PmgMACC^^GNpSTlC and MOLECULAR STUDIES ON TrieHosantfies cCioica "Roxb., Triefiosantfies cucumerina L. Chapter VI dioica Roxb. & Trichosanthes cucumerina L.

Introduction 'Patola' is used in the traditional system of medicine for curing wide array of disorders (Raghunathan & Mitra, 1982). It is one of the ingredients of ayurvedic preparations like Gulgulutiktakam kasayam, Mahatiktaka ghrtam, Mahatiktakam kasayam, Vijrakam kasayam, etc. Ayurvedic text recognizes two kinds of'Patola'' namely small fruited 'Patola'' and large fruited 'Svadupatola\ In different parts of , sources of this drug is vary for example in north India used as 'Patola'' where as in south India T. cucumerina used. This creates ambiguity in correct identity of 'Patola'. Non bitter species T. dioica and variety T. cucumerina var. anguina are edible and occasionally admixtured with bitter variety. This has necessitated the development standardization parameters for correct identification of these species. 6.1 Taxonomic details 6.1.1 Trichosanthes dioica Roxb., Fl. India 3: 701.1832; Jeffrey in Kew Bull. 34:798.1980; Chakrav. in Fasc. Fl. India 11: 114.1982; K. M. Rao in Singh et al., Fl. Maharashtra State 2:66.2001; Renner & Pandey in PhytoKeys 20:92.2013 Vernacular names: Bengali - Potol; English - Pointed ; Gujarati- Patal; Hindi- Parwal; Karmada- Kaadu-padavala; Malayalam - Patolam; Marathi - Perval; Sanskrit - Putulika, Patolah, Kulakah, Pandukan, Karkasacchadan, Rajiphalah, Panduphalah, Rajanama, Amrtaphalah, Viryagarbha pratanah, Kusthaha, Kasamuktidah; Tamil - Kombu-pudalai; Telugu - Kommupotla Large, dioecious climbers, stem softly pubescent 5-angIed twine branched, with trifid tendrils. simple, petiolate, I -4 mm long, slender, striate, shortly villose-hirsute. lamina 6-12 x 3-9 cm, cordate, ovate to oblong, acute, sinuate or dentate, rigid, sparsely scabrous, basal lobes narrow round, 1.5-2.0 cm deep. Tendrils slender, short, trifid. Flowers white, male flowers in pairs, one flower shorter than the other, calyx tube up 3-4 cm long, subcylindric, petals oblong, fimbriate; anthers free, subsessile, oblong, 3.5 x 1.5 mm. Female flowers solitary; ovary oblong, villose, petals similar to male flowers. 5-8 x 2-4 cm, spindle aped, glabrous, smooth, green with white stripes when young. Scarlet red at ripening. Seeds ovoid, black. Flowering and fruiting: July- September Distribution in India: Cultivated throughout country for its fruits. Chapter VI Thchosanthes dioica Roxb. & Trichosanthes cucumerina L.

6.1.2 Trichosanthes cucumerina L., Sp. PI. 1008.1753; Chakrav. in Fasc. Fl. India 11: 112. 1982; K. M. Rao in singh et al., Fl. Maharashtra State 2: 66. 2001. T. cucumerina L. var. anguina (L.) Haines, Bot. Bihar Orissa 388.1923; Jeffrey in Kew Bull. 34:797. 1980; Renner & Pandey in PhytoKeys 20: 91. 2013. Annual, monoecious, climbers to 5 m (to 8 m in var. anguina); stem 1.5-5 cm diameter, branched, softly pubescent, grooved. Tendril 3 branched. Leaves simple, unlobed, or sub palmately 3-7 lobed; petiole 2-12 cm; blade membranous, pubescent, few times faintly scabrous, subcircular or broadly reniform in outline, 5 - 20 x 5 - 25 cm, base cordate with broad sinus, apex acute. Flowers white, male flowers in raceme sometimes with co-axillary a solitary male flower or female flower, raceme 8 - 25 cm log, pubescent, pedicel 5-20 mm long. Bracts sub-persistent, membranous, ellipsoid, without gland, entire or rarely with few lobes. Pedicel of male flowers 5-20 mm, receptacle tube 15 - 20 mm long, apex 3-5 mm diameter, sepals linear, 2-3 mm long, margin entire, petals ovate- oblong, fimbriate, 6-10 mm long, threads 10 mm long, synandrium 2- 3 mm long, filaments short. Female flowers: pedicel 5-12 mm long; ovary elliptic- oblong, 3-10 mm long, hairy, petals as described in male flowers. Fruits, ovoid- oblong, narrow toward apex, 2.5 - 6 x 1.5 - 4 cm, green when young turning bright orange to red. Seed pale or dark brown, flat, elliptic-oblong, 6-18x4-9 mm, margin broad, edge undulate.

la. wild, delicate, leaves 3- 15 cm diameter; fruits 2.5 - 6 cm, beaked, 9-10 seeded; Seeds 6-10 mm long var. cucumerina

lb. Plants cultivated, robust, leaves 8- 25 cm diameter; fruits elongated, snake like, some times coiled, 35- 150 cm long, containing many seeds; seed 16-18 mm long var. anguina Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. a. var. cucumerina Annual, growing in wild, stem delicate, 1-2 cm in diameter, stem and petiole with or without scattered hairs. Petiole 2- 6 cm long. Fruits 2.5- 4.6 cm, containing few (up to 8-10) seeds; pulp bitter. Seed oblong, 6- 10 mm long. Vernacular names: Bengali - Ban-patal; Hindi - Jangli-chahinda; Marathi - Patola; Sanskrit - Patolah, Amrtaphalah, Kashlhhanjan; Tamil - Adavi-poda; Telugu - Adavi-patla, Chedupotla Flowerig and fruiting: June- October Distribution in India: Warmer parts of the country in forest edges, scrub, disturbed, open areas b. var. anguina (L.) Haines, Bot. Bihar Orissa 388.1923; Jeffrey, Jeffrey in Kew Bull. 34:797. 1980 sub-perennial, cultivated; stem 2-5 cm diameter, grooved or angular. Petiole 2- 12cm long. Fruits long, snake- like, 30- 150 cm long or more, containing up to 50 seeds, pulp rather sweet. Seed 14-18 mm long Vernacular names: Bengali - Chachinga; Hindi- Chachinda; Marathi - Padwal; Sanskrit - Chachinda, Svadupatola; Tamil - Pudal; Telugu - Lingapotla Flowering and fruiting: May - August Distribution in India: Widespread in cultivation

6.2 Collection and voucher deposition Male and female plants of TD were collected from Bardoli and Anand (Gujarat). Both the varieties of TC i.e. TCC and TCA were collected from different parts of Maharashtra (Table 6.1 & Map 6.1). Different crude drug markets were screened and eight market samples were procured from Akola, Dhule, Kolhapur, Pune, Mumbai, Ratnagiri (Maharashtra), Anand (Gujarat) and Lucknow (Uttar Pradesh) under name of Patolah / PotoU Padval /Ran padval /Jangli padval (Map 6.1). Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Table 6.1 Details of collection Species Date of Locality Herbarium Repository collection number Vouchers AHMA T. dioica 28/09/2010 Anand 25488, 25489 WP-087, 088, 30/09/2010 Bardoli 27026, 27027 F-170

15/09/2009 Shivapur 27001 WP-086, T. cucumerina 27/08/2010 Akola 27002 F-169 var. cucumerina 26/08/2011 Kankawali 27096 27/08/2011 Amboli 27023 T. cucumerina 04/08/2009 Junnar 27412 WP-89 var. anguina 26/08/2009 Kankawali 27477 F-171 24/08/2011 Kolhapur 27003

Map 6.1. Area of collection and market survey (Map was prepared using DIVA GIS Version 2) Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

