neuromuscular blocking action similar to tubocurarine with grater potency and less toxicity. Plant is also used for heart problems, kidney stones, kidney infections and pains, asthma, arthritis and muscle cramps.[5–7] The genus Cissampelos is highly specialized for the richness of a diverse array of biologically active bisbenzylisoquinoline alkaloids. The quantity and composition of the alkaloids found in the stem, leaves and roots of Cissampelos pareira L. seem to differ between plants from different regions and of various countries.[6] This chemophore diversity may be the result of great genetic diversity of the species, which warrants study of this taxon from different geographical regions.[1,6] Since, it is an important traditional medicine used for the treatments of a wide variety of ailments world over, a systematic botanical evaluation is lacking barring a few isolated studies[8–12] on discrete parts of the plant. The present study aims to delineate the histomorpho diagnostic proile of the stem, root and leaves of Cissampelos pareira, a Kerala habitant and analyze the quantitative, luorescent and powder microscopic peculiarities to support its pharmacobotanical characterization. Figure 1. Cissampelos pareira Linn. supported on trees or into the crowns of trees. The stem is slender, lexible, and twines for support and reach a maximum diameter of 1 cm. The leaves are membranous, simple, alternate, palmately 4–7 nerved, slightly peltate, insertion of petiole slightly away from the margin of the blade. Lamina when full grown is dark green, sparsely silky-hairy above, light green and densely velvetypubescent or silky hairy beneath hence known as velvet leaf. Lamina is broadly ovate, 2–12 cm × 4.5–12 cm, base rounded or cordate, apex obtuse or notched. The petiole is pulvinate at both ends, 4–7 cm long. Flowers are small, unisexual, dioecious, green in color. The small staminate and pistillate inlorescences are borne in leaf axils. Male inlorescences are in short umbels of 1–3 together and 10–12 cm long. Female inlorescences are in pendulous spikes, 7–10 cm long. The fruits are round partially covered by a rounded bract, red-orange hairy drupes, 4–5 mm in diameter and one seeded. The seeds have horseshoe shape. The family is well known by the curved seed, an allusion to the morphology of the seed found in many of the genera, that look like the fourth form of the moon hence the common name ‘‘moonseed’’ family.[13] MATERIALS AND METHOD Plant material Cissampelos pareira is a perennial, twinning and climbing shrub ( Figure 1) having 2–5 m along the ground and 58 METHODS Cissampelos pareira Linn. was collected from the Pathanamthitta District of the State of Kerala and identiication Phcog J | Dec 2012 | Vol 4 | Issue 34 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. was done using Gamble’s Flora of Presidency of Madras. Fine hand sections of lamina, petiole, stem and root and epidermal peels were taken using standard procedures and stained with aqueous Safranin 1% and mounted in glycerin. The investigation on macroscopic, micromorpho diagnostic proile, analysis of quantitative, luorescent and powder microscopic properties of the specimens were undertaken in the Drug Standardization Laboratory of the Government Ayurveda College, Thiruvananthapuram and where voucher specimens were deposited. Microphotographs of sections and powder analysis were made by using Olympus Microscope (Model CX 41; Tokyo, Japan) with CCD camera 2 mega pixel and quantitative measurements were taken using Olympus Image-Pro Plus, version 5.1 software. The number of epidermal cells, stomatal number, stomatal index were calculated per square millimeter of leaf area from intercostal areas of fresh leaves and vein islet number and vein termination number were calculated from cleared leaves as deined by Salisbury.[14] Palisade ratio was determined based on Wallis[15] and size of Guard Cell Area (GCA) was estimated following Franco’s formula.[16] For analysis of the luorescence properties of crude drug, the solvents of HPLC/Chromatographic Grade procured from Merck and Qualigens Fine Chemicals, India were used. Fluorescence analysis was carried out in UV light (256 nm & 366 nm) using Camang UV apparatus. The descriptive terms of the anatomical features used here as per Hickey[17] Metcalfe and Chalk[18] and Carlquist.