Results Part 1:

4.1 Cissus quadrangularis

Family: Vitaceae

Synonym: Vitis quadrangularis (L) Wall. Ex. Wight

Vernacular names:

English: Adamant Creeper or Edible Stemmed Vine Hindi: Hajra

Sanskrit: Asthisanhara

Tamil: Pirandai

4.1.1 Morphological characters

This is a perennial vine, wide spread in dry parts of India in seashores, coastal regions, scrub jungles, fences of agricultural fields and in uninhabited areas. They grow up-to 500 m MSL It is fleshy much rambling shrub climbing over bushes, thorny trees and fences. The mature stem is dark green in color. New leaves and branches appear in rainy season. During summer, the plant is mostly devoid of leaves. The stem is easily broken at the nodal regions. Stem is glabrous, much contracted at nodes and with a waxy coating. It also attains pink color in some parts of the plant. The matured and older stems have a layer of ash brown colored bark and loosing their quadrangular shape to more or less round The fleshy inner region of the plant produces irritant over the skin and to tongue. The internodal region is 7-12 cm in length. Occasional formation of aerial roots from nodes during rainy season is also observed. It is opposite to tendril, uniform in thickness (2-2.5 mm) and white to pink to blurry brown to black in color. Petiole is 0.7-4.0 cm in length, glabrous, green and with a long trench on the adaxial surface running throughout. Leaves are simple, alternate, glabrous, early caducous, usually broader than length, attains 3.0-6.0 x 4.5-7.2 cm in size. Cordate to rounded, base truncate or cuneate, tip acute to acuminate and margin toothed. Sometimes the leaves are 3-5 lobed. More number of lobed leaves occurs in plants grown in drier areas. Lateral nerves are 3-6 paired.

Tendrils are unbranched, green, perennial, glabrous and slender, when young. However, they are strong on maturity. The length of the tendril is 10-17 cm. A node with a small bract is present in the middle of the tendril. The bract is ovate and acute. White, tiny, globular and ephemeric exudates are seen all over the plant body. In leaf, their number is more on abaxial side. They are observed frequently during rainy season and in young parts. Flowers in umbels, found from April to July. Flowers arise axillary in short and stout peduncles branched 3-4 times. Peduncle is up to 3 cm in length, pedicel up to 1.5 cm in length, green and glabrous. Petals 4, valvate, yellowish in color, exerted portion is reddish to pink in color, ovate to oblong, 3 mm in length, tip acute and hooded at the apex. Stamens 4, filaments slender, 2 mm in length, anther oblong and dehisce longitudinally. Disc small, erect and adnate at base of the ovary. Ovary 2 celled, 1 mm, ovules 2 in each cell, style subulate, 1 mm, and stigma small and simple. Fruits globose, green, and turn red on ripening.

Variant I

This variant is described as a variety named as. ‘Sadurappirandai' in Siddha literatures (meaning square-stemmed). This type is the most common and available throughout India and described in all the Indian Floras available. The distinct character of this type is the presence of quadrangular stem with winged comers (Fig: l). The internodes on four sides invaded or depressed deeply in the middle and the comers' are exerted with sharp reddish brown to black colored margins.

Variant II

This variant is the variety described in Siddha literatures as 'Uruttuppirandai' (meaning round stemmed). The plant is characterized by the presence of wingless stem (Fig. 3). It is found rarely in selected areas like Seithur Hills near Rajapalayam of Viruthunagar district, Tamilnadu. The internodes are four sided and depressed slightly four sides. Comers are blunted.

Variant III

This variant is mentioned to be a very rare plant in Siddha literature. It is known as 'Olaippirandai'. (Meaning flat-stemmed). The internodes of the stem are flat or strap shaped or two-sided (Fig. 5 A). Three sided internodes are also seen occasionally in a plant. The comers are pointed with reddish brown to black color. The internodal portion of stem is convex on both sides, one side being more convex. Rarely, tendrils are branched (Fig. 5 B). This type is not found in wild. However, it is grown in gardens of private practitioners and institutions.

