K. Hemamalini et al., Jour. of Sci. Res. in Phar. 2012, 1(2), 5-7

Journal of Scientific Research in Pharmacy Research Article Available online through ISSN: 2277-9469 www.jsrponline.com

Phytochemical and Anticholinesterase of Leaves of Different Herbs on Frog’s Experimental models

K. Hemamalini1, Anurag Bhargav2, Dr. Uma Vasireddy3, Ch. Lavanya1, B. Murali Krishna1, B. Sathya Shirisha1 1Teegala Ram Reddy College of Pharmacy, Meerpet, Hyderabad, 2Principal and Professor, Lords International College, Alwar, Rajastan, 3H.O.D.and Professor - Pharmacology, TRR College of Pharmacy, Meerpet, Hyderabad,

Received on: 04-04-2012; Revised on: 05-02-2012; Accepted on: 10-04-2012

ABSTRACT

The present study reports the preliminary phytochemical, anticholinesterase activity of leaves of the of Tabebuia rosea, pubesens and Gymnosporia emarginata. The shade dried powder of Tabebuia rosea (Bignoniaceae), Solanum pubesens () a nd Gymnosporia emarginata (Celastraceae) was subjected to successive extraction using the solvents (Petroleum ether and Methanol) in the increasing order of polarity. Thus the prepared extracts were subjected to the preliminary phytochemical analysis. The extracts were investigated for Anticholinesterase activity in green frogs which was compared with standard drug Neostigmine (1mg/ml). The parameter measured for Anticholinesterase activity was ciliary movement in frog’s buccal cavity and degree of contraction of frog’s rectus abdominus muscle. Results revealed that the metha nolic extract of the leaf showed significant anticholinesterase activity.

Key words: Tabebuia rosea, Solanum pubesens and Gymnosporia emarginata, Phytochemical and anticholinesterase.