[19] RESULTS AND DISCUSSION Microscopic evaluation of leaves The leaf of Cissampelos L is microphyll, which consists of an average length of 4.5 cm and width of 5.2 cm. Leaves have no characteristic taste and odour. Histo-anatamical characteristics of leaf revealed, dorsiventral differentiation with adaxial and abaxial epidermis. Lamina was lat and much reduced in dimension bearing numerous long, slender uniseriate clothing trichomes (65.7–159.7 µm × 14.2 µm). Mid rib region was slightly raised on the adaxial side with broadly semicircular on the abaxial side (Figure 2). Midrib composed of epidemics, collenchyma, mesophyll and vascular bundle. Just below the epidermis of the mid rib lies a patch of subepidermal collenchymas, 3–4 cells wide. Elongated tanniferous ideoblast are present scattered amongst the collenchymas, which correspond to patterns recorded for members of Menispermaceae C. hirta and C. mucronata by wet.[20] Phcog J | Dec 2012 | Vol 4 | Issue 34 Figure 2. Cissampelos pareira Linn. T.S. of Lamina with midrib (×10). A chlorenchyma zone consisting of 1–2 layers are located beneath the collenchymas. Parenchymatous ground tissues (6–7 layers) occupy the large area collateral vascular bundle lies in the middle of the ground tissue with the xylem lies in the adaxial and phloem on abaxal side. Patches or group of 3–5 cells of sclerenchyma are distributed around the vascular strand. Epidermis is uniseriate in both surfaces, composed of compactly arranged oval to rectangular cells with moderately cuticularized (>3 qm) outer walls. Cells of the lower epidermis are remarkably small. Epidermal cell of the midrib are comparatively smaller in size than those of the lamina. Some of the epidermal cells of the midrib are provided with long uniseriate clothing trichomes, identical to that of lamina (Figure 3d). Starch grains are distributed in plenty in epidermal as well as in mesophyll. The size of individual upper epidermal cell was found to be 28.7 µm to 53.2 µm in length and 16.3 µm to 24.4 µm in width) and size of the upper epidermal cells area was found to be ranged between 1800.2 µm2 to 2803.5 µm2. The mesophyll is divided into palisade and spongy tissues (Figure 2a). A single row of palisade cells in adaxial side (Figure 2a & 2c ). Paliside cells are somewhat elongated, compactly arranged and illed of plenty of chloroplasts (Figure 2e) and the size of the individual palisade ranges from 19.9 µm to 29.8 µm in length and 9.1 µm to 11.6 µm in width. Central portion of lamina was occupied very lacunar spongy tissues composed of 2–3 layers. Cells of the spongy parenchyma are lobed and cells are interconnected (Figure 2d). Vascular bundles of the veins embedded in the mesophyll tissues. Both epidermis bear uniseriate, bicellular trichome. The basal cell is short, having conspicuous lumen and elongated terminal cell which tapers towards the apex. The palisade ratio was found to be about 3. Epidermal characters The epidermis is hypostomatic, adaxial epidermis is devoid of stomata and cells are irregular in shape. 59 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Figure 2a. Cissampelos pareira Linn. T.S. of Lamina (×40). Anticlinal walls of upper epidermal cells are wavy (Figure 3 & 3a) and cells are sinuate to wavy on the lower epidermis (Figure 3b & 3c). The wall of outer epidermal cells were moderately thickened cuticularised (>3 µm). Stomata are anomocytic conined to lower epidermis (Figure 3b & 3c). The mean length and breadth of stoma was observed as 21.7 µm × 13.2 µm and Guard cell area (GCA) was found to be 224.9 µm2. The mean number of adaxial epidermal cells per square millimeter area of the leaf was observed as 737.7 and that of abaxial epidermal cells was 1817.4. Mean density of abaxial stomata per square millimeter area of leaf was found to be 243.6 and stomatal index was found to be 11.8 (Table 1). Figure 2c. Cissampelos pareira Linn. T.S. of Lamina: a portion of upper epidermis enlarged (×40). Figure 3. Cissampelos pareira Linn. Surface features of adxial epidermis (×10). Figure 2d. Cissampelos pareira Linn. T.S. of Lamina: a portion of lower epidermis enlarged (×40). Figure 2e. Cissampelos pareira Linn. Showing palisade and spongy parenchyma cells (×40). 