4.1.2 Description of the useful parts

Tender shoots of C. quadrangularis comprise the young stem, leaves and tendrils. They are pale green in color and shining. Tendrils are slender. Ephemeric exudates are present. On drying, the stem shrinks longitudinally. Fracture is fibrous surface texture is rough, yellowish green to pale green in color and taste is uncertain but irritant to over the tongue. Powder and macerated tender shoots show fibers, sticks of calcium oxalate vessel elements with spiral thickenings and thin walled parenchyma cells.

4.1.3. ANATOMICAL CHARACTERS

The following anatomical descriptions apply equal to three variants, unless otherwise stated.

TS of Stem

In ground plan, the TS of stem of the three variants show three zones, cortex, vascular bundles and broad pith (Fig. 2,4 and 6). The outline of the stem varies from gibbose to dumbbell depending upon the variants. Epidermis is single layered with cuticle and stomatal openings (Fig. 8, 6 and 24). The epidermal cells are small, radially elongated, varied in shape and 4-7 x 1-15 m in size. Cortex is differentiated into outer chlorenchyma of 2-4 layers and inner parenchyma of 3-6 layers. Chlorenchyma cells are round to polygonal in shape with intercellular spaces, compactly arranged and ranges from 10-15 m in diameter. Small stomatal cavities are observed. Parenchyma cells are round to polygonal in shape, ranging from 10-15 m in diameter and have intercellular spaces. Large size mucilage cells ranging from 25-48 m in diameter are frequently observed in the cortex. Patches of collenchyma are present at the comers of the stem. Primary vascular bundles are present in-groups of 3-5 at the comers. Endodermis is not distinct. Smaller secondary vascular bundles appear along the inter-fascicular cambium forming a ring.

Vascular bundle is collateral, open and exarch. Sclerenchyma cap consisting of cells of 3-5 m in diameter is present above the phloem of each bundle. Vessel elements are large and 7-25 m is diameter. Ground tissue present between corner vascular bundles consists of smaller and compact cells of varying in shape and sizes (4-8 m). Pith is parenchymatous present at the center and occupying most of the area. These cells are round to irregular polygonal in shape and 15-25 m in diameter. A number of large mucilage cells (size: 25-48 m) are present in the pith. Raphides of calcium oxalate crystals are also present. They are 30 x 75 m in size (Fig 17).

On observing the development of secondary growth, it is noticed that the cambial ring formed is connecting the vascular bundles. Secondary vascular bundles are formed one on each side of the stem, four in four sided stems and two in two sided stems. Second set of secondary vascular bundles is formed in-between the primary bundles and first formed secondary vascular bundles and thereby giving the appearance of beads.

Variant I

In TS, the stem has a dumbbell shaped outline with slight modifications. The four comers are exerted forming the wing of the stem (Fig 2, 7 and 8). It has three cellular zones as in the general pattern. However, the mature stem is distinguished by the presence of a patch of sclerenchymatous cells at the four comers below the epidermis.

Variant II

In TS, the outline of the stem is nearly rectangular with blunt comers (Fig. 4, 15 and 16). Sclerenchyma is absent in the cortex.

Variant III

The outline of the stem is mostly two sided (Fig. 24B) and rarely three sided The shape varies from spindle or gibbose (Fig. 6A) to top (Fig. 6B). In mature stem, sclerenchyma cells are present in the cortex, along the comer.

Petiole

In TS, petiole shows the following distinct zones, epidermis, cortex, collenchyma and vascular zone embedded in parenchymatous ground tissue (Fig. 9). It is more or less circular or pentagonal in outline. A notch is present on the adaxial side with two flanks. Towards the apical region of the petiole, this notch is absent.