6.3 Pharmacognosy 6.3.1 Macroscopy Comparative investigation of TD and both the varieties of TC were carried out. In case of TD, both female and male plants were studied. Similarities in both plants are highlighted in bold (Table 6.2 and Figure 6.1). Table 6.2 Macroscopic characters of TD and TC TD TCC TCA Occurrences • Extensively cultivated •Wild • Extensively cultivated Throughout the warmer part of India Plant • Dioecious • Monoecious Aerial part Stem • 3-6 m long, climbing. • 4-5 m long. • 5-8 m long. promintly grooved. distances distances sturdy, branched. between inter- between inter- pubescent, with node 10- 12 cm node 15-17 trifid tendrils, cm distances between • Climbing, slender, grooved, inter-node 3-4 cm branched, pubescent, with trifid tendrils Leaves • Petiole 2-5 mm. • Petiole 2.2 - 3 • Petiole 5 - 7 channeled, shortly cm. cm. villose-hirsute. Lamina 9-13 x Lamina 10- lamina 3- 12 x 3-9 10-13, with 5-7 15 X 12- 18, cm, cordate, ovate- lobed with 3- 5 oblong, sparsely lobed scabrous, apex • Petiole cylindrical, villose- hirsute. acute, margins Leaf orbicular- reniform. dentate, basal lobed margins denticulate, apex acute. narrow round, 0.5- villose-hirsute on both side, 1.5 cm deep, palmate venation villose-hirsute, palmate venation. Continued on next page. Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Table 6.2 Continued Male • Peduncles, 0.8- 1.5 cm. • Length of peduncle • Length of flowers paired, flowers white, 8-15 cm; Pedicels peduncle 10- single on each of each flower 25 cm; peduncles, one short 0.5- 2 cm; Calyx Pedicels of and other long, calyx tube 1.5-1.8 cm each flower tube 4-5 cm long. long 2.5- 3 cm; subcylindric, teeth Calyx tube approximately 2mm 2.5- 3 cm long, reflexed, linear. • Peduncles in pair, single flowered, later dilated, petals oblong. become racemed, 8 -15 flowered, fimbriate, anther free. pedicels erect, bracteate, filiform. subsessile, oblong. calyx tube dilated at apex, apex 2.5 3.5- X 1.5 mm mm across, teeth approx. 2 mm long, reflexed, petals across 8-9 x 3 mm, oblong, 3 nerved, staminal filaments slender, across 2mm long; anthers ovoid, pistillode 15-17 mm Female • Peduncles 5- 8 mm • Peduncles 5- 20 • Peduncles 3- 5 flowers long, flower white. mm; ovary mm; solitary; ovary oblong. narrowly fusiform ovary villose, petals oblong, elongated fimbriate same as • Flowers white, solitary, style 16-18 mm explained in male long, filiform, ovary villose-hirsute flower Fruits • Fruits, 5-8x2-4 cm. • Fruits, with stalk, 5- • Fruits very long oblong- ovoid. 6 X 3.5-4 cm. up to 1.5- 2 m. spindale shaped, ovoid, conical/ cylindrical. glabrous, smooth. beaked, green often twisted. prominent remnant of with white stripes surface petals at one end. when young smooth, often green when young and become orange to 7-8 white. become red at maturity scarlet red on stripes along maturity the length Seed •5-8x5- 6mm, 5 mm • 11- 12 X 6-6.5 mm • 14-17 X 7-9 mm across, thick, globose. and 4 mm thick. and 3-5 mm black in colour ovate- oblong. thick, oblong, compresses. finely undulate, truncate/ rugulose. immarginate, base undulate, apex attenuate round or obscurely truncate, base attenuate Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Figure 6.1 Comparative macroscopic characters of T. dioica and T. cucumerina [var. cucumerina and var. anguina] A-F: T. dioica (A. Habit; B. Flowering twig; C. Female flower; D. Male flower; E. ; F. Seeds); G-L: T. cucumerina var. cucumerina (G. Habit; H. Fruiting twig; I. Male flowers; J. Female flowers; K. Fruit; L. Seeds); M-R: T. cucumerina var. anguina (M. Habit; N. Fruiting twig; O. Male flower; P. Female flower; Q. Fruit; R. Seeds)

6.3.2 Microscopy The detailed microscopy of TD and both varieties of TC were carried out. The results of both the species were as follows. Microscopic studies of TD Stem: The TS of the stem is pentagonal in outline covered with trichomes; collenchymatous tissues located underneath the epidermis, which being broader at ridges; followed by a small continuous ring of chlorenchymatous tissue which encircles the large ring of sclerenchymatous tissue; vascular bundles arranged in two circles, five in each circle situated alternate to each other; in general, vascular bundles of inner circle are larger and variable in size compared to that in the outer Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. circle. Central large pith is parenchymatous, in older stages, stem acquires wide lumen at the centre (Figure 6.2 A). TS in detail shows a layer of epidermis cells rectangular to oval in shape covered with thick cuticle bearing glandular and non glandular trichomes (described in detail in leaf) 3-5 rows of collenchymatous cells arranged underneath the epidermis, which become 3 - 8 layered at the elevations; followed by a ring of 1 - 3 layers of chlorenchymatous cells encircling the ring of 2 - 8 rows of thick- walled, penta to hexagonal sclerenchymatous cells; conjoint, bicollateral vascular bundles arranged in two circles in parenchymatous ground tissue; vascular bundles from inner ring are larger and more variable in size, xylem vessels are well developed, and of variable sizes; at maturity, central large parenchymatous pith may show large lumen; simple starch grains occasionally embedded in parenchymatous cells throughout the section (Figure 6.2 B). Leaf: The TS of leaf passing through midrib is convex at lower side and elevated at upper side with the patch of collenchyma; single large meristele embedded in the parenchymatous ground tissue, rarely two or three of smaller size, the larger one is situated in the centre encircled by the sclerenchymatous band; collenchymatous cells underlying the lower epidermis. Lamina being dorsiventral showing palisade cells underneath the upper epidermis and mesophyll at lower side; different types of trichomes present on both the epidermis (Figure 6.2 C). Detailed TS shows upper and lower epidermis covered with cuticle, epidermal cells are cubical, occasionally radially flattened; bear both non glandular and glandular trichomes present on upper as well as lower epidermis, non glandular trichomes being long, multicellular with pointed apices; glandular trichomes are multicellular stalked or sessile, stalk occasionally as long as non glandular trichomes, multicellular stalk and unicellular head, occasionally filled with yellowish brown content. Simple trichomes with pedestal base and cystoliths, lignified; Collenchymatous patch of 5 - 8 celled thick, present underneath the ridge of the upper epidermis at midrib region, while it is only 3-4 layered underneath the lower epidermis and getting discontinuous at lamina region. The bicollateral meristele of various sizes lies in the center, the lowest being bigger in size and is composed of radially arranged xylem vessels associated with tracheids, fibers, and medullary rays. Except the upper most meristele lying underneath the collenchymatous tissue, all other meristeles are encircled by a continuous band of Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. sclerenchyma fibers. One to three layers of pahsade cells lie underneath the upper epidermis at lamina, mesophyll cells lie underneath the lower epidermis occupying major area of lamina. Occasionally, smaller vascular bundles traversing the mesophyll tissue (Figure 6.2 D & E). Quantitative epidermal study: No differences were observed in the leaves of male and female plants. In both, anomocytic stomata were recorded on adaxial and abaxial surfaces. The observations are tabulated in (Table 6.3). Table 6.3 Epidermal study Epidermal Characters TD (per sq.mm)

Stomatal Adaxial surface 12.0 ±2.0 number Abaxial surface 28.0 ± 4.0 Stomatal index Adaxial surface 04.2 ±0.1 Abaxial surface 14.6 ± 0.2 Trichome Adaxial surface 12.0 ±0.2 index Abaxial surface 08.0 ± 0.3 Vein-islets number 04.2 ±0.1 Vein termination number 08.0 ± 0.2 Values are mean ± SD of six replicates

Petiole: TS of the petiole is shallowly pear- oval in outline with a groove at abaxial side giving wavy nature to the section; the circular ring of collenchymatous tissue lies underneath the epidermis; the sclerenchymatous tissue capped the individual vascular bundle; about 8-10 bicollateral meristeles arranged in ring around central ground tissue (Figure 6.2 F). Detailed TS shows single layer of epidermis, cells of epidermis are rectangular to radially elongated oval bearing glandular and non glandular trichomes like that of leaf; 3-5 rows of collenchymatous cells lie underneath the epidermis forming a continuous ring around the parenchymatous cortex, some collenchymatous cells replaced by chlorenchymatous cells occasionally; about 8-10 bicollateral meristeles arranged in the ring, lower two are smaller and underdeveloped lies at groves present at abaxial side; each one being capped with 2 - 5 celled sclerenchymatous cells; central pith showing larger parenchymatous cells. Simple starch grains embedded in parenchymatous cells (Figure 6.2 G). Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Fruit: TS of immature fruit is circular in outline; outer epidermis rarely covered with trichomes, followed by epicarp which encircles the hypodermis embedded with scattered bicollateral vascular bundles, six placentas attached with two immature seeds (Figure 6.2 H). Detailed TS shows a layer of epidermis occasionally covered with trichomes similar to that of described in leaf, at maturity, detached from epidermis; cells of epidermis being cubical, occasionally being slightly oval or elongated; upper 2 - 4 layers of pericarp consists of chlorenchymatous cells, the groups of sclerenchyma cells arranged isolated or in groups observed in mature fruit, finally forming a ring around the hypodermis; a zone of 6 - 10 layered parenchymatous hypodermis followed by large cortex; many small conjoint, bicollateral fibro-vascular bundles scattered distributed in the hypodermis; in parenchyma from central region, isolated sieve-tubes distributed occasionally; vascular bundles also observed near the placenta; simple starch grains embedded in parenchymatous cells; maximum 6 seeds attached with parietal placentation, one to each placentum (Figure 6.2 I).