60 Figure 3a. Cissampelos pareira Linn. Surface features of adaxial epidermis (×40). Phcog J | Dec 2012 | Vol 4 | Issue 34 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Table 1. Quantitative Microscopy of Cissampelos pareira Linn. Parameters Stomata length (µm) Stomata width (µm) *Guard cell area GCA (µm2) Epidermal cell density/mm2 (upper) Epidermal cell density/mm2 (lower) Stomatal density/mm2 (lower) Stomatal index Vein-islet number/mm2 Vein- termination/mm2 Palisade ratio Size of areoles (mm) Mean value 20.5 11.4 183.5 737.7 1817.4 243.6 11.8 3.6 4.5 3 0.06 Range 18.1–22.7 7.7–13.7 109.5–244.3 642.9–793.5 1531.2–1897.3 218.7–268.5 12.5–12.3 3.5–3.9 4.3–4.9 2.75–3.75 0.05–0.07 * Franco’s formula. Figure 3b. Cissampelos pareira Linn. Abaxial epidermis with stomata (×40). Venation pattern Petiolate simple leaves with entire margins had observed actinodromous venation under low (×2) magniication. Areolation was well developed. Areoles are small, area of areoles ranges from 0.055 to 0.073 mm and areoles are pentagonal in shape. Within the areoles terminal vein-endings was absent (Figure 5). The free ending ultimate veins of the leaf are branched one time or two. Marginal ultimate venation was imbrial (Figure 5a). The minor venation pattern viz., the mean number of vein islet number/mm2 of leaf was found to be 3.6. Veinlets termination number/mm2 were found 4.5 and average size of areoles was observed as 0.06 mm. The number of areoles/mm2 was found to be 1.7; when critically analyzed microscopically. Figure 3c. Cissampelos pareira Linn. T Abaxial epidermis with stomata (×10). Figure 3d. Cissampelos pareira Linn. Abaxial epidermis showing trichomes (×40). Phcog J | Dec 2012 | Vol 4 | Issue 34 Petiole In cross sectional view, the petiole is circular in outline (Figure 4). Epidermis is single layered with moderately thickened cuticle (>3.1 µm). Some of the epidermal cells are provided with long uniseriate, bicellular (Figure 4b), non-glandular trichomes (320.4 µm × 14.1 µm). A chollenchyma zone consisting of 4–5 layers are located beneath the epidermis, which is followed by 2–3 layered parenchymatous tissues. Seven free vascular bundles (Figure 4) are arranged as in ring in the middle of the ground tissue. A strand of thick walled sclerenchyma ibres forms an arc on the outside of each vascular bundle (Figure 4a). The sclerenchyma ibres are about 5–6 cells broad in each bundle and is about 1–2 broad in the interfasicular area. Xylem vessels are aligned in rows of 4–5 and phloem lies on adaxal side. The cells of parenchymatous ground tissue are large, hexagonal or pentagonal in shape which contains numerous calcium oxalate crystals (dimensions 20.4–36.8 µm × 16.8–20.5 µm). 61 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Figure 4. Cissampelos pareira Linn. T.S of Petiole (×4). Figure 4a. Cissampelos pareira Linn. T.S of Petiole: portion of vascular bundle enlarged (×10). Figure 5. Cissampelos pareira Linn. Cleared leaf showing areoles, vein-islets and vein termination (×2). Figure 5c. Cissampelos pareira Linn. Cleared leaf showing venation of the margin (×2). Microscopic evaluation of stem The T.S of young stem in microscopic view presents a circular out line with a smooth and undulate surface (Figure 6). Epidermis is single layered composed of rectangular cells, outer wall of cells are cuticularised (<3.2 µm). Some of the epidermal cells are provided with long uniseriate, bicellular trichomes (182.2–333.9 µm in length and 13.2–14.5 µm in width). A chollenchyma zone consisting of 2 layers are located beneath the epidermis, followed by 2–3 layered parenchymatous layers. Cortex is composed of thick-walled ligniied ibers forming a cap arching over vascular strands and a few layers of large, thin-walled parenchyma cells enclosing the secondary phloem (Figure 6a). Figure 4b. Cissampelos pareira Linn. Surface of petiole: showing trichome (×10). 