The epidermis is single layered with cuticle. The epidermal cells are barrel shaped and 20-28 X 12-20 m in size. Cortex is made-up of 4-8 layers of parenchyma cells and size varying from 20-45 m. 2-3 layers of cells are chlorenchymatous in nature. Next to cortex is the collenchyma zone. It is 3-5 layers thick, continuous along the petiole in the basal end, but it is restricted as a patch below the vascular bundles in the middle and in the tip of the petiole (Fig. 26 and 27). Vascular bundle is exarch, open, and collateral with a collenchymatous cap. Vessel elements are large up to 40 m in diameter. If the numbers of bundles are five, they are restricted to five comers of the petiole. Inner to this is the ground tissue, which is made up of parenchyma cells with clear intercellular spaces. Cells are 25-58 m in size. Large sized mucilage cells and raphides of calcium oxalate crystals are frequently observed in the pith and cortex.

Leaf midrib

In TS, midrib is broader on abaxial side and has a small protuberance in the adaxial side. The midrib is broader at the basal portion of the leaf and narrowing towards the tip of the leaf. The midrib has the following zones, outer epidermis, followed by collenchyma tissue, parenchymatous ground zone and centrally located vascular zone (Fig. 11, 18 & 25). The epidermal cells in the abaxial surface and adaxial protuberance region are smaller (8-15 x 10-20 m) and square to rectangular. 2-6 layers of collenchyrna cells are present in the abaxial and adaxial surfaces. These cells are compactly arranged, small, 12-28 m and polygonal to round in shape. In-between these are the ground tissue made up of parenchyma cells. These cells are large, thin walled with clear intercellular spaces and 30-52 m in diameter. Mucilage cells of 30-52 fl in size are frequently observed in the midrib (Fig. 18).

Vascular bundles are centrally located. The number of vascular bundles varies from basal to apical regions of the leaf, single in the apical, 3-4 in the middle and 4-6 in the basal regions. A collenchymatous cap is present over each vascular bundle. Bundles are open, collateral and exarch. Vessel elements are large. Raphides are rarely present. Stomatal openings are observed on the abaxial surface.

Leaf lamina

In TS, leaf lamina is homogenous with epidermis on both surfaces and mesophyll cells (Fig 10, 19 & 28). The thickness of the lamina is variable ranging from 40-120 m. The epidermal cells are rectangular to barrel shaped with a thin cuticle. The cells are 12-40 x 8-11 m in size. Stomatal openings are observed on both the surfaces. Mesophyll is made up of round to irregular polygonal cells with clear intercellular spaces. Chlorophyll pigments are observed in the mesophyll of both surfaces. Sometimes they are present in the middle region also. In some instances, mesophyll cells present in the upper surface are columnar shaped (Fig. 28). Large sized mucilage cells are frequently observed which is 110-145 m in size. Calcium oxalate raphides are present (Fig 19, 20, 29 and 30). They are 30-70 m in length. Surface view of the epidermis show polygonal shaped cells with straight margins (Fig. 13, 14, 22 and 23). Striations of epidermal cells are common in Variant I (Fig. 14) but absent in other two variants. Stomata are of anomocytic type.

Tendril

In TS, tendril is circular in outline. The internal structure is differentiated into epidermis, parenchymatous cortex, collenchymatous zone, vascular bundles and pith (Fig. 12, 21 and 31). Epidermis is single layered with cuticle. It encloses 4-7 layers of cortex. It is made up of polygonal and compact parenchyma cells. These cells are 15-23 m in size. Medium sized mucilage cells and raphides are also observed. Inner to the cortex is collenchyma zone of 3-5 layers in thick and the size of cells are ranging from 15-25 m. Inner to this is the 2-4 layers of parenchyma cells, which are polygonal in shape. Vascular bundles are more than 6 in number, 8 being most common. As secondary thickening proceeds, all vascular bundles are inter-connected by the inter-fascicular cambium and forming a ring. Vascular bundles are exarch, open and collateral. Vessel elements are up-to 18 m in diameter. Pith is made up of polygonal shaped parenchyma cells without intercellular spaces and ranging from 18-49 m in size. Mucilage cells and raphides are occasionally present in pith.