INTRODUCTION Gymnosporia emarginata (Willd.) Thw. With synonyms Tabebuia rosea (Bertol.) DC. Belonging to Family Celastrus emarginatus willd. Maytenus emarginta (willd.) Ding Hou., Bignoniaceae. Commonly known as “Pink Trumpet Tree” can grow up to Gymnosporia montana (Roth) Benth. belongs to family celastraceae, is 15 meter and well known for its beautiful flowers. The timber is widely an evergreen, Dioecious tree, scandent shrub or small tree that tolerates used for general construction and carpentry in many European various types of stresses of the desert, locally known as “Chinni, Danthi, countries. The fruits are green, long and bean pod- like with a length of Sinni, Danti, Chini tuppa, Chinni chettu, Goddali-cippa” in Telugu, 20-40 cm (8-16 inch). The fruits turn dark brown when ripe and contain “Thorny staff tree” in English. The species is globally distributed in flat, heart- shaped seeds with tiny wings. The graceful beauty is a treat Paleotropics. Within India, it is common in dry scrub forests throughout, for the eyes, but the tree has medical uses as well. Tea made from the particularly on poor soils in Central and Western peninsular India. The leaves and bark is known to have fever-reducing effect [1]. Taheebo is genus Maytenus distributed in drier parts of Central, South-Western reported to be an astringent, anti-inflammatory, antibacterial, antifungal, and North-Western India. It grows in moderately fertile, moist but well- diuretic and laxative [2-6]. drained soil in full sun with midday shade. Develops new leaves from with synonyms Solanum semicoalitum June to August. Traditionally species of Maytenus has been used for Bitter, , ortega, fever, asthma, rheumatism and gastrointestinal disorders worldwide. luteoalba, Cyphomandra luteoalbum belongs to family Solanaceae, Habit: Recently some biomolecules from Maytenus species has Shrubs 1-3m tall. Stems moderately to densely pubescent with been reported to be active against HIV-Protease [7] Carcinoma and eglandular unbranched or dendritically branched hairs and also with leukemia [8]. Ulcers and MDR (Multi Drug Resistance) [9]. Various parts short-stalked glands; tips of shoots sometimes densely glandular. of this plant contain immense medicinal properties which are mentioned Sympodial structure: Sympodial units’ 6-many foliate. under: Leaves: Leaves simple, chartaceous, elliptic or ovate-elliptic, Fruit: The fruits are used in medicines to purify blood [10]. moderately to densely pubescent with eglandular unbranched or Leaf- Pulverized leaves of Maytenus emarginata are given in dendritically branched hairs and often also with short-stalked glands ; milk to children as a vermifuge [11]. A decoction of the leafy twigs is used base cuneate to slightly decurrent; margin entire; apex acute to as a mouthwash to relieve toothache. Ash of leaves used to heal up sores acuminate; petioles 1-4.5cm, densely pubescent with hairs like those of and wound gives cooling effect. The leaves are burnt and mixed with the stem. ghee to form an ointment used to heal sores [12]. The tender leaves Inflorescences: Inflorescences 4-10(-15)cm, unbranched, are chewed raw in the treatment of jaundice. forked, or sometimes further branched, with ca. (5-)10-25 flowers, all Stem: Tender shoots of the plant help for mouth ulcer. The flowers perfect, the axes moderately to densely puberulent with bark is ground to a paste and applied with mustard oil to kill lice in the eglandular unbranched and/or dendritically branched hairs. hair. Root- Used in gastrointestinal troubles, especially dysentery [13]. Flowers: Flowers with the calyx radius 3-4 mm, deltate Fruits are capsule, berry, 6-12mm long, leaves are thick, abruptly narrowed into acuminate tips, moderately puberulent with coriaceous and usually longer than 40mm, apex rounded, alternate on glandular and eglandular hairs. Corollas 2-3 cm in diameter, the radius young branchlets, fasciculate on older ones, Bark is pale brown, smooth, 11-20mm, stellate, chartaceous, purple (rarely white). cracked, flowers are bisexual or sometimes functionally unisexual, Fruit: Fruits 1.5-2 cm in diameter, globose, obtuse at apex, actinomorphic, white or cream coloured 5-7mm in diameter. Male orange or red when ripe, glabrous; stone cell aggregates absent. flowers- stamens slightly shorter than petals, stigmas absent, disc green, Seeds: Seeds angled, smooth to rugose. 5- lobed, Distribution: Andean slopes from southern Ecuador to Female flowers: Staminodes shorter than stamens of male southern peru; gravelly or rocky slopes and cliffs to moist river valleys; flower, Ovary 3-locular, green, style as long as ovary. Roots are Tap root, 2200-3300m. cream yellow in colour. Phenology: Flowering in August through March. Fruiting in Since the plants has shown wide medicinal properties these all months except july. plants was selected for our study. In the present study, we report the anticholinesterase activity of leaf part of various plants. *Corresponding author: MATERIALS AND METHOD K. Hemamalini Plant material: Associate Professor, The leaves of Tabebuia rosea, Solanum pubesens and Teegala Ram Reddy College of Pharmacy, Meerpet, Hyderabad. Gymnosporia emarginata were collected from the local area of *Email: [email protected] Rajampets, Kadapa, Andhra Pradesh, India and authenticated by Dr. C

Journal of Scientific Research in Pharmacy 2012, 1(2) 5-7 K. Hemamalini et al., Jour. of Sci. Res. in Phar. 2012, 1(2), 5-7