62 T.S of the mature stem shows eight vascular bundles arranged in a ring (Figure 6b). Adjacent vascular bundles are separated by wide bands of parenchymatous vascular Phcog J | Dec 2012 | Vol 4 | Issue 34 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. rays. The vascular bundles are collateral, dispersed around the parenchymatous ground tissues. A strand of thick walled sclerenchyma ibres forms a cap/arc on the outside of each vascular bundle (Figure 6c). These sclerenchyma ibres or extraxylary ibers are later jointed laterally to form a regularly indented ring. The sclerenchyma ibres are about 6 cells broad in each bundle (Figure 6d) and contain plenty of starch grains. Between the secondary phloem and ring of extraxylary ibers, develop a parenchymatous zone composed of 7–8 cells wide. The development these large thin walled parenchyma cells in between the cortical sclerenchyma ibre and secondary phloem appears to be a characteristic feature of the stem of C. pareira (Figure 6d). The parenchyma are polygonal in shape, cells of second row are comparatively larger in size. These parenchyma cells are 2–3 times larger in size than that of the cells of pith and cortical cells and about 2–3 times larger than that of the cells of sclerenchyma ibre. Primary phloem are crushed and collapsed, formed tangential bands of ceratenchyma (Figure 6d). Deposition of simple and compound starch grains (Fig. 15) and prismatic calcium oxalate crystals (dimensions 15.7–20.3 µm × 9.4–14.8 µm) in sclerenchyma ibers are a common feature. The calcium oxalate crystals found in secondary phloem cells are comparatively larger in size (41.4–64.2 µm in length and 35.5–42.1 µm in width). Xylem occupies a small portion of the stem. Vessels are mostly solitary, circular or polygonal in shape; vessels with wide lumen (diameter 40.3–54.3 µm) are co-occurred with vessels bearing narrow lumen (19.4–32.5 µm). Mean diameter of the vessel lumen was found to be 37.8 µm. Vessel cluster of two was seldom occurred. Xylem vessels with spiral and pitted thickenings (Figure 6f) were observed. Intervessel pitting was found in alternate position (Figure 6f) with pit aperture diameter of about 3.2 µm. The mean number of vessels per square millimeter of wood Figure 6. Cissampelos pareira Linn. T.S of young stem (×4). Phcog J | Dec 2012 | Vol 4 | Issue 34 Figure 6a. Cissampelos pareira Linn. T.S of young stem: a portion enlarged (×10). Figure 6b. Cissampelos pareira Linn. T.S of mature stem (×2). Figure 6c. Cissampelos pareira Linn. T.S of mature stem: a portion enlarged (×10). 63 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. was found to be 327.6; when interfacicular area and pith were included. Deposition of starch grains and prismatic calcium oxalate crystals in tyloses are a common feature. The bulk of the xylem was constituted by ibers (Figure 6e). Vascular rays are non-ligniied and many cells wide (13–20). Centrally placed pith composed of large, polygonal parenchyma cells. Pith cells contain prismatic crystals of calcium oxalate (Figure 6a). Microscopic evaluation of root Root is cylindrical, slightly curved, long and narrow, highly bitter in taste. Bark is brownish to dark grey in color, surface rough, longitudinally striated with furrows and ridges. When cut, root appears starchy white internally. Figure 6d. Cissampelos pareira Linn. T.S of mature stem showing extraxylary ibres (×40). Figure 6e. Cissampelos pareira Linn. T.S of stem: a portion enlarged (×40). Figure 6f. Cissampelos pareira Linn. R L.S of stem showing vessel thickening (×40). 64 Distinct 10–12 radiating vascular stripes alternating with broad medullary rays in the cross section of root resemble a wagon wheel with spokes appearance (Figure 7); 2–3 vascular streaks particularity elongated and converges at the centre, a few vascular streaks bring about only the halfway from the periphery to the centre and most recently formed ones are too short (Figure 7). Conspicuous non ligniied cork is composed 8–10 layers. Outer 2–3 layers are thick walled cells illed with dark brown content, inner cork composed of 6–7 layers of thin walled rectangular, empty cells and a narrow band of parenchyma towards the interior. The cork zone has a strand of thick walled sclerenchyma, which forms a broken ring on the outside of each vascular strand (Figure 7a). As secondary growth progresses, stone cells