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Preliminary study on the immunomodulatory effect of methanol extracts from leaves and flowers of Ilex glabra. mouse model

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  • Save Advances in Life Sciences 2012, 2(6): 170-173 DOI: 10.5923/j.als.20120206.04 Preliminary Investigation for Immunomodulation of Methanolic Extracts of Leaves and Flowers of Pongamia Glabra Vent. in Mice Model Sanjeev Heroor1,*, Arunkumar Beknal1, Nitin Mahurkar2 1Dept. of Pharmacognosy and Phytochemistry, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India 2Dept. of Pharmacology, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India Abstract Evaluation of the immuno modulatory activity methanolic extracts of leaves and flowers of Pongamia glabra Vent. in cyclophosphamide induced myelosuppressed mice. Methanolic extracts of leaves and flo wers of Pongamia glabra Vent. at the doses of 250mg/kg and 500mg/kg p.o. were ad ministered for 13 days to normal adult albino mice of either sex and cyclophosphamide (30mg/kg i.p.) was in jected on 11th,12th and 13th days 1 hour after the administration of the treatment with extract. On 14th day blood was collected by retro o rbital puncture and the activity was evaluated by determining the RBC, Hb%, Platelet, total WBC and differential counts. Methanolic extracts of leaves and flowers of Pongamia glabra Vent. showed dose dependant highly significant(p<0.001) counteracting effect to cyclophosphamide induced reduction in total WBC, DLC and plate let counts and significant (p<0.01) effect to that of reduction in RBC counts and Hb%. The significant immunostimu lant activity of the methanolic ext racts of Pongamia glabra Vent. leaves and flowers at the doses of 250mg/kg and 500mg/ kg p.o. in cyclophosphamide induced myelosuppression may be attributed towards the presence of bioactive components, viz.- saponins, sterols, tannins and flavonoids in the ext racts. Keywords Pongamia Glabra Vent., Cyclophosphamide, Immunomodulatory, Myelosuppression 1. Introduction Modulation of immune responses to allev iate the diseases has been of interest for many years in Ayurveda under the concept of ‘Rasayana’ (Chemical rejuvenators)[1]. Immuno modulation is a p rocedure which regulates the immune system of an organis m by suppression and stimulat ion of the cells and organs of the immune system[2]. Immunostimu lation in a drug induced immunosuppression model can be said to be true immunomodulation[3]. Certain agents called immuno modulatory agents have the ability to normalize or modulate pathophysiological p rocesses[4]. A nu mb er o f med icinal p lants such as Oci mu m ind icu m, Withania somnifera, Tinospora cordifolia, Mangifera indica, et c.[5] h av e b een s yst emat ically s creen ed fo r t heir immu n o mod u lato ry act iv it y. Cy clop hosph amide is an alky lating agent widely used in anti- neoplastic therapy of a variety of cancers such as ly mphoma, myelo ma and chronic ly mphocyt ic leu kemia[6]. Cyclophosphamide b rings up general depletion of immune competent cells by acting on both cyclic and intermitotic cells[7]. Cyclophosphamide * Corresponding author: (Arunkumar Beknal) Published online at Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved induced immunosuppression (myelosuppression) is reported to cause various types of infections[8]. Pongamia glabra Vent. (Syn. Pongamia pinnata) Fam. Legu minosae (Figure 1), co mmonly called in reg ional language as Karanja, a tree found all over India bearing imparipinnate leaves and pinkish wh ite colored flowers[9]. Leaves of the plant are reported to contain a bitter alkaloid. The ancient folklore uses of the plant claimed are as follows–The juice of the leaves is used to treat diarrhea, cough, dyspepsia, flatulence, leprosy and gonorrhea[10]. Leaves are used as antiparasitic and insect repellant[11]. In review of the wide range of med icinal applications, the present study was aimed to screen methanolic extracts of leaves and flowers of Pongamia glabra Vent. for immuno modulatory activity in cyclophosphamide induced immune suppressed albino mice. 2. Materials and Methods 2.1. Plant Material Leaves and flowers of Pongamia glabra Vent. were collected fro m local areas of Gu lbarga, No rth Karnataka and a voucher specimen has been deposited at the departmental herbariu m. The plant was identified and authenticated by Dr. Srinathrao, Pro f. and HOD, Dept. o f Botany, Gu lbarga 171 Advances in Life Sciences 2012, 2(6): 170-173 University, Gu lbarga and Ref. No. GUG/ BOT/ Herbariu m/ 2008-09/ 09. The mentioned parts of the plant were dried and powdered and the powdered drug was first defatted with petroleum ether (40-600C) and extracted with methanol using Soxh let apparatus for 48h to obtain methanolic ext racts of leaves and flowers of the plant respectively. The filtrates of the extracts were concentrated to dryness under reduced pressure using a rota flash evaporator.The yields of the methanolic extracts of leaves and flowers were 10.28g m (14.68%w/ w) and 43.68g m (55.96%w/w) respectively. 