Seed: The TS of seed is oval testa showing outer parenchymatous cells covered with epidermis, followed by layer of stone cells; small layer of parenchymatous cells and nucellus which being filled with oil globules (Figure 6.3 J). Detailed TS of testa shows outermost layer of compactly arranged, thin-walled palisade-like 1 - 2 layered cells with different lengths; occasionally, cells embedded with starch grains; followed by 10 - 17 layered thick-walled stone cells filled with yellowish brown content; followed by thick-walled, 2-4 layered compactly arranged radially elongated stone cells; followed by 1 - 3 layered of aerenchymatous cells with large air spaces encircles a collapsed cell layer. At hilar region, palisade-like cells are larger and arranged in 2 - 4 layers (Figure 6.3K). T3 g. » (U

O GO H

o CO H

lU Q- '^ o Q H d a o u V5 c o u e u

ui, •a a O 0) oo Q H ^^ •a <<

Q (U 3 OX) Q o -2 a .o -o E <*- o 00 H s >i Q o '« U S u

O C/5 u CO •4—t H ^ D- . o u Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Microscopic studies on powder of TD Organoleptic evaluation is presented in Table 6.4. Table 6.4 Organoleptic characters of powder Colour Odour Taste Feel Vegetative parts Green Not specific Bitter Rough, fibers Fruit Greenish yellow Not specific Sweet Granular

Microscopic studies of powder sample of vegetative parts and fruits were carried out and results are presented in Figure. 6.3. Vegetative parts: Powder shows fragments of upper and lower epidermis of leaf with anomocytic stomata; upper epidermis of leaf showing two layered palisade cells and trichome; glandular and non glandular trichomes; fragments of vessels and tracheids having pitted and spiral thickenings; fragments of elongated or polygonal thick-walled stone cells and sclereids. Fruits: Powder shows fragment of thick-walled, polygonal cells of pericarp in surface view showing stomata; palisade-like, elongated cells of testa in transverse view embedded with simple starch grains; fragment of polygonal thick-walled sclereids from seed embedded with starch grains; fragment of polygonal thick- walled, compactly arranged cells of testa in surface view; tricolporate pollen grains with very thin pollen wall; simple, oval to round starch grains with central hilum and prismatic crystals of calcium oxalate scattered as such. Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Figure 6.3 Structural details of powder of TD A. Upper epidermis with stomata and trichome base; B. Lower epidermis with trichome; C. Trichomes type; D. Sclerenchyma; E. Stone cells; F & G. Endospermic and nucellar cells; H. Testa cells; I. Vessels; J. Septate and non- septate fibres; K. Upper epidermis; L. Surface view of testa, M. Pericarp in surface view; N. Pollen grains; O. Simple starch grains; P. Prismatic crystals of calcium oxalate.

Microscopic studies of TC Comparative microscopic studies were carried out for both the varieties of TC. However, very few cellular differences were observed in both the varieties which mentioned below in bracket with bold. Stem: TS of the stem is pentagonal in outline (deep ridges and furrows in var. anguina) covered with trichomes; collenchymatous tissues located underneath the epidermis at the ridges; followed by a continuous ring of chlorenchymatous tissue which encircles the ring of sclerenchymatous tissue; vascular bundles arranged in Chapter VI Trichosanihes dioica Roxb. & Trichosanthes cucumerina L. two rows, five in each row situated akernate to each other; members of inner row are variable in size compared to that in outer row. Central large pith is parenchymatous; acquire large lumen at the centre in older stages (Figure 6.4 A & E). TS in detailed shows a layer of conical or tangentially elongated cells of epidermis covered with cuticle bearing glandular and non glandular trichomes similar to that on leaf; 2-4 rows of collenchymatous cells arranged underneath the epidermis, become 5-10 layered at the elevations; occasionally (except from the elevations) collenchymatous cells are replaced by chlorenchymatous cells at ridges which are 2 - 6 layered; followed by the ring of 4-7 rows of thick-walled, penta- to hexagonal sclerenchymatous cells; conjoint, bicollateral vascular bundles arranged in two rows in parenchymatous ground tissue; vascular bundles from inner ring are larger and more variable in size, xylem vessels are well developed; at maturity, central large parenchymatous pith may show large lumen; simple starch grains occasionally present in parenchymatous cells (Figure 6.4 B & F). (In var. cucumerina, swollen portion of stem shows excessive growth of parenchyma and irregularly scattered vascular bundles). Leaf: The TS of leaf passing through midrib is dorsiventral, largely convex at lower side and slightly elevated at upper side; 3 vascular bundles of various sizes embedded in the parenchymatous ground tissue, the central vascular bundle is larger and mature; collenchymatous cells underlying the lower epidermis and elevated part of upper epidermis (Figure 6.4 C & G). Lamina is dorsiventral flattened showing single layer of palisade underneath the upper epidermis and mesophyll underneath the lower epidermis; different types of trichomes present on both the epidermis (Figure 6.4 D & H). TS shows upper and lower epidermis covered with thin cuticle, epidermal cells are radially flattened to rectangular and square; both non glandular and glandular trichomes present occasionally on upper and lower epidermis, former trichomes being long, multicellular, occasionally unicellular with pointed or obtuse; latter are rare, multicellular stalked or sessile, stalks occasionally as long as non glandular trichomes, head is multicellular, globose; mostly with yellowish brown content. At the mid- rib the elevated portion 5-12 layered collenchymatous patch present, while it is 3 - 4 layered underneath the lower epidermis and getting discontinuous at lamina region. About 3-6 bicollateral vascular bundles traversed in parenchymatous ground tissue, larger one placed at the centre, other smaller placed just above of it; 2 - 4 layered Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. parenchymatous cells, covering the phloem of the vascular bundles, are smaller than other ground tissue. One layered, (multilayered in van cucumerina) small palisade cells lie underneath the upper epidermis of lamina, mesophyll cells lie underneath the lower epidermis occupying major area of lamina (Figure 6.4 D & H). Quantitative epidermal study: In both the varieties anomocytic stomata were recorded on abaxial surfaces. The results are presented in Table 6.3. Table 6.3 Comparative epidermal study Epidermal Characters TCC (per sq.mm) TCA (per sq.mm) Stomatal Adaxial surface - - number Abaxial surface 24 ± 2.0 12 ±2.0 Stomatal index Adaxial surface - - Abaxial surface 25 ± 1.0 17± 1.0 Trichome index Adaxial surface 03 ± 2.0 7 ±2.0 Abaxial surface 07 ±2.0 11 ±2.0 Vein- islets number 15 ±2.0 20 ±2.0 Vein termination number 23 ± 2.0 33 ±2.0 Values are mean ± SD of six replicates Petiole: The TS of the petiole is pentamerous (shallow in var. cucumerina) in outline with a groove at abaxial side; shallow ridge has a patch of collenchymatous tissue; the sclerenchymatous tissue encircles the individual vascular bundle; about 8-12 meristeles arranged in a ring around central ground tissue (Figure 6.5 I & O). Detailed TS shows single layer of epidermis, cells of epidermis are rectangular to elongated oval bearing glandular and non glandular trichomes like that of leaf; 3 - 5 rows of collenchymatous cells lie underneath the epidermis forming a continuous ring around the parenchymatous cortex, some collenchymatous cells replaced by chlorenchymatous cells occasionally; about 9 - 11 bicollateral meristeles arranged in the ring, lower two are smaller and underdeveloped; each being capped with 2 - 5 celled, thick-walled sclerenchymatous cells; central pith showing larger parenchymatous cells. Simple starch grains embedded in parenchymatous cells (Figure 6.5 J & P). Fruits (stalk fruits are only present in TCC and described as follows): TS of fruit stalk is irregularly circular in outline covered with trichomes, a continuous ring of collenchymatous tissue encircles the ring of sclerenchymatous tissue, 6 - 8 Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. meristeles arranged in circle; each meristele capped with curved band of sclerenchymatous cells when young; central ground tissue being parenchymatous. TS in detailed shows a layer of epidermis covered with cuticle and with glandular and non- glandular trichomes similar to that described in leaf; followed by 3 - 6 rows of collenchymatous cells, some cells embedded with chlorophyll pigment; followed by 2 - 5 layered penta to hexagonal sclerenchymatous cells; conjoint, bicollateral vascular bundles traversed in parenchymatous ground tissue; pith cells are polygonal, thin-walled rarely filled with simple and compound starch grains. Fruit: In structural anatomy fruits of both the varieties show similarity. TS of immature fruit in both the varieties is roughly circular in outline; outer epidermis covered with trichomes, followed by epicarp encircling the hypodermis embedded with scattered bicollateral vascular bundles, six placentas attached with two immature seeds (Figure 6.5 K & Q). TS in detailed shows a layer of epidermis covered with trichomes similar to that described in leaf, detaching at maturity of fruit; cells of epidermis cubical, occasionally slightly oval, upper layer of pericarp consists of chlorenchymatous cells, the groups of sclerenchyma cells arranged either isolated or in groups in mature fruit, finally forming a ring; a zone of 6 - 10 layered parenchymatous hypodermis (5-7 layered in var. anguina) followed by large cortex; many small conjoint, bicollateral fibro-vascular bundles scattered in the hypodermis; in parenchyma from central region, isolated sieve-tubes distributed occasionally; vascular bundles also observer near the placenta; simple starch grains embedded in parenchymatous cells; maximum 6 seeds attached with parietal placentation, one to each placenta (Figure 6.5 L & R). Seed: TS of seed is oval in outline with irregular margin showing shallow ridges and furrows, testa showing outer palisade-like elongated cells in var. anguina (however, in var. cucumerina outer parenchymatous cells covered with epidermis), followed by many layers of stone cells; small layer of parenchymatous cells and nucellus filled with oil globules (Figure 6.5 M & S). Detailed TS of testa shows outermost layer of compactly arranged, thin-walled palisade-like 1 - 3 layered elongated, penta- to hexagonal cells with different lengths; occasionally, some cells embedded with starch grains; followed by 10 - 16 in var. anguina were as 7 - 13 layered in var. cucumerina, thick-walled, pitted parenchyma cells showing intercellular spaces, some cells filled with simple starch grains and prismatic crystals of calcium oxalate, outer 1 - 2 rows of cells being filled with Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L. brownish content; followed by thick-walled, 2 - 4 layered compactly arranged stone cells, the inner layer of which is larger and radially elongated; 1 - 3 layered aerenchymatous cells with large air spaces encircles a collapsed cell layer. At hilar region, palisade-like cells are larger and arranged in 3 - 4 layers (in var. anguina stone cells are larger and more in number), compactly embedded with starch grains, pitted parenchyma cells reduced in number and arranged in 4 - 6 layers. (Figure 6.5 N & T). < Hu XI U c •a « 'E c^ u c3 O ^ XI J= >, 0 (u«- o M X a. u (U M > Q. O X H t+- :M 0 0 C/2 JO H 'u -0 I s ^ CM a •I ^ ^ •a -4-^ s c c/: « !+- s 0 s S o iM •S 0 tin" s 13 a •^ o 0 w C/u2 0 0 ^ U u H 'sii •2 'u X > •a '•C o « 'E uss t*- a ca S 3 00 •^^ H < so a u ^ O o -5 ^_* o u VI Q X Q d O O c« > 3 =a exi b u E 4-