are embedded within the broken cortical sclerenchyma strands and form a complete ring (Figure 7a). Stone cells are rectangular to pentagonal in shape, walls are striated, pitted with wide lumen (Figure 7b). Vascular rays are very prominent (13–19 cells wide) and occupy the major portion of the root. Vessels are solitary, circular, elliptical or polygonal in shape. The Diameter of vessel lumen ranges from 18.6 µm to 60.3 µm with a mean diameter of 40.2 µm. Some of the xylem vessels contain prismatic crystals of calcium oxalate (Figure 7d); It ranges in size from 7.4 × 11.6 µm to 24.7 × 42.2 µm. Secondary xylem tissues and vascular rays contain plenty of simple and compound starch grains (Figure 7d). Some of the vessels are completely occluded with the tyloses and illed with brown colored contents (Figure 7c). The vascular rays of the root are composed of thin-walled, unligniied cells. The vessels are with bordered pits and intervessel pitting was in alternate position, pit aperture about 3.15 µm in diameter (Figure 7e). Mean number of vessels per square millimeter of the secondary xylem and excluding the area of vascular rays was found to be 227.4 (ranges from 171.6 to 263.7/mm2). The bulk of the wood is constituted by ibers. Wood parenchyma is apotracheal, diffuse type (Figure 7e). Phcog J | Dec 2012 | Vol 4 | Issue 34 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Figure 7. Cissampelos pareira Linn. T.S of root (×4). Figure 7c. Cissampelos pareira Linn. T.S of root: central portion enlarged (×10). Figure 7a. Cissampelos pareira Linn. T.S of root: a portion enlarged (×10). Figure 7d. Cissampelos pareira Linn. T.S of root: interfacicular parenchyma showing starch grains (×40). Figure 7b. Cissampelos pareira Linn. T.S of root: a portion enlarged (×40). Figure 7e. Cissampelos pareira Linn. R.L.S of root (×10). Phcog J | Dec 2012 | Vol 4 | Issue 34 65 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Wood anatomy of the Menispermaceae has been studied by different investigators.[13,18–23] The rays in the Menispermaceae have been interpreted by many as being medullary.[12,17] Eames and MacDaniels[24] of the view that, the wide rays present in stem and root are vascular rays, originated from the interfascicular cambium. Medullary rays were present at the beginning of the primary growth and limited to occupying a small extension between adjacent vascular bundles. Thereafter, intrerfasicular cambium develop between the adjacent vascular bundles exclusively gave rise to thin walled parenchymatous vascular rays. Another noteworthy feature of histology of the stem of the taxa of Menispermaceae is the presence of extraxylary ibers. According to Solereder[25] the extraxylary ibres in the cortex of the stem contribute to the formation of pericycle. Pericycle forms a continuous ring, undulating and arching over the vascular bundles and providing mechanical strength. Mennega,[13] Carlquist[19] and Tamaio[23] have reported that two types of histology coexist in members of Menispermaceae; the ligniied rays are more common with the successive cambia type. Successive cambia was absent in C. pareira and only non-ligniied rays ware found in all the specimens analyzed. The anatomical features of the specimen viz., presence of moderately large xylem vessel lumen, greater abundance of axial and radial parenchyma and less xylem ibre, slerenchymatous ibrers in stem cortex, plenty of simple and compound starch grains may confer greater lexibility and increase in their climbing, conduction and storage performances. These histological peculiarities of C. pareira might be represented as important facets of their life history as lianas, to use external mechanical support to sustain their weight and hence provide resistance to the twisting and girdling. Figure 8. Cissampelos pareira Linn. Powder microscopy of leaf showing trichomes (×4). Figure 8a. Cissampelos pareira Linn. Powder microscopy of leaf showing trichomes (×10). Powder microscopy The dried leaves, root and stem of C. parereia were analyzed for powder characteristics. Leaf powder was dark green in color and has no characteristic taste and odor. Microscopic examination showed fragments of leaf epidermis with uniseriate trichomes (Figure 8 & 8a). Stem powder is light brown, and has no characteristic taste and odor. Root powder is brown colored, highly bitter in taste. Stem and root power contain abundant pyramidal calcium oxalate crystals (Figure 8b & 8d) and ligniied ibers with pointed ends (Figure 8f) simple and compound starch grains (Figure 8g). Fragment of parenchymatous tissues of the cortex (Figure 8e) and ibres (Figure 8c) were also observed. 