J. Res. Educ. Indian Med., Jan.-March, 2009

PHARMACOGNOSTICAL STUDIES ON THE FRUITS OF CYMBALARIA FENZL EX NAND. T.R.SHANTHA,1 G.VENKATESHWARLU,2 M.J.INDIRA AMMAL3 AND K.GOPAKUMAR4 Regional Research Institute (Ayu.), C.C.R.A.S., Departmet of AYUSH, Ministry of Health & F.W. Government of , Jayanagar, Bangalore - 560011 Karnataka (India)

Abstract : The present communication deals with the pharmacognostical and preliminary phytochemical studies on the fruits of Momordica cymbalaria with Ayurvedic name Kuduhunchi which has been described in Rajnighantu (Narahari).This of considerable economic importance has been recommended mainly for its antidiabetic properties. Its green fruits are used as a vegetable. No study reports are available on the microscopical and phytochemical studies of the fruits. Hence, the present attempt has been undertaken to investigate the microscopical and preliminary phytochemical studies. The study revealed the presence of abundant protein bodies, oil globules, simple starch grains, glandular and non-glandular trichomes, highly lignified stone cells and bicollateral vascular bundles. Keywords: Momordica cymbalaria, Medicinal , Pharmacognosy. Introduction faculty), ruchikara (inducer of taste), rakta Momordica cymbalaria is a perennial and anila doshakara (causetive factor for monoecious trailing plant with large turnip blood and nervous disorders). Drug has got shaped tuberous rootstock. Stem slender, 5 many synonyms like Pratipatraphala, Bahuphala, angled, leaves 5-7 angled flowers white or Shreephalika, Kandalata, Karavi, Shubravee, yellow male flowers in short racemes, female Kshudrakaralika and Kshudrakaravalli flowers solitary, long peduncle, fruit pyriform (V.S.Togunasi et al., 1977). or fusiform, 8- ribbed. Plant is not cultivated but occurs mostly in bushes along the bank of Literature review revealed that watercourses and in ledges, which commonly Momordica cymbalaria is a known Ayurvedic found in Deccan and Carnatic southwards to drug, which introduced by Narahari in his Thirunalveli in Tamilnadu. Locally the fruits of Rajnighantu and this drug has not found a place Momordica cymbalaria is known as Karsikayi in any other nighantus (V.S.Togunasi et al., in Kannada, Athalakkai in Tamil ( Kirtikar 1977). It has been sub classified under and Basu et al., 1993) Kattupaval in Phalasaaka varga and Moolakadi varga with Malayalam and in Marathi it is known as various local names and has ascribed the Kadavanchi, Kuduhunchi. (Anonymous, 1962; synonyms as mentioned which depict different V.S.Togunasi et al., 1977). characteristics of utmost important for facilitating its identification. The studies have The fruits are katu (pungent), tikta been carried out on Athalakkai or Kuduhunchi (bitter) in rasa, deepanada (enriching digestive with respect to chemical composition and