t^ Hu C3 •d c (4- u o GO U f- U H (X •s =« •a O ij 1/3 u T3 e Hu rt . r a 'k' c :^ C 3 OJ (^ 0) O I o C>0 s '•3 H OJ a ^ :M . r -s 03 •T3 o; 1) , O a c/^ o i-

X G 1 .E

t4_ tmt> - •*•' o 8 as C/1 C8 H -J o c U :^ < i/i . r. 1) -4—' a \C o u v O u -^^ 3 H) (/) a. H b 0) H T3 o (/) c« a H XI o U ~> C/) <« H MiM GO <: w 3 m^r- Hu ^ Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Comparative microscopic studies of TCC and TCA powder The results of powder of organoleptic evaluation of TCC and TCA are presented in Table 6.4. Table 6.4 Organoleptic characters of TCC and TCA powder Test TCC TCA VP VP Colour Green Green Creamish yellow Yellow to brown Odour Bitter Not specific Sweet Bitter Taste Bitter Not specific Sweet Bitter Feel Rough, fibrous Rough, fibrous Rough Rough VP- vegetative parts; F-fruits Both the varieties shared common cellular characters with some dimensional differences showed in the Figure 6.6. The detailed characters observed in both the powder are as follows. Vegetative part: The powder of both varieties show fragments of upper and lower epidermis from leaf with anomocytic stomata and wavy cell walls; leaf fragment shows single layered palisade cells with trichome; trichomes glandular and non glandular, similar as describe in leaf section; fragments of vessels and tracheids showing pitted and spiral thickening; groups of polygonal thick-walled stone cells. In TCC leaf trichomes, thin walled, irregular in shape and without flattened disk at base, however, some trichomes are slightly conical and end with acute apex. On the other hand, in TCA leaf trichomes with slightly blunt apex. In both the species epidermis shows straight anticlinal cell wall pattern at adaxial and curved at abaxial surfaces with irregular epidermal cell wall shape and anomocytic stomata exclusively on lower surfaces. Fruit: Powders of both the varieties commonly show groups of polygonal thick- walled stone cells; palisade-like, elongated cells of testa in transverse view embedded with simple starch grains; fragment of polygonal thick-walled, compactly arranged cells of testa in surface view; tricolporate pollen grains with very thin pollen wall; simple, oval to round starch grains with central hilum (In TCC powder prismatic crystals of calcium oxalate scattered as such). Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Figure 6.6 Comparative powder analysis of TCA and TCC TCA and TCC: A & B. Lower epidermis of leaf showing stomata and trichome base; C. Different types of trichomes; D. Fragment of upper epidermis with trichomes, layer of palisade cells; E. Spiral, pitted vessels and tracheids; F. Septate and non septate fibres; G. Pitted parenchyma; H. Fruit epicarp; I. Stone cells; J. Fragment of pericarp; K. Fragment of testa; L. Endospermic and cotyledonous cells embedded with oil globules; M. Simple starch grains; N. Pollen grains; O. Prismatic crystals.

6.3.3 Proximate analysis: The results of proximate analysis are complied in Table 6.5. No significant differences were observed in analysis except foaming index and amount of carbohydrate in both the species. n — r-1 fN (^) —; o O o o o o o o o o o O o o o -H -H -H -H -H -H +1 -H -H +1 +1 -H +1 -H -H -H o o o +1 m — — in ^ in CN oo " O o oo r-j o -- — r-- O oi o o yn — 00 oi in oo O O o o o o —

r) — ro (^1 o o o o o o o o O C o o o o o o o -H -H -H +1 +1 -H +1 -H +1 +1 -H -H -H +1 -H -H -H ri o o O O oo r\i -- in -* oc o o in in r-1 o O oo — -* in oo ON oo o oo OO o o o O — r--) o in — o o o —

•—I m o o o o o O o O o o o o o o o O O +1 •M -H +1 -H +1 -H -H -H -H -H -H +1 -H M -H iri o O CN) o ^ — o +1 r- — in in o O ^ o oo OO NO o r^ ON ON in in oo CN) — o o oo — o O o ^ o — — Tj-