Madhava Chetty, Head of the Department of Botany, S.V. University, reach the spot is noted. Buccal cavity was washed with normal saline, Tirupathi, Andhra Pradesh, India. and then above test was repeated for the standard drug neostigmine and test drug. Preparation of plant extracts: Rectus abdominus muscle contraction method: The leaves of various plants like Tabebuia rosea, Solanum The frog was pithed and laid on its back on the frog dissecting pubesens and Gymnosporia emarginata were shade dried and powdered. board and the four limbs were pinned. The skin on the abdomen was About 1 Kg of dry powder was extracted in the petroleum ether by removed and the rectus abdominus muscle was exposed. The muscle continuous hot percolation using Soxhlet apparatus. The extraction was was prepared and a thread was tied to the bottom and top of the each continued for 72 h. The petroleum ether extract was filtered and muscle preparation before detaching the muscle from the body of the concentrated to a dry mass by using vacuum distillation. Subsequently, frog. The preparation was mounted in upright position in organ bath the dried powder was extracted with methanol. The solvents were containing frog ringer solution under a tension of 1g. The tissue was evaporated to dryness and then the residue of different plant extracts relaxed for 45 min with the wash of tissue with fresh quantum of ringer were obtained was taken for the experiment. four times. The contractions of the rectus abdominus muscle due to the increasing dosed of acetylcholine were recorded using either simple side Experimental animals: way or frontal writing lever. The methanolic extracts of the different Swiss albino mice 20-22g of either sex were purchased from plants was mixed with the acetylcholine and the contractions and NIN, Hyderabad, India. They were housed in polypropylene cages in a relaxation were recorded till a maximum response was reached. The controlled room temperature 22± 1ºC and relative humidity of 60-70. height of the response was measured in mm and a dose –response curve They were kept under standard conditions of 12/12 h light and dark was constructed. cycle. The animals were maintained with standard pellet diet and water ad libitum. The animals were acclimatized to laboratory condition for Statistical Analysis: seven days before commencement of experiment. The experimental The results were expressed as the mean + SEM for each protocol was subjected to the scrutiny of the Institutional Animal Ethical group. Statistical differences were evaluated using one way analysis of Committee and was cleared by the same before starting. variance (ANOVA) followed by Dunnett’s test. Results were considered to be statistically significant at p< 0.001 Phytochemical evaluation: All the extracts screened for the presence of various RESULTS AND DISCUSSION secondary metabolites like alkaloids, carbohydretes, flavonoids, proteins using standard methods [14]. The extracts after phytochemical investigation showed the presence of following active principles, in Petroleum ether extract Acute oral toxicity studies: showed the presence of Fixed oils and fats. And in Methanolic extract Acute oral toxicity study of methanolic extract of various showed the presence of Carbohydrates, Reducing sugar, Alkaloids, plant extract was carried out in swiss albino mice of either sex 20-22 g Phytosterols, Tanins, Flavones. according to OECD guidelines No. 423. Different plant extracts at different doses upto 300 mg/kg p.o was administered and animals were Acute toxicity Studies: observed for behavioral changes, toxicity and mortality upto 48 h [15]. The results of acute toxicity studies indicate that none of the extracts studied showed any behavioral changes, toxic reaction and Anticholinesterase activity: mortality even after 48 h. The extract was found to be safe at the dose of Ciliary movement method: 3000mg/kg. The frogs were decerebrated and pinned to the frog board on its back separately as standard and test. Each group consists of 6 frogs. Anticholinesterase activity: The lower jaws of frogs were pinned to the abdomen, cutting sufficiently Comparative study of Anticholinesterase activity has been the buccal cavity and the openings of the oesophagus were wetted by conducted for methanolic extracts of Tabebuia rosea, Solanum pubesens irrigating it with normal saline. The two parts were fixed i.e. from a point and Gymnosporia emarginata among them Tabebuia rosea showed well in the lower jaw to the beginning of the oesophagus. Poppy seeds were pronounced Anticolinesterase activity by increasing Ciliary movement as placed at premarked spot in the jaw and time taken by the object to inferred by decrease in the time for a poppy seed to move on the lower reach the beginning of the oesophagus was noted. This experiment was jaw of green frog as shown in the Table. 1. done in standard followed by test separately. A few drops of acetylcholine 1mg/kg was added in buccal cavity and after 10 min poppy seeds were placed at premarked spot in the jaw and time taken by it to

Table 1: Anticholinesterase activity of the methanolic extract of leaves of Tabebuia rosea, Solanum pubesens and Gymnosporia emarginata by the measurement of ciliary movement of poppy seeds in lower jaws of green frogs. Drugs Distance Time in sec Normal saline (6% w/w) 1cm 274.16 + 5.25 Ach (1mg/ml) 1cm 226.6 + 2.118 Neostigmine (10mg/ml) 1cm 185 + 3.619* Methanolic Extract of Tabebuia rosea 1cm 60.83 + 2.509* Methanolic Extract of Solanum pubesens 1cm 320.83 + 7.370* Methanolic Extract of Gymnosporia emarginata 1cm 416.66 + 4.420* Values are expressed as mean + standard deviation (n=6) in each group, statistical *significance (p<0.001) one way ANOVA followed by Dunnett’s test.

It is confirmed by the contraction of rectus abdominis muscle the height of construction in the dose response curve as shown in Table. which increased with the methanolic extracts as inferred by increased in 2. Table 2: Anticholinesterase Activity of the Methanolic extract of leaves of Tabebuia rosea, Solanum pubesens and Gymnosporia emarginata by measurement of contraction of rectus abdominis muscle preparation in frogs. Conc. Of Measurement of Conc. Of Measurement of Measurement of Measurement of Ach contractions (cm) Ach+Methanolic extract Contractions (cm) T.R Contractions (cm) S.P Contractions (cm) G.E 6µg 0.48+0.065 6µg 0.841+0.0376****** 0.36 + 0.042* 0.366 + 0.033* 9µg 0.81+0.069 9µg 1.275 + 0.031****** 0.633+0.0423***** 2.3+0.03073****** 30µg 2+0.187 30µg 1.7 + 0.058* 1.433+0.0614****** 0.616+0.602****** 100µg 2.6+0.268 100µg 2.475 + 0.044* 3.083+0.0475****** 1.4 + 0.0581****** 300µg 3.16+0.203 300µg 3.216 + 0.0479* 3.566 + 0.033*** 1.8 + 0.0729*** Values are expressed as mean + standard deviation (n=6) in each group, statistical Significance * p<0.5,**p<0.1, ***p<0.01, ****p<0.02, *****p<0.05, ******p<0.001 one way ANOVA followed by Dunnett’s test.