4.1.4. Quantitative Microscopy

Quantitative microscopical values like stomatal index, vein islet number and vein termination number of the three variants are given in Table I. Stomatal index value of leaf lower surface is higher than that in upper surface in all the three variants. Highest value in average stomatal index is observed in the upper and lower leaf surfaces of Variant I. Lowest average stomatal index value is observed on upper surface of Variant III and lower surface of Variant II.

4.1.5. Analytical Values

Analytical values like, total ash, acid insoluble ash, loss on drying, solubility in alcohol and water and successive extractive values in petroleum ether, benzene chloroform, alcohol and water are given in Table - II. Total ash value acid insoluble ash value and loss on drying are highest in Variant I, lowest in Variant II and medium in Variant III. Solubility percentage in alcohol and water are highest in Variant III and lowest in Variant II. In general, successive extraction value in petroleum ether, benzene, chloroform, alcohol and water are highest in Variant III.

4.1.6. Study of Powder

Behavior of powdered tender shoots of the three-variants of C. quadrangularis on treatment with different chemical reagents and their fluorescent behavior are given in Table III and Table IV respectively. It shows both similarities and dissimilarities between the variants. Distinct among dissimilarities is the behavior of shoot powder in NaOH and iodine solution.

4.1.7. Qualitative phytochemical studies

Quantitative phytochemical analysis for alkaloids, carbohydrates, tannins and phenols, flavonoids, gums and mucilage’s, fixed oils and fats and saponins in extracts of tender shoots of three variants of C. quadrangularis are given in (Table V). It is to be noted that petroleum ether and alcoholic extracts of Variant-I gave positive results for all the four reagents for alkaloids. Alcoholic extract of Variant III also reveals positive for all the alkaloid tests. The three variants show negative results for flavonoids. Color and physical consistency of extractives by different solvents reveal that Variant III has distinct characteristics. Thus, petroleum ether, benzene and chloroform extractives of Variant I and II are green and sticky whereas Variant III is yellow and oily, brownish yellow and sticky and brown and oily respectively. Extractives in alcohol are brown and oily semisolid in Variant III. Water extractives of the three variants show distinct features. Variant I being brown and oily Variant II brown and granular and Variant III being brown and sticky. The results of qualitative phytochemical tests show both similarities and dissimilarities.

4.1.8. Quantitative estimation of Vitamin C

Ascorbic acid (Vitamin C) is estimated in dry powder of tender shoots of three variants. Ascorbic acid content is found to be more in Variant III (4.5 mg/gm) than Variant I (3.4 mg/gm) and Variant II has least percentage (1.9 mg/gm).

Table I: Quantitative microscopical values of C. quadrangularis L.

Sl. No.	Parameters Studied	Variant I	Variant II	Variant III
1	Stomatal Index Upper surface Lower Surface	3.05-3.79-5.08 3.75-5.61-6.61	2.08-3.04-4.34 2.66-3.76-6.00	1.15-3.00-4.48 2.22-4.55-6.73
2	Vein islet number	2.56-2.65-3.50	2.00-2.48-3.70	1.75-2.28-3.00
3	Vein termination number	5.16-7.71-10.9	5.0-7.18-11.70	5.25-7.56-10.1

Table II: Analytical values of tender shoots of C. quadrangularis L.