(^1 CM o\ o o o o O o o o o o o o o O o o o +1 -H -H +1 -H +1 -H -H -H -H -H +1 +1 -H -H +1 +1 -^ o o •O oo oo NO NC oo ir-i r-i o in in NO NO in in OO oo — o in X o o o '— o od o

m m CNl o o O o O O C5 o O o o o o o n o o +1 +1 -H -H +1 -H +1 +1 +1 -H +1 +1 -H -H o ON o o o CM CNI in "'^ NO oo O o o in oo ^ r~- o MO (N 00 (N in 1^ CM' r-i *-! r-i o o o NO CM o O o — r-J —

m CNI — O o o O o o o o o o O o o O o o -H +1 -H -H -H -H -H -H -H -H -H -H -fl +1 s; 4^ +1 -H oo o o in in NO in oo oo _« ON oo in ON o o 00 NO o NO o in CNI > CM in VI o in o in oo' o CD V ^ — o o NO CM o o NO •*—' VI O o CL >^ « U C

o c I •I ^ ^ i ~£i) >; E M vi I ^ b£ ^-^ 4> OJj *< it. ^^^ u -. Vi &£ U VI IS .s w c s S5 O • art c 3 o ^ o IT/ e ,—1 •fc- '3 ^ 1/3 •a '3 ^ « O Vi B i 3 S5 C5 O o -a O O a: 3 « H 0. tb CZJ c U &. U. < > oi SI U i-

> Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

6.3.4 Phytochetnical studies A. Qualitative assessments (Micro-chemical tests) The results of different tests carried out to assess presences of different phyto- constituents are presented in Table 6.6 Table 6.6 Qualitative assessments of phyto-constituents in powders Powder + Reagent Reaction TD TCA TCC Inference VP F VP F VP F Dragendorffs reagent Reddish + + + + + + Alkaloid brown Wagner's reagent Reddish brown Diluted HCI + Mayer's Creamy reagent colour Ninhydrin Purple + + + + + + Amino- colour acid Sudan III Red colour + + + + + + Fats 80% v/v H2SO4 Orange or + + + + + + Flavonoid yellow colour 1% picric acid + drop of Red colour + + + + + + Glycosides 10% aq. Sodium carbonate Acetic acid + FeCb + 2 Blue colour + + + + + + Glycosides drops cone. H2SO4 Phloroglucinol + HCI Red colour + + + + + + Lignin Ruthenium red Red colour + + + + + + Mucilage Millon's reagent Brick- red + + + + + + Protein colour Benedict's reagent Green- + + + + + + Reducing brick- red sugar colour Iodine Blue colour + + + + + + Starch Molybdophosphoric acid Blue colour + - + Steroides & + - Terpenes 1% Vanillin 50% Blue colour + - + Steroides & phosphoric acid + - Terpenes 5% FeCb Black/dark + + + + Tannins green + + (- Absent; + Present) Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

Secondary metabolites viz. tannins, phenols, flavonoids, glycosides, steroids and alkaloids are present in all the parts. The test of steroides & terpenes found positive for vegetative part of TD and both vegetative as well as fruits of TCC. B Quantitative assessments The results of estimation of total tannins, polyphenols, flavonoids and alkaloids are presented in Table 6.7 Table 6.7 Quantitative assessment of phyto-constituents Group of TD TCA TCC chemicals ^V? F VP F VP F (mg/g) 48.50 ± 93.40 ± 38.30 ± 97.50 ± 62.50 ± 91.20 ± Tannins 0.1 0.1 0.1 0.1 0.1 0.1 37.10± 47.10± 37.10± 65.10± 56.40± 72.30± Polyphenols 0.1 0.1 0.1 0.1 0.1 0.1 00.35 ± 00.34 ± 00.21 ± 00.49 ± 00.28 ± 04.33 ± Flavonoids 0.1 0.1 0.1 0.1 0.1 0.1 00.20 ± 00.32 ± 00.14 ± 00.33 ± 00.16 ± 00.34 ± Alkaloids 0.1 0.1 0.1 0.1 0.1 0.1 Values are mean ± SD of six replicates

6.4 Chromatographic fingerprinting The extracts were subjected to preliminary TLC standardization. The maximum/ better separation of phyto-constituents from petroleum extract was observed in Toluene-Chloroform-Acetone (5.0:4,5:0.5, v/v/v/v) where as methanol extract show good separation in Chloroform: methanol (9.5:0.5, v/v) (Figure 6.7). Plates were observed under 254 nm, 366 nm and derivatized with with anisaldehyde sulphuric acid reagent (heating at 105°C for 5- 10 min). The numbers of bands along with the respective Rp values were recorded (Table 6.8). Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

254 nm 366 nm After derivatization

^

2^1^-H

Figure 6.7 Comparative TLC profile of petroleum ether and methanol extracts A-F: Petroleum ether extract, G-L: Methanol extract, Tracks 1 (VP) & 2 (F)- TD; 3 (VP) & 4 (F)- TCC; 5 (VP) & 6 (F)- TCA Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumer'ma L.

Table 6.8 HPTLC fingerprinting of petroleum ether and methanol extracts

Extract TD TCA TCC VP F VP F VP F Petroleum ether extract 254nm 0.12, 0.19, 0.31,0.76 0.41, 0.41,0 0.15, 0.33, 0.57, (4*) 0.64, .69, 0.41, 0.52, 0.65, 0.69, 0.58 0.69, 0.68, 0.70, 0.78, (3*) 0.85 0.83, 0.80(5*) 0.91(5*) (4*) 0.87(6*) 366nm o7T8^ ^A9, 0.90(1*) 0.63(1*) 056 0.83 0.22, 0.22, (1*) (1*) 0.87(3*) 0.62, 0.79, 0.88 (^ Derivatization aT2^ oT9^ 0.31,0.76 (X4K (L58 0.15, 0.33, 0.57, (4*) 0.64, (2*) 0.41, 0.52, 0.65, 0.69, 0.69, 0.68, 0.70, 0.78, 0.85 0.83, 0.80(5*) 0.91(5*) (4*) 0.87(6*) Methanol extract 254nm 'QA2, O712^ 0.12,0.34, 02^ 07l2^ 0.12, 0.34, 0.34, 0.36,0.52 0.34, 0.21, 0.21, 0.39, 0.39, 0.67,0.85 0.36, 0.39, 0.35, 0.45, 0.45, (6*) 0.52, 0.42, 0.45 0.52, 0.48,0.54 0.67(5*) 0.51, (4*) 0.67, (6*) 0.59, 0.85 (7*) 0.62 (7*) 366nm 0.34, 0.34, 0.33,0.42, 0.18, 0.28, 0.22, 0.39, 0.39, 0.42,0.48, 0.32, 0.32, 0.32, 0.45, 0.43, 0.64,0.72, 0.40, 0.36, 0.32, 0.89(4*) 0.45, 0.81,0.89 0.64, 0.42 0.38, 0.89(5*) (8*) 0.72,0.89 (4*) 0.42 ifi^ (5*) Derivatization 0.22, 0.19, 0.31,0.42, 0.31, 0.33, 0.33, 0.32, 0.29, 0.52,0.67, 0.52, 0.83 0.42, 0.34, 0.31, 079,0.83 0.67, (2*) 0.58, 0.42, 0.42, (6*) 0.78,0.83 0.83 0.52, 0.54,0.83 (5*) (4*) 0.67, (6*) 0.79, 0.83 (8*) * Total number of bands Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L.

6.5 HPTLC method for quantitative estimation of bitter constituent: Cucurbitacin B The mobile phase Chloroform: methanol (9.5:0.5, v/v) gave optimized result with sharp, symmetrical, and well resolved peak of Cucurbitacin B at RF 0.52 from other components of the samples extract (Figure 6.8). A linear relationship was obtained between response (peak area) and amount of Cucurbitacin B in the range 500 - 2500 jig; the correlation coefficient was r = 0.99694, (y = 7.843+0.06092*X). The amount of Cucurbitacin B was found in vegetative parts and fruits of both species are documented in Table 6.9. The results showed that fruits of TD, vegetative part and fruits of TCA were devoid of Cucurbitacin B.