1. Research Assistant (Botany) 2. Research Officer (Ay.) 3. Research Asstistant (Chemistry) 4. Assistant Director 2 Shantha et al. utilization of the wild edible vegetable are under way to elucidate the mechanism of Momordica tuberosa, and it has been identified antidiabetic activity of M. cymbalaria. and major nutrients were assessed. It has been observed that Athalakkai contains higher Methodology amounts of calcium, potassium, sodium and Fresh fruits of Momordica cymbalaria vitamin C than the bitter gourd. were collected from the surroundings of Ananthapur (A.P), in the month of August. For It was also observed that it had high microscopical studies free hand sections of fresh crude fiber (6.42g/100g) content. The nutrient fruits were cut cleared with chloral hydrate contents of Athalakkai are more when solution and water stained with safranin compared to bitter gourd. The calcium content according to the prescribed methods. of Athalakkai is 3 times higher than bitter gourd. (Johansen D.A, 1940; Wallis.T.E., 1967). Iron content is same as that of bitter gourd A drop of HCl and Phloroglucinol was used to (S.Parvathi et al., 2002) The content of detect the lignified cells in the cut sections and potassium in Athalakki is also two times higher in the powder drug (Wallis.T.E., 1967). than in bitter gourd and β-carotene content in Photomicrographs were taken by Nikon digital Athalakki is negligible. G. Azeemuddin et al., camera. Powder of the dried fruit was used for (1966) has carried out the work on seed fat of chemical analysis. Physico-chemical studies and the plant contains a conjugated triene acid as preliminary phytochemical screening of the drug that of the α oleostearic acid which is a was carried out Anonymous (1998). The characteristic of seed fats of Momordica genus. fluorescence behavior of the powdered drug in (G. Azeemuddin, et al.,1966). B.Kamaeswara different solutions towards the ordinary and Rao et al. (1999) have carried out the studies ultraviolet light was carried out (Chase and of the fruit powder on blood glucose and other Pratt R., 1949). Thin Layer Chromatography biochemical parameters in alloxan induced studies of the Petroleum ether at 60°-80°C, diabetic rats and they have concluded that fruit chloroform and ethanol, aqueous extracts were have beneficial effects on the the blood glucose carried out in various solvents at 30°C using level as well as improving hyperlipidaemia due precoated silica gel GF 254 plate as adsorbent. to diabetes. B.Kameswara Rao et al. (2001) (Igon Stahl, 1969). have carried out antihyperglycemic activity of M. cymbalaria fruits in alloxan diabetic rats Results and Discussion and concluded that there is a fall in the blood Macroscopically fruits are 2 to 2.5cm glucose of diabetic rats and no effect in normal long, pyriform or broadly fusiform narrowed in rats. to the curved peduncle, fruits dark green,8 ribbed, sparsely hairy. Seeds 4-6mm long broadly B.Kameswara Rao et al. (2001) have ovoid slightly compressed, strophiolate, not carried out the studies on evaluation of margined testa polished and shining, dark brown antidiabetic effect of M.cymbalaria fruits in coloured, tender fruits are dark green in colour alloxan diabetic rats and stated that aqueous and matured fruits are light yellow coloured, fraction of M. cymbalaria fruits has beneficial taste is bitter, smell agreeable, not characteristic effects on blood glucose levels as well as (PLATE I, II, III, IV). improving hyperlipidemia due to diabetes through Microscopically upper most region of the an increase in insulin secretion and further fruit show furrows and ridges with Hexagonal pharmacological and biochemical investigations to Octagonal structure in out line Phytochemical Studies on Momordica cymbalaria 3

Momordica cymbalaria (Fruit) Microscopical Characters:

PLATE 1 PLATE 11 FIG 1 FIG 2

T.S of the fruit 10x X10 x. Epicarp and mesocarp region with vascular bundles Fruits Fruits and Seeds . 10x X10x

PLATE III PLATE IV FIG 3 FIG 4

T.S. of the fruit showing furrows and ridges. Glandular trichome. Seeds Single fruit 10x X10x Macroscopical Characters 10x X10X