66 Figure 8b. Cissampelos pareira Linn. Powder microscopy of stem-crystals in parenchyma cells (×10). Phcog J | Dec 2012 | Vol 4 | Issue 34 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Figure 8c. Cissampelos pareira Linn. Powder microscopy of stem showing ibres (×10). Figure 8d. Cissampelos pareira Linn. Powder microscopy of stem showing prismatic crystals (×10). Figure 8f. Cissampelos pareira Linn. Powder microscopy of root showing-starch grains, crystals and ibres (×10). Figure 8g Cissampelos pareira Linn. Powder microscopy of root showing-starch grains (×40). Fluorescence analysis Figure 8e. Cissampelos pareira Linn. Powder microscopy of stem-parenchyma cells (×10). Phcog J | Dec 2012 | Vol 4 | Issue 34 The use of luorescence can be very useful adjunct to botanical study, since it is an easy test to verify certain identiications of the crude drug. The powdered crude drug extracts were taken in a series of solvent systems with increasing polarity as follows; petroleum ether, cyclohexane, toluene, benzene, ethylacetate, chloroform, acetone, ethyl alcohol and methanol. All the extracts were iltered through Whatman ilter paper and then analyzed under UV light (long and short). Specimens were recorded as either luorescent (with color and intensity) or not luorescent as on their responses under UV light. The results obtained are presented in Table 2. 67 Sudhakaran M.V: Histo-morphological, luorescent and powder microscopic characterization of Cissampelos pareira Linn. Table 2. Fluorescence properties of the extract of root, stem and leaves of Cissampelos pareira Linn in various solvents. Solvent under UV (254 nm) Aqueous Methanol # Ethyl Alcohol # NF S , L, R, W NF S , L, R, W NF S, L , R, W Acetone* NF S , R, L (light) pink W Chloroform* NF R S, L (light) pink W Ethyl acetate* NF S , R (light) pink L, Benzene # NF S, R ,W (light) pink L Toluene # NF S, R, W (light) pink L, Cyclohexane # NF S , L. R, W Petroleum ether # NF S , L. R, W under UV (366 nm) (color & intensity) NF S , L , R , W NF S milky white R, W green L (light) pink S, milky white R, orange L, W (light) pink S, milky white R, orange L, W (high) pink S , (light) pink R, orange L, W (medium) pink S NF R, orange L (medium) pink S, (light) pink R, orange L, W (light) pink S, R, W, NF L (medium) pink S L, W, NF R, (light) pink S,L W, NF R * from MERCK: # from Qualigens ; NF= not luorescent R = extract of root, S= extract of stem, L =extract of leaf, W = extract of whole plant CONCLUSION The present macroscopic and histo-anatomical observations of stem, root and leaves of Cissampelos pareira thus provides useful information for quality control parameters for the crude drugs. Powder, quantitative and luorescence standards put forth could tag on valuable information as identifying parameters to substantiate and authenticate the phytomedicine. Abbreviation used in the igures are: A = areola; abx = abaxial; abx.e = abaxial epidermis; adx = adaxial, adax.e = adaxial epidermis; b.c = basal cell; cam = cambium; ck, cr = cork, co = cortex; col = collenchyma; cern = ceratenchyma; crl = crystal; cut = cuticle; cyl = cystoliths; ep, epi.c = epidermal cell; epi = epidermis; f = foot cell; gu.c = guard cell; gt = ground tissue; la = lamina; l. up = lower epidermis; mdr = medullary rays; pal = palisade cell; ph = phloem; pi = pith; scb = sclerenchymatous bundle sheath; spa, spo = spongy parenchyma; s.co = secondary cortex; s.ph = secondary phloem st = stomata; sto = stone cell; str = starch grain s.xy = secondary xylem; tri= trichome; t.c = stalk cell; xy = xylem; u.epi = upper epidermis; tyl = tylosis; vb= vascular bundle; VI = vein islet; VT veinlet termination; wd = wood. 68 REFERENCES 1. Wang W, Heng-Ghang W, Zhi-Duan C. 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