Substance: CUCURBITACIN B ® 254 nm

Regression via height Linear Y . 7 843 + 0 06092 "X r - 0 99694 sdv - 4 39 Regression via area Linear Y - 50 58 + 1 705 * X r - 0.99721 sdv - 4 46

Track Vial Rf Amount Height X(Calc) Area XfCalc) SamDielD/Rsmark

1 1 0.52 500 00 |jg 33.97 792 39 2 1 0.52 1000.00 Mg 7176 1826 64 A 3 1 0 52 1 500 mg 102.32 2686 44 " 4 1 0.62 2 000 mg 131.81 3530 70 5 1 0 62 2.500 ma 156 24 4202 19 6 1 0.52 32.63 450.00 Mq 933.35 517 84uq 7 1 8 1 0 52 56.44 781.37 Mg 1835 95 1.047 mg 9 1 0 52 70.44 1 028 mg 2154 47 1 234 mg 10 1 1 ^ B 1

Figure 6.8 HPTLC estimation of Cucurbitacin B A. Calibration data for Cucurbitacin B Standards (1-5); TD (VP: 6; F: 7); TCC (VP: 8; F: 9); TCA (VP: 10; F: 11); B. HPTLC plate scanned at 242 nm (1-5); TD (VP: 6; F: 7); TCC (VP: 8; F: 9); TCA (VP: 10; F: 11) Chapter VI Trichosanthes dioka Roxb. & Trichosanthes cucumerina L.

Table 6.9 Estimation of Cucurbitacin B Cucurbitacin B (%) VP 1.12 ±0.04 TD F Absent VP 2.34 ±0.08 TCC F 3.64 ±0.05 VP Absent TCA F Absent Values are mean ± SD of six replicates

6.6 Antioxidant potential The antioxidant potential of vegetative part and fruits of TD, TCC and TCA was determined by using different spectrophotometric assays. The results of these studies are express in IC.soin |ig/ml (Table 6.10). "^ -^

rl r-n * iri * O C5 o o O +1 +1 +1 +1 +1 .—. O m to r-i GN r^ in oo oo H od NO r-i u r-i u O * *

o o CD o +1 +1 +1 +1 -H in rl r'n (N NO in o iri od ON' > ^ r-- * * * CNJ * CNI o o o o C5 +1 M -H +1 m VC +1 m — CNJ ON o ^ 0^ ON ON od m o < ^ iri U V H * * * ts o O o O O o +1 -H +1 +1 c 03 — oo ON OO o Cl. > r-- in 'c CO * * * in m o o o o o C3 o OO c~- +1 -H -H +1 -H '-5 (N ^ "^ (N CNJ NO c ^ o ON CNI K r-1 Q ri

* * CJ CM * CN) CJ O o o o -H -H -H -H -H oo ON CNJ tso cu od O oo in u > NO

ON ON ON ON CJ e -a o \o NO 13 u CJ « o ^ o CD -H 'x -H -H -H -H "5. B lO oo OJ _o c o ON in i^ ^ od od NO s G3 CN) « o o > |> en _> a- u o %-» 0) X « S5 O C3 So u o o BX) 'o a o — -H -a C E S '5c o u O c X £ c O O c^ DO in o o < o o c O en C c o 'x Q. X U 3 o C3 1/3 _o < C CQ o c c/) o O 'x 'x c -. > -o o o o 4> •5 P- u Vi '5 'a. 'C p ex CO a- o C3 U Q H C/0 < H CJ > Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

6.7 Molecular profiling using ISSR markers TD (Male and female plant); TCC and TCA samples were collected from different locations of Maharashtra and Gujarat. Identified plant samples were used to develop comparative molecular profiles using ISSR markers. Initially, total 94 primers (UBC 807-900) were screened for their ability to amplify DNA fragments using these four samples. Out of the 94 primers tested, 68 primers did not amplify any fragments; 10 produced either faint or no bands upon a highly smeared background and remaining 16 generated band patterns. Based on the clear, scoreable and reproducible band patterns, 10 primers were selected for further profiling work (Table 6.11). Table 6.11 ISSR primers selected for profiling of TD and TC

Primers Sequences No. of Bands 808 5'-(AG)nC-3' 8 809 5'-(AG)nG-3' 4 812 5"-(GA)nA-3' 6 813 5--(CT)nT-3- 6 818 5'-(CA)nG-3' 7 836 5--(AG)nYA-3' 4 842 5"-(GA)nYG-3" 5 887 5'-DVD(TC)n-3' 8 888 5'- BDB (CA)n-3' 12 889 5"-DBD(AC)n-3" 6 Total 66

Y=(C,T); B=(C,G,T)(i.e not A); D=(A,G.T) (i.e not C); V=(A,C. G) (i.e not T)

A total of 66 bands were scored from the selected 10 primers. The profiles generated by selected markers did not show any polymorphic/ differentiating bands in male and female plant profiles of TD. However, all the selected polymorphic primers contained di-nucleotide repeats and generated distinct band profiles for TD and both the varieties of TC. It was possible to distinguish between TD and TC (TCC and TCA) based on the profiles. The amplification profiles generated by primers UBC 808, 887. 888 and 889 are shown in Figure 6.9. The sizes of the amplified products ranged from 400 to 1815 bp. The number of bands varied from 4 (UBC 809 & UBC 836) to 12 (UBC 888). Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

Figure 6.9 ISSR amplification profiles of TD and TC samples A. 808; B. 887; C. 888; D.889 M- Low range DNA ruler plus; TD: l(Male) and 2 (Female); TCC: 3; TCA: 4

Among these primers UBC 812, 822, 887, 888, 889 produced specific bands which clearly distinguish the closely related varieties of TC i.e. TCA and TCC from each other. The specific bands generated using specific primers profiles are presented in Table 6.12 Table 6.12 Diagnostic markers for identification of both varieties of TC Markers showing specific bands

TCA UBC 8127,8*; 883M474< UBC 822960*; UBC 887950* TCC UBC 812624*; UBC 822456*;UBC 888632*; UBC 889685* * Molecular weight in base pairs (bp) Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

To validate the presence of species specific bands, 3 sets of well identified TCC and TCA samples collected from different locations were extracted and used for study. The primer profiles generated by UBC 812, 822, 888, 889 are presented in Figure 6.10. These results confirmed the presence of diagnostic bands and showed that UBC 812, 822, 888, 889 primers could be used as species specific primers for correct identification and distinguishing these two varieties.

Figure 6.10 ISSR amplification profiles showing specific bands in TCC and TCA A. 812; B. 822; C. 887; D.889; M- Low range DNA ruler plus; TCA: 1, 3, 5 ; TCC: 2, 4, 6 (Amboli, Akola, Kankawali, Kolhapur, Junnar)

6.8 Investigations of market samples Market survey: Eight market samples were processed for pharmacognostic identification. Moisture and foreign matter percentage were recorded and is presented in Table 6.13. Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

Table 6.13 Detection of moisture and foreign matter Market Moisture (%) Foreign matter (%) Akoia 12.84 ±0.04 03.12 ±0.01 Dhule 06.41 ±0.03 20.12 ±0.12 Kolhapur 03.12 ±0.02 03.10 ±0.03 Mumbai 05.19 ±0.06 07.12±0.13 Pune 02.14 ±0.02 03.12 ±0.02 Ratnagiri 08.47 ± 0.03 14.15 ±0.01 Surat 11.17±0.02 12.14±0.03 Lucknow 03.41 ±0.04 04.47 ±0.01

Samples from Akola and Surat contained higher amount of moisture while samples from Dhule, Mumbai, Ratnagiri and Surat exceeded the limit of % foreign matter. Except sample form Pune market all the remaining samples were of low quality and should be rejected as per pharmacopoeial norms. However, since the aim of present study was to establish the correct identity, these samples were processed for macroscopic and microscopic study 6.8.1 Macroscopy: All the collected crude drug samples were in semi granular or fine powdered form (Figure 6.11). The detailed macroscopic characters of these powders are presented in Table 6.14. Due to powdery nature of the samples macroscopic characters were of limited use to established correct identity of these samples. Hence, all the samples were further processed for microscopic analysis. 6.8.2 Microscopic observations: The detailed microscopic characters of powders are presented in Table 6.15. On the basis of these primary microscopic observations the samples from Kolhapur, Pune, Surat and Lucknow were from vegetative parts of plants. On the other hand samples from Akola, Dhule, Mumbai and Ratnagiri contained dried fruit parts. On the basis of microscopic characters samples from Dhule may belong to TCC Sample from Ratnagiri was admixture of TCC and MC fruits, where as sample from Surat was completely replaced by other species which could not be identified. For the confirmation of the above results all the samples were further processed for chromatographic examinations by using HPTLC. Chapter VI Trichosanthes dioicci Roxb. & Trichosanthes ciicumerina L