FIG 5 FIG 6 (Figs.1 and 4) Outer most layers, epicarp made up of epidermis which is single layered, covered by thick striated cuticle. Some of the epidermal cells show both glandular and non-glandular type of trichomes (Fig.3). The non-glandular SCL trichomes are unicellular to multi cellular and glandular trichome is single headed. Epidermis is followed by mesocarp, made up of 2 to 5 T.S. of the fruit upper region10x X10x Portion enlarged.showing sclerenchyma band layers of paranchymatous cells, where some of Showing continuous band of sclerenchyma. 10x 40x the cells show simple rounded starch grains. The seed of develops from Mesocarp is followed by continuous band or 3 a bitegmic crassinucellate ovule but the inner to 5 layers of sclerenchymatous tissue and this integument disintegrates early in seed is followed by a zone of rounded to tangentially differentiation. The seed coat is thus derived elongated thin walled paranchymatous cells from the outer integument, the inner integument (Figs. 5 and 6). In this region, bicollateral is narrow, and the outer is wide. After vascular bundles are present under each furrows fertilization the outer integument further and ridges. Apart from the main vascular increases in thickness by periclinal divisions and bundles, few small vascular bundles are seen differentiates in to several distinct layers scattered above or side by the main vascular (Fig.13). The epidermis of the seed coat that bundle and centre portion is occupied by thin is outer testa consists of collapsed epidermis walled compactly arranged parenchymatous and hypodermis, consisting of several layers of cells (Figs. 2,4,7,8.9.10,11). compactly arranged lignified stone cells of T.S. of the fruit through middle region different size and shape which are irregular shows the same structures as that of the upper with highly thickened walls (Figs. 14 to 17). region of the fruit except in the internal Beneath the hypodermis, thin walled 1 to 6 structures of the seed. layers of parenchymatous cells are present with 4 Shantha et al.

FIG 7 FIG 8 FIG19 FIG 20 MESO VBS COTY COTY

Vascular bundle enlarged. Epicarp and mesocarp enlarged.. 10x X 40x 10x X 40x T.S of the seed region 10x X 10 x Cotyledon portion enlarged.10x X 40x

FIG 9 FIG 10 FIG 21 scattered vascular tissues representing OG only xylem elements of helical thickenings. Beneath this layer, inner epidermis of the T.S of the fruit along with seed portion. (Middle Vascular region enlarged.10x X 40x. Cotyledon portion showing region)10x X10x. oilglobules.10x X 40 X

FIG 11 FIG 12 EPI CU outer integument with nucellar and endosperm PAR cells occurs and this is followed by cotyledon MESO region with many layers of thin walled compactly SG arranged parenchymatous cells abundantly loaded with oil globules and protein bodies

(starch grains).All most all cells are heavily .Enlarged portion of epicarp and mesocarp.10x X40 x Mesocarp portion showing starch grains. 10xX40x loaded with oil globules and protein bodies FIG 13 FIG 14 (Figs. 18 to 21).

Macerate: - Macerate of the fruit along with the seed shows following structures (Figs. 1 to 10). 1) Polygonal parenchyma cells.

T.S.of the seed showing outer integument, Portion enlarged showing ( stone cells parenchyma and cotyledon region of seed ) (outer integument ). 2) Abundant oil globules. 10x X 10x 10x X 40x Hypodermis portion enlarged. 3) Thin walled elongated parenchyma cells FIG 15 FIG 16 and stone cells. 4) Irregularly shaped lignified stone cells. 5) Helical to spiral xylem vessels. 6) Helical xylem vessel. 7) Thin walled, parenchyma cells. 8) Elongated sclerenchyma fibers.

Parenchyma portion enlarged. Parenchyma region showing vascular strands. 9) Elongated, thin walled to rectangular FIG 17 .FIG 18 parenchyma cells. 10) Helical xylem vessel and parenchyma cells. COTY Powder Microscopy (Figs. 1 to 5) Powder is dark green in colour with an agreeable odour; taste slightly bitter and

Parenchyma region with vascular bundle.. Cotyledon portion.10x X 10x mucilaginous in nature. When it is treated with Explanation for the abbreviations used: COTY = Cotyledon, CU = Cuticle, EPI = Epidermis, MESO = Mesocarp, SG = Starch grain, PAR = Parenchyma VBS = Vascularbundle, SCL = Sclerenchyma, OG = Oilglouble. Phytochemical Studies on Momordica cymbalaria 5

Macerate

FIG 1 FIG 2 FIG 7 FIG 8

Parenchyma with starch grains Oilglobules.10x X 40x. Parenchyma Fibers

FIG 3 FIG 4 FIG 9 FIG 10

Stonecells 10x X 40 x Stonecells Parenchyma Powder study Xylem vessels

FIG 5 FIG 6 FIG 1 Powder Study FIG 2

Xylem vessels. and parenchyma cell10x X 40 x xylem vessels and parenchyma Fragments of different tissues Parenchyma

FIG 3 FIG 4. chloral hydrate, water and glycerin, under the microscope following different fragments of structures were observed. 1. Fragments of different tissues with thin walled parenchymatous cells, starch

grains etc. Parenchyma Oilglobules(lipid glouble)

2. Fragments of parenchyma cells. FIG 5 3. Fragments of thin walled parenchyma cells. 4. Abundant oil globules. 5. Fragments of epidermal cells with non- glandular trichomes.