Journal of Scientific Research in Pharmacy 2012, 1(2) 5-7 K. Hemamalini et al., Jour. of Sci. Res. in Phar. 2012, 1(2), 5-7

CONCLUSION 7. Yadav P., Sarkar S., Bhatnagar D. Lipid peroxidation and antioxidant enzymes in erythrocytes and tissues in aged diabetic rats. Indian J. Exp. Biol. 1997: 35; 389–392. Leaf extracts of Tabebuia rosea, Solanum pubesens and 8. Acherekar S., Kaklij G.S., Kelkar S.M. Hypoglycemic activity of Gymnosporia emarginata exhibited a significant anticolinesterase Eugenia jambolana and ficus bengalensis: mechanism of activity in experimental frogs. Methanolic extract of Tabebuia rosea action. In vivo. 1991: 5; 143–147. exhibited relatively better anticolinesterase activities than methanolic 9. Aderibigbe A.O., Emudianughe T.S., Lawal B.A. extract of Solanum pubesens and Gymnosporia emarginata. The Antihyperglycemic effect of Mangifera indica in rat. difference in the evaluated activities could be due to the number and also Phytother Res. 1999: 13; 504–507. quantity of phyto – chemical constituents present in the extracts. 10. Khanna P., Jain S.C., Panagariya A., Dixit V.P. Hypoglycemic activity of polypeptide- p from a plant source. J. Nat. Prod. REFERENCE: 1981: 44; 648–655. 11. Shibib B.A., Khan L.A., Rahman R. Hypoglycemic activity of 1. Gentry A. H., A Synopsis of Bignoniaceae Ethnobotany and Coccinia indica and Momordica charantia in diabetic rats: Economic Botany, Annuals of Missouri Botanical Garden. depression of the hepatic gluconeogenic enzymes glucose-6- 1992: 79(1); 53-64. phosphatase and fructose-1, 6-biphosphatase and elevation 2. Abbott B. J, Hartwell J. L, Leiter J , Perdue R. E and Schepartz S. of liver and red-cell shunt enzyme glucose-6-phosphate A., Screening data from the cancer chemotherapy national dehydrogenase. Biochem. J. 1993: 292; 267–270. Service Center screening laboratories. XL, Plant extracts., 12. Vats V., Grover J.K., Rathi S.S. Evaluation of antihyperglycemic Cancer Res. 1967: 27(Suppl.); 190-345. and hypoglycemic effect ofTrigonella foenum-graecum Linn, 3. Hartwell J. L., Plants used against cancer; a survey, Lloydia. Ocimum sanctum Linn and Pterocarpus marsupium Linn in 1968: 31; 71-170. normal and alloxanized diabetic rats. J. Ethnopharmacol. 4. Bastein J.W., Pharmacopeia of Qollahuaya Andeans, J. 2002:79; 95–100. Ethnopharmacol. 1983: 8; 91-111. 13. Rai V., Iyer U., Mani U.V. Effect of Tulasi (Ocimum sanctum) 5. Arenas P., Medicine and magic among the magic the Maka leaf powder supplementation on blood sugar levels, serum Indians of Paraguayan Chaco, J. Ethnopharmacol. 1987: 21; lipids and tissue lipid in diabetic rats. Plant Food For Human 279-295. Nutrition. 1997; 50: 9–16. 6. Almedia E. R, da Silvia Filho A. A, dos Santos E. R and Lopes 14. Farnsworth NR, Biological and phytochemical screening of C.A.C., Antiinflammatory action of lapachol, J. plants, J Pharm. Sci. 1966: 55; 225. ethnopharmacol. 1990: 29; 239-241. 15. Ghosh MN. Fundamental of Experimental Pharmacology. Second edn. Calcutta: Scientific book Agency. 1984; 153- 158.

Source of support: Nil, Conflict of interest: None Declared

Journal of Scientific Research in Pharmacy 2012, 1(2) 5-7