Figure 6.11 Market samples of ^Patola I Ranpatola'' A. Akola; B. Dhule; C. Kolhapur; D. Mumbai; E. Pune; F. Ratnagiri; G. Sural; H. Lucknow

Table 6.14 Macroscopic characters of market samples Samples Colour Texture Taste Akola Brown to yellow Oily granular, rough Bitter Dhule Brown to yellow Oily granular, rough Bitter Kolhapur Light green to straw Fibrous, smooth Bitter Mumbai Blackish brown Granular, rough, Bitter Pune Light green to straw Fibrous, smooth Non bitter Ratnagiri Blackish brown to yellow Granular, rough, Bitter Surat Dark green Fibrous, rough Non bitter Lucknow Green to straw Fibrous Bitter Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

Table 6.15 Microscopic characters of market samples , c >- Samples Characters of powder Akola Group of hexagonal thick walled collenchymatous cells; polygonal thick-walled stone cells, elongated cells filled with simple starch grains; group of compactly arranged stone cells; starch grains simple and prismatic crystals of calcium oxalate Dhule Hexagonal, thick walled collenchymatous cell layer with anomocytic stomata; small groups of polygonal thick-walled stone cells, transverse layer of testa with palisade-like, elongated cells filled with simple starch grains; group of compactly arranged stone cells; starch grains simple, oval to round with central hilum, and prismatic crystals of calcium oxalate Kolhapur Small fragments of epidermis with anomocytic stomata and wavy cell walls; disturbed trichomes; fragments of vessels and tracheids showing pitted and spiral thickenings; groups of polygonal thick- walled stone cells and prismatic crystals Mumbai Squarish to rectangular thin-walled epidermal cells with stomata; glandular trichomes with multicellular head; small groups of chlorenchymatous tissue with crystals of calcium oxalate; starch grains; small fragment of palisade cells with thick cuticle, small fragment of isodiametric ells; sclerenchymatous thick walled; starch grains, aleurone grains and prismatic crystals Pune Small fragments of epidermis with anomocytic stomata and wavy cell walls; disturbed trichomes; fragments of vessels and tracheids showing pitted and spiral thickenings; groups of polygonal thick- walled stone cells Ratnagiri Squarish to rectangular thin-walled epidermal cells with stomata; glandular trichomes with multicellular head; small groups of chlorenchymatous tissue with crystals of calcium oxalate; starch grains; small fragment of palisade cells with thick cuticle, small fragment of isodiametric ells; sclerenchymatous thick walled; starch grains, aleurone grains and prismatic crystals Surat Fragment of upper and lower epidermis of leaf showing angular straight epidermal cell walls wdth the eglandular trichomes and anomocytic stomata; trichomes two-celled, with cystolithic appendages, curved pointed apical cell and 8-10 flattened basal cells; fragments of vessels and tracheids showing pitted and spiral thickenings Lucknow Fragments of upper and lower epidermis of leaf with anomocytic stomata, wavy epidermal cell walls and trichomes; trichomes glandular and non glandular; fragments of vessels and tracheids showing pitted and spiral thickenings; fragments of elongated or polygonal thick-walled stone cells and sclereids Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

6.8.3 Quantitative determination of Cucurbitacin B from crude drug samples All the market samples were processed for estimation of Cucurbitacin B by HPTLC method. All conditions and parameter for HPTLC were followed as described under section (4.3.5). The calibration curve was obtained for Cucurbitacin B in the concentration range of 500 |jg - 2 mg. The respective regression equation was y = 7.843 +0.06092*x (r= 0.99). The results confirm the presence of Cucurbitacin B in the market samples Akola, Dhule, Kolhapur, Ratnagiri and Lucknow and are documented in Table 6.16. The remaining samples from Mumbai, Pune, and Surat market were found to be devoid of Cucurbitacin B (Figure 6.12).

wInCATS Planar Chromatography Manag«r

Substance; CUCURBITACIN B C3> 2S4 nm Regress on via heigh Linear Y - 7 843 + 0 06092 * X r - 0 99694 sdv - Regress on via area Linear Y - 50 58 + 1 705 • X r- 0 99721 Sdv - Track V •1 Rf Amount hMaht X(Calc) Are* xrcaici Sam Dial D/Ramark , 0 52 500 00 MQ 33 97 792 39 2 0 52 000 00 M9 71 76 1826 64 3 0 52 1 500 mg 102 32 26B6 44 4 0 52 2 000 mg 131 81 3530 70 5 6 0 52 32 63 450 00 pg 933 35 517 84 jjg 7 0 52 70 44 t 028 mg 21 54 47 1 234 mg 8 0 52 55 44 781 37 Mg 1835 95 1 047 mg g 10 11 12 0 52 41 1 74 2 750 mg 17871 27 2 750 mg 13 0 52 19 79 45O.00 M9 532 74 450 00 Mg 14 0 52 336 23 2 750 mg 13019 80 2 750 mg 15 16 17 0 52 18 00 450 DO Mg 607 27 450 00 M9 B 18 19 0 52 353 26 2 750 mg 14213 16 2 750 mg

Figure 6.12 HPTLC analysis of market samples A. Calibration plot obtained from Cucurbitacin B Standards (1-4); TD (F: 6; VP: 7); TCC (F: 8; VP: 9); TCA (F: 8; VP: 9); Market samples (12: Akola; 13: Dhule; 14: Kolhapur; 15: Mumbai; 16: Pune; 17: Ratnagiri; 18: Surat; 19: Lucknow) B. HPTLC profile at 242 nm Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

Table 6.16 Cucurbitacin B in market samples Market sample Cucurbitacin B (%) Akola 2.750 ±0.510 Dhule 2.450 ±0.454 Kolhapur 1.248 ±0.241 Ratnagiri 0.831 ±0.085 Lucknow 1.082 ±0.375 Values are mean ± SD of n==6 replicates. On the basis of microscopic characters and HPTLC profile it is confirmed that samples from Dhule was of fruits while that from Kolhapur was of vegetative part of TCC. Sample from Ratnagiri was admixture of TCC and MC fruits, where as Surat sample was replacement by unidentified species. Identity of remaining four samples could not be established due to insufficient diagnostic microscopic characters as well as non specific chemical profile as many of closely related species shows similar chemical profiles. Therefore these samples were further processed for molecular fingerprinting. To establish the correct botanical identity of crude drug resources, four species of genus Trichosanthes species along with Momordica charantia (MC) were collected from field, identified and voucher specimens were deposited in AHMA (Table 6.17). The tender leaves of authentic samples were stored in Liquid nitrogen and further used for DNA extraction and analysis. Table 6.17 Collection details of Genus Trichosanthes L. Species Locality Date Voucher no. T. dioica Roxb. [Male] Anand 28/09/2010 25493 r. rf/o/ca Roxb. [Female] Anand 28/09/2010 25495 T. cucumerina var. anguina (L.) Kankwali 26/08/2009 21 All Haines T. cucumerina \2ir. cucumerina Kankwali 16/09/2009 27006 T. tricuspidata \.o\ir.[Ma\t\ Kolhapur 24/08/2011 27410 T.tricuspidata\.o\ir.[Ytra2i\t\ Kolhapur 24/08/2011 27414 Momordica Charantia U Bardoli 01/09/2011 27461 Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

6.8.4 Molecular profiling Four species of Trichosanthes including commonly adulterated species Momordica charantia along with four market samples were screened by selected five primers for comparative DNA profiles. Genuine as well as market samples were successfully amplified by selected five primers and showed clear and reproducible band pattern. Further, only well-separated, reproducible and intense bands that were observed in all the three independent amplifications were selected for scoring (Figure 6.13). The representative amplification and profiles are presented in Figure 6.14. Analysis of dendrogram: The dendrogram based on the amplification profiles showed following correlations: Market sample collected from Lucknow showed 65% similarity with genuine TD and Akola sample shows 70% similarity with genuine TCC. The sample collected from Pune formed separate clade and clubbed with TCA and shows 26% similarity. However, sample from Mumbai showed 100% similarity with MC. The molecular profile confirms the trade of other Trichosanthes species and MC under the name of "Patola/ Ranpadval" in Maharashtra as well as some other parts of country.