Trichome -nonglandular Powder study with different reagents 1. Powder + water No change 2. Powder + Iodine Blue colour 3. Powder + Sudan III solution Light pink

4. Powder + Fecl3 Solution No change 5. Powder + Conc.H2SO4 Black 6. Powder + Conc. HCl Black 6 Shantha et al.

Diagnostic Characters lignified, with different shapes of cells 1) Presence of glandular and non-glandular like irregular to square shaped. types of trichomes. 5) Presence of abundant oil globules in the 2) Presence of Bi collateral vascular bundles cotyledon region. in mesocarp region. 6) Presence of simple starch grains in the 3) Presence of 5 to 6 layered thick band of mesocap region and in the cotyledon sclerenchymatous tissue in mesocarp region. region of the fruit. 7) Presence of 6 to 8 ribbed, sparsely hairy 4) Presence of compactly arranged 6 to 10 dark green fusiform fruits. layered stone layer cells, which are highly 8) Presence of polished and shining, brown colored testa of the seed. Histochemical tests

Section Reagent Change in Test for Inference colour (1) Section (Mesocarp and Iodine solution Blue Starch ++ Ctyledon region) (2) Section Sudan III solution Pink Oil globule ++ (Cotyledon region) (3) Section Ferric chloride No change Tannin _ _ solution (4) Section Chloral hydrate No change Crystals _ _ solution (5) Section Phloroglucinol, Majentha Lignin + + HCl + Alcohol

Table 1. Physicochemical and Preliminary phytochemical analysis 1 % Foreign matter Less than 2 2 % Loss on drying at 1100C. 8.29 3 % Ash content 19.96 4 % Water soluble ash 10.87 5 % Acid insoluble ash 0.10 6 % Extractive values: a. Petroleum ether 7.49 b. Chloroform 2.03 c. Ethanol 10.68 7 % Solubility at room temp. a. Ethanol 10.90 b. Water 21.90 8 %Extractable matter (Hot) 27.50 9 % Fat content 6.68 10 pH value 5.72 11 Swelling index 8ml/gm 12 Foaming index Less than 100 13 Inorganic constituents (Qualitative) Carbonate, sulphate, chloride, phosphate Calcium Magnesium, Sodium, Potassium. 14 Organic constituents (Qualitative) Alkaloids .glycosides, triterpenes, steroids , saponins, flavanoids, sugar. Phytochemical Studies on Momordica cymbalaria 7

Table 2. Organic Constituents compounds. The fruit showed more amounts of water soluble components than alcohol Constituents Aqueous Ethanol soluble components. extract extract In phytochemical investigation the Alkaloids +ve +ve percentage yield of Ethanol extract was found Tannins -ve -ve to be more than when compared to Petroleum ether extract (Table 1). Steroids -ve +ve The qualitative investigation test Saponins +ve +ve performed in the two extracts (ethanol and Sugars -ve -ve aqueous) revealed the presence of alkaloids, steroids, saponin, triterpenoids and flavanoids Triterpinoids -ve +ve etc. the fruits of Momordica cymbalaria Flavanoids +ve -ve containing organic constituents can be considered as to be responsible for curing wide Resins -ve -ve range of diseases like Diabetes. Medicinal plants usually contain alkaloids, saponins etc Physico-chemical and Preliminary phyto- as their major constituents (Table 2). chemical analysis The dried powder of the material was The total ash value and water soluble initially defatted with petroleum ether (60-80oc) ash value of Momordica cymbalaria fruit in a soxhlet apparatus and successively powder were found to be more in crude drug. extracted with chloroform and ethanol. The Ash value is a measure of the quality and extracts were filtered while hot and solvent purity of the crude drug. Alcohol and water removed by distillation .The percentage of yield soluble extractive values were determined to of the extract were calculated (Table 3). find out the amount of water and alcohol soluble