Sl. No	Parameters studied	Variant I	Variant II	Variant III
I	Total ash value	11.9	10.0	13.2
2	Acid in-soluble ash value	5.7	3.5	4.9
3	Loss on drying	91.0	87.0	90.0
4	Solubility % in
	Alcohol	6.0	5.0	7.0
	Water	36.6	26.8	55.7
5	Extractive values in
	Petroleum ether	2.86	3.30	5.0
	Benzene	1.003	1.243	3.179
	Chloroform	0.365	0.477	0.375
	Alcohol	18.637	13.015
	Water	1. 795	24.922	26.250

Table III: Behavior of powdered tender shoots of three variants of C. quadrangularis on treatment with different chemical reagents

SI. No.	Powder + Reagent	Variant I	Variant II	Variant III
1	Powder as such	Yellowish green	Green	Green
2	P + Conc. H2SO4	Dark Brown	Blackish green	Blackish brown
3	P + Conc. HCl	Yellowish red	Reddish brown	Reddish brown
4	P + HN03	Yellowish red	Reddish brown	Reddish brown
5	P + Acetic acid	Brownish green	Dark green	Pale green
6	P + 10% NaOH	Yellow	Grey	Green
7	P+ 1N HCl	Yellow	Green	Green
8	P + Iodine solution	Brown	Grey	Green
9	P + 5% Ferric chloride	Brownish black	Green	Dark green

Table IV: Fluorescent behavior of tender shoots of three variants of C. quadrangularis L.

Sl. No.	Powder + Reagent used	Variant I		Variant II		Variant III
		Visible light	UV light	Visible light	UV light	Visible light	UV light
1	Powder as Such	Yellowish green	Dark gray	Green	Grey	Green	Grey
2	P + 50 % HNO3	Dark brown	Dark yellowish green	Yellowish brown	Green	Yellowish brown	Yellowish green
3	P+ 1N HCl	Brown	Yellowish green	Grey	Green	Grey	Green
4	P + 1N NaOH in water	Yellowish green	Green	Light green	Yellowish green	Green	Green
5	P+ IN NaOH in alcohol	Yellowish green	Green	Green	Dark green	Green	Dark green

Table V: Preliminary phytochemical analysis on tender shoots of variants of C. quadrangularis L.

Sl No.	Test for	Reagents Used/ Methods adopted	Extracts of
			Petroleum ether			Benzene			Chloroform			Alcohol			Water
			Var I	Var II	VarIII	Var I	Var II	VarIII	Var I	Var II	VarIII	Var I	Var II	VarIII	Var I	Var II	VarIII
	Color & Physical consistency		Green Sticky	Dark Green Sticky	Yellow oily	Green sticky	Greensticky	Brownish yellow sticky	Green sticky	Green sticky	Brown oily	Brown oily semisolid	Brownish oily semisolid	Dark brown sticky	Brown oily	Brown granular	Brown sticky
1	Alkaloids.	Picric acid	++	+	+	+	+	+	+	+	+	+	+	+	-	+	+
		Dragondroff	+	-	-	-	-	+	-	-	-	+	-	+	-	-	-
		Mayer's	+	-	-	-	-	-	-	-	-	+	-	+	-	-	-
		Wagner's	+	-	-	-	-	+	-	-	-	+	-	+	-	-	-
2	Carbohydrates	Molish's	-	-	-	-	-	-	-	-	-	+	+	+	+	-	-
		Fehling's	-	-	-	-	-	-	-	-	-	-	-	+	-	-	-
		Benedicts	-	-	-	-	-	-	-	-	-	+	+	+	+	-	-
3	Tannins &	Ferric chloride	-	-	-	-	-	-	-	-	-	-	+	+		-	-
	Phenols	Gelatin	-	-	-	-	-	-	-	-	-	-	+	+		+	+
		Lead acetate	-	-	-	-	-	-	-	-	-	-	-	+	+	+	+
4	Flavonoids	Shinoda's test	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
5	Gums& Mucilage’s	Alcoholic Precipitation	-	-	-	-	-	-	-	-	-	-		-	+	+	+
6	Fixed oils & fats	Spot test	+	+	+	+	+	+	-	-	+	+	+	+	-	-	-
7	Saponins	Foam test	-	-	-	-	-	-	-	-	-	+	+	-	+	+	+