Figure 6.13 Comparative ISSR profiles for identification and authentication M- Low range DNA ruler plus; TD (I.Male; 2.Female); 3. TCC; 4. TCA; TT (S.Male; 6.Female); 7. MC; 8. Akola; 9. Lucknow; 10. Pune; 11. Mumbai Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

Ttichostviihi's diowo (Miitct

Trkhosanthcs dioica (fcinaio

- Lticknow

* Trichosanthes cucumerina

.A kola

" Trichosanihes anguinu

' Pune

Trichosanthes iricuspuiaia (Male)

Tnchosanthes ihcusfntfaUi i\-cnv,\\c\

MomorcUca charanlUf

Mtimbai

! .fn)

Similariiv cootTtciem

Figure 6.14 Dendrogram of Trichosanthes genuine and market samples

6.9 Discussion 6.9.1 Conventional pharmacognosy: A useful tool for identification Ayurvedic drug, "Po/o/a" is recommended in the treatment of liver disorders, spleen, hemorrhoids, skin related diseases etc. Two different species viz., Trichosanthes dioica and T. cucumerina (var. cucumerina and anguina) are recognized under the name of ''Patola". Due to common synonyms of this drug, the correct identity has become a prime subject. Hence, above selected Trichosanthes species were studied by conventional pharmacognosy combine with chromatography and molecular techniques. TD is a dioecious plant; both male and female individuals are differentiated only at flowering stage while both the varieties of TC are monocious and can be easily differenced from TD. In microscopic study of TD, male and female individuals do not show any structural differences. In leaf epidermal study, anomocytic stomata at adaxial and abaxial surface has observed which in agreement with previous study (Raghunathan & Mitra, 1982; AH & Hemaid, 2010; Singh et al., 2010 & 2011; Chauhan, 2011). For comparative study of both the species, female individual of TD was used. TD and TC showed differences in their cellular arrangement as: leaf petiole of TD has pear shaped with homy outgrowth Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L at abaxial side; TC (both varieties) have oval petiole with shallow furrow. A single large bicollateral vascular bundle found in leaf of TD whereas one large and two small vascular bundle in TC. In seed testa of TD, pitted parenchymatous layer is absent. Further, cellular arrangement in vegetative part and fruit of both varieties of TC (TCC & TCA) does not show differences except prismatic crystals are present in fruit and seed of TCC. The detail microscopy of TCC was previously carried out by Shah & Shah (1967) and revised by Raghunathan & Mitra (1982) which is supported by present work. In comparative leaf epidermal study, TCC showed trichomes with acute apex where as slightly blunt apex trichomes observed in TCA. These observations are in support of previous results (Beevy & Kuriachan, 2009; Ali & Hemaid, 2010; Adebooye et al., 2012). Proximate analysis of both the species has showed significant differences. The trend of foaming index was observed as TD < TCA < TCC. The swelling index of vegetative parts and fruits of TCA was two times higher than TCC and TD. TCC show highest carbohydrate and vitamin C content. Previously, the study on proximate analysis was reported only on TCC by Raghunathan & Mitra (1982).Reported contents of total ash and acid insoluble ash of TCC were 13.45 and 0.45% for stem and leaf, respectively and 7.95 and 0.4% for fruit and seed, respectively. However results obtained in present study are not comparable with the report. Growing conditions and mineral availability through soil may be responsible for these changes (Kano & Goto, 2003). Alkaloids, flavonoids, glycosides and tannins were present in both species. Steroids and terpenes were found only in vegetative part of TD and vegetative part, fruits of TCC. The trend of tannins, polyphenol and flavonoids content were found as: TD < TCA < TCC. These results are in agreement of previous report (Sandhya et al., 2010; Toshniwal et al., 2013)

Cumulatively, conventional pharmacognosy study gives diagnostic characters which may serve as a primary step for identification of these two species. However, to confirm the correct identity at variety level, analysis using modem pharmacognostic tools is required. 6.9.2 Evaluation of phytochemical references standard The chromatographic profile of petroleum ether and methanol extracts of both the species showed differences in banding pattern. The optimized chromatographic conditions for TLC profile of TD gave better separation of phyto- constitutes than previous reported one (Kumar et al., 2012). TLC fingerprinting of Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L

TC varieties showed differences in band pattern. Several phytoconstitutes were reported from both the species. HPTLC quantification of lupeol and stigmasterol was reported from TD (Hussain et al., 2012). However, lupeol and stigmasterol are also present in both of the varieties of TC. The reported HPTLC method could not differentiate them from each other. Further, HPTLC and HPLC methods are reported for estimation of Cucurbitacin B from methanol extract of TD leaves and fruit juice of TCC (Samanta, 2012; Kongtun et al., 2009). However, there are no reports available on estimation of PRS from others parts of TD and TCC. To overcome this /imitation, chemica/ compound Cucurbitacin B responsibfe for toxicity and bitterness was estimated by HPTLC method. Vegetative parts and fruits of TCA and fruits of TD were completely devoid of Cucurbitacin B. On the other hand, 1.12 ± 0.04% of Cucurbitacin B was observed in vegetative parts of TD and 2.34 ± 0.08 and 3.64 ± 0.05% from vegetative parts and fruits of TCC, respectively. Comparative TLC fingerprinting and analysis of Cucurbitacin B may be useful parameter for quality inspection.

6.9.3 ISSR marker: Species specific markers for identification In present study, 94 ISSR primers were screened and 10 markers produced polymorphic DNA profiles between TD and TC (TCC & TCA). Further, primers UBC 812, 822. 887, 888 and 889 were useful to differentiate between both the varieties. Further, validation using samples from multiple locations confirmed that these bands would be useful as diagnostic markers to identify and differentiate these two varieties. To our knowledge, this is a first report on the application of ISSR markers for identification and authentication of these plant species. 6.9.4 Evaluation of antioxidant activity In present work, antioxidant activity of methanol extracts of vegetative parts and fruits of both species have investigated by chemiluminescences assay along with radical scavenging activity. Radical scavenging activity has been reported on fruits of TD and TC (Adebooye, 2008; Shivhare et al., 2009; Dixit & Kar, 2009; Tapkir et al., 2013). However, medicinally important vegetative parts of these plants were not studied so far. Hence, comparative evaluation of multifunctional biological antioxidant activity of vegetative parts and fruits of both species was undertaken. Fruits of TCC showed significantly higher chemiluminescence and radical scavenging activity followed by its vegetative parts. The trend of activity was as Chapter VI Trichosanthes dioica Roxb. & Trichosanthes cucumerina L follows: TCC fruit > TCC vegetative parts > TCA fruits > TCA vegetative parts > ( TD fruit > TD vegetative parts. Further, the activity of all the plant extracts was^J - comparatively less than standard ascorbic acid and Trolox. The major groups of phytochemicals tannin, polyphenols, vitamin C and E have been suggested as a natural source of antioxidants. These compounds were estimated from both the species may contribute to the total antioxidant activity. In addition, presence of "''' J Cucurbitacin B in TCC may be correlated with increased antioxidant potential (Tannin-Spitz et al., 2007). 6.9.5 Investigation of market samples Identification parameters developed by conventional and modem pharmacognostic methods were applied for investigation of market samples. All market samples were in semi granular or fine powdered form so macroscopic observations were not useful. The microscopic characters revealed sample from Dhule was similar to TCC fruit powder; where as sample from Ratanagiri was admixture of TCC and MC fruits. Sample from Surat was leaf material other than Trichosanthus species and MC. Further, these samples were used for HPTLC studied for chemical profiling. The samples of Dhule, Ratngiri previously identified on microscopic characters along with samples from Akola, Kolhapur and Lucknow were found to be containing Cucurbitacin B. To establish correct identity of samples from Akola, Kolhapur, Pune, Mumbai and Lucknow, molecular study was carried out with four common species of genus Trichosanthes and adulterant MC. Sample from Kolhapur do not produces amplification. Reaming samples were further used to for amplification. Selected 10 ISSR primers including five diagnostic markers (UBC 812, 822, 887, 888, and 889) were used for analysis. The dendrogram study confirms that sample from Akola belong to TCC, Pune belong to TCA, Mumbai belong to MC and sample from Lucknow belong to TD. Thus, the present study confirms different Trichosanthes species are traded under common name ''Patola I Kadoo patolam I Ran Padwar and supports previous reports (Raghunathan & Mitra, 1982; Sivarajan & Balachandran, 1994). Developed standards are useful in investigation of adulteration in market samples and establishment of correct identity.