Table 3. Thin Layer Chromatography

Sl. Extractive Adsorbent Solvent system Viewed in Rf. Value No U-V Chamber 1 Under U-V Petroleum-ether Silica gel 60 F of 365 nm 0.11,0.21, 254 Benzene 60-80°C pre coated sheets 5% con.H2SO4 0.32,0.38 in methanol 2 Under U-V Toluene: Silica gel 60 F of 365 nm Chloroform 254 Ethyl acetate 0.16,0.38, pre coated sheets 5% conc.H SO (93:7) 2 4 0.46 in methanol 3 Under U-V Butanol:Acetic 0.16,0.28, Silica gel 60 F of 365 nm Ethanol 254 acid:Water 0.49,0.56, pre coated sheets 5% con.H SO 5:1:4 2 4 0.80,0.93 in methanol 4 Under U-V Butanol:Acetic Silica gel 60 F of 365 nm 0.16,0.32, Aqueous extract 254 acid:Water precoated sheets 5% con.H SO 0.56,0.75 5:1:4 2 4 in methanol 8 Shantha et al.

Table 4. Fluorescence studies Sl. Sample + Reagent Ordinary light U.V. Long wave U.V. Short wave No. 365 nm 254 nm 1 Powder as such Olive green Grey Olive green Fluorescence 2 Powder + Water Dull green Dull grey Dull green Fluorescence 3 Powder + 1N. HCl Yellowish green Mustard yellow Dull green 4 Powder + 1N. Na OH Dull green Mustard yellow Dull green 5 Powder + 1N. NaOH Yellowish green Bluish grey Dull green in MeOH Fluorescence 6 Powder + 50% KOH Yellowish green Dull grey Dull green Fluorescence

7 Powder + 50% H2SO4 Yellowish green Mustard yellow Dull green

8 Powder + Con. H2SO4 Dark brown Grey Dull green

9 Powder + 50% HNO3 Dull green Mustard yellow Dull green

10 Powder + Con. HNO3 Dull brown Black Dull green 11 Powder + Acetic acid Yellowish green Mustard yellow Dull green 12 Powder + Iodine water Yellowish green Black Dull green

The concentrated petroleum ether, Momordica cymbalaria recommended mainly chloroform, ethanol and aqueous extract of the for its antidiabetic properties and green fruits fruit were subjected to T.L.C. Studies. Equal are used as a vegetable.Phytochemical amounts of these extracts were loaded on the screening has revealed that fruits consist of T.L.C. Plates. The best separation was achieved alkaloids, tannins,aminoacids, proteins,fats and using Benzene, Toluene: Ethyl acetate and BAW phenols and contains higher amounts of calcium, as a mobile phase .After developing; the plates potassium, sodium and vitamin C. were viewed under U-V Light and in 5% Pharmacognostical studies revealed the con.H2SO4 in methanol showed the presence presence of abundant oil globules,glandular and of spots. The R.f values were calculated and nonglandular trichomes,simple abundant starch given in the Table 3. Fruit powder was grains, thick walled elongated irregularly shaped moistened with different solutions to study lignified stone cells and macroscopically fruits fluorescence and viewed under ordinary light are 2 to 2.5cm long, pyriform or broadly fusiform and U-V light having wave length of 250 nm narrowed into the curved peduncle fruits dark and 365 nm (Table 4). green, 8 ribbed sparsely hairy. Some of the pharmacological and biochemical investigations Table 4 contains observations on Fluorescence through literature review studies have shown studies. that fruits have antidiabetic, hypolipidemic and antihyperglycemic activity in alloxan diabetic Summary and Conclusion rats. All these studies will be of immense use The pharmacognosticl and physico in carrying out further research and revalidation chemical studies carried out on the fruits of of its use in Ayurvedic system of medicine. Phytochemical Studies on Momordica cymbalaria 9

T. L .C pattern of Momordica cymbalaria Plate-I Plate-II

0.46

0.32

0.38

0.21 0.16

0.11

petroleum ether extract- chloroform extract

Plate-III Plate-IV 0.93

0.93 0.80

0.56 0.80 0.56 0.49 0.49

0.28

0.28 0.16

0.16

Alcohol extract (before acid spray) Alcohol extract (after acid spray) 10 Shantha et al.

Acknowdgements diabetic rats. Journal of Ethnopharmacology. The authors are thankful to the Director, 67, 103 -109. C.C.R.A.S for encouragement and financial 9. B.Kameswara Rao, M.M.Kesavulu and support. C. Apparao (1966) : Evaluation of antidibetic References effect of Momordica cymbalaria fruit in 1. Anonymous (1962) : The Wealth of India - alloxan diabetic rats. Fitoterapia. 74. pp 7-13. Raw materials. Vol.VI, pp 412 to 413 CSIR, 10. B.Kameswara Rao, M.M.Kesavulu and C. New Delhi. Apparao (2001) : Antihyperglycemic activity 2. Anonymous (1998) : Quality control of Momordica cymbalaria fruit in alloxan Methods for Medicinal Plants Materials. diabetic rats. Journal of Ethnopharmacology, WHO, Geneva. 78 67-71. 3. G. Azeemuddin and S.D. Thirumala Rao 11. K.R. Kirtikar and B.D. Basu (1993) : Indian (1966) : Seed fat of Momordica tuberosa or Medicinal Plants. Bishan Singh and Luffa tuberosa. Current Science. August 26, Mahendra Pal Singh, Dehradun. Vol.II. pp pp 100. 1137. 4. Chase and Pratt R (1949) : Fluorescence of 12. A.K.Nadkarni (1976) : Indian Materia powdered vegetable drugs with particular Medica. Papular Prakashan Pvt. Ltd., Bombey. reference to development of a system of pp 755. identification. J. Am. Pharm. Assoc. Sci. Ed. 13. S. Parvathi and V.J.F. Kumar (2002) : Studies pp 38:324-331. on chemical composition and utilization of 5. Igon Stahl (1969) : Thin Layer the wild edible vegetable athalakkai Chromatography: A Laboratory Handbook. Momordica tuberosa. Plant Foods for Springer Verlag Student Edition, Springer Human Nutrition. 57. pp 215- 222. Verlag, Berlin. pp 52-86,127-128,900. 14. Roma Mitra (1985) : Bibliography on 6. Iyengar M.A (1950-1975 ) : Bibliography Pharmacognosy of Medicinal Plants. of Investigated Indian Medicinal Plants NBRI, Lucknow. Manipal. Power Press, Manipal. pp 15. V.S.Togunasi, B.S.Venkatram and 11,114. S.N.Yoganarasimhan (1977) : Disscussion 7. Johansen D.A. (1940) : Plant Micro- on the origin and identification of Technique. MCGraw Hill New York. Kuduhunchi. A hither to unknown Ayurvedic pp 183-203. drug introduced by Rajanarahari. Jour. Res. Indian Med. 12:1. pp118 to 121. 8. B.Kameswara Rao, M.M.Kesavulu, R.Giriand C. Apparao (1999) : Antidibetic 16. Wallis.T.E (1967) : Text Book of and hypolipidemic effects of Momordica Pharmacognosy. 5th ed. T. A. Churchill cymbalaria Hook fruit powder in alloxan London. pp- 571-582.

Address for correspondence: Dr T. R. Shantha, Regional Research Insitute (Ay.), Govt. Central Pharmacy Annexe, Ashok Pillor, Jayanagar, Bangalore - 560001 Karnataka (India). E-mail: [email protected]

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