2.4. Acute toxicity studies Acute toxicity studies were carried out as per OECD guidelines by 425 method (#26)[14]. The animals did not show mortality at the dose of 5000mg/kg and hence its 1/10th dose i.e., 500mg/kg and 1/20th dose i.e., 250mg/kg p.o. were used as the therapeutic doses for the methanolic extracts of the study undretaken. 2.5. Test Samples Weighed quantities of the test extracts were suspended in 1% sodium carbo xy methyl cellu lose to prepare a suitable dosage form[15].The control an imals were ad min istered an equivalent volume of sodium CM C vehicle. 2.6. Drugs Cyclophosphamide was used as a standard immunosuppressant, Cyclo xan® (Biochem–pharmaceutical industries Ltd. Mu mbai) containing 200mg – cyclosphosphamide, was purchased fro m the market and dilutions were made using sterile water fo r in jection as labeled on the marketed product. Figure 1. Image of Pongam ia glabra Vent 2.2. Preliminary Phytochemical and Thin Layer Chromatography studies The methanolic extracts of leaves and flowers were subjected for preliminary qualitative chemical tests and the presences of major phytoconstituents were confirmed by TLC studies[12, 13]. ● TLC profile for flavonoids Solvent system: ethyl acetate: formic ac id: glacia l acetic ac id: water (10:1.1:1.1:2.6). Detection: UV 365(blue fluorescent spots). ● TLC profile for alkalo ids Solvent system: toluene: ethyl acetate: diethylamine (7:2:1) Detection: wagner’s reagent (brown colored spots). ● TLC profile for steroids Solvent system: petroleu m ether: acetone (7:3). Detection: anisaldehyde: sulphuric acid reagent (pink to red colored spots) ● TLC profile for saponins Solvent system: chloroform: g lacial acetic acid : methanol: water (6.4:3.2:1.2:0.8). Detection: anisaldehyde: sulphuric acid reagent (pink co lored spots) 2.3. Animals Swiss albino mice of either sex, weighing 25-30g housed in standard atmospheric conditions were used. They were ma intained by feeding with standard rodent diet and water ad libitum. The study was approved by Institutional Animal Ethical Co mmittee, Ref. No. HKECOP/IA EC/ 45/ 2011-12. 2.7. Cyclophos phami de Induced Myelosuppression[16] Animals were divided into six groups of six animals each. Group I served as control group and received the vehicle (1% sodium CM C) for 13 days. Group II (Cyclophosphamide group) received the vehicle (1% sodium CM C) fo r a period of 13 days and on 11th, 12th and 13th days was injected with cyclophosphamide(30 mg/kg i.p.). Groups III and IV were administered methanolic extracts of leaves of the plant at the doses of 250mg/kg and 500mg/ kg p.o. daily for 13 days respectively. Similarly Groups V and VI were ad min istered methanolic extracts of flo wers of the plant 250mg/kg and 500mg/kg p.o. daily for 13 days respectively. All the groups III, IV, V and VI were injected with cyclophosphamide (30mg/kg i.p) on the 11th, 12th and 13th days, 1 hour after the administration of the respective treat ment. Blood samples were drawn on 14th day of experiment by retro orbital puncture and hematological parameters were studied for RBC, Hb %, p latelets, total W BC counts and differential leucocytes counts (DLC). 2.8. Statistical Analysis Data were exp ressed as mean ± SEM and differences between the groups were statistically determined by analysis of variance fo llo wed by Dunnet’s test. 3. Results and Discussion The preliminary phytochemical screening and TLC studies of the methanolic extracts of leaves and flowers indicated the presence of carbohydrates, glycosides, steroids, saponins, flavonoids, alkalo ids and tannins and in addition the presence of proteins was observed in the extract of flowers. Results are represented in Table 1. Sanjeev Heroor et al.: Preliminary Investigation for Immunomodulation of M ethanolic 172 Extracts of Leaves and Flowers of Pongamia Glabra Vent. in M ice M odel Cyclophosphamide at the dose of 30mg/kg. i.p. caused a significant reduction in total WBC count, differential leucocyte counts and platelets along with marg inal reduction in RBC and Hb % co mpared to control g roup (Group I). Methanolic ext racts of leaf and flowers at the doses of 250mg/kg and 500mg/kg p.o. showed dose dependant statistically highly significant (p<0.001) increase in total WBC, DLC and platelets and significant increase in RBC and Hb% (p <0.01) co mpared with cyclophosphamide group (Group II). However the methanolic extract of flowers showed more significant effect than the methanolic e xtract of leaves. Results are represented in Table 2 and 3. Table 1. Thin Layer Chromatography results of Pongamia glabra Vent. leaves and flowers methanolic ext ract s Flavonoids Alkaloids Steroids Saponins Extract Leaf Ext. Flower Ext. Rf value 0.57, 0.65 (2 sp ot s) 0.62, 0.72 (2 sp ot s) Rf value 0.94 (1spot) 0.57 (1spot) Rf Value 0.61,0.63 ,0.68,0.76 (4 sp ot s) 0.63,0.66, 0.72,0.78, 0.81 (5 sp ot s) Rf value 0.55, 0.62 (2 sp ot s) 0.71, 0.78 (2 sp ot s) Table 2. Effect of methanolic extracts of Pongamia glabra Vent. leaves and flowers on cyclophosphamide induced myelosuppression in mice Animal gr. n=6 I (Cont ro l) RBC (106/mm3 ) 6.232± 0.070 II (Cyclophos phamide) III(Leaf Ext. 250mg/kg ) IV(Leaf Ext. 500mg/kg) V (Flower Ext. 250mg/kg) VI(Flower Ext. 500mg/kg) 5.023± 0.056 5.345*± 0.187 5.560*± 0.197 5.452*± 0.200 5.583*± 0.130 Hb (g%) 9.683± 0.101 8.250± 0.136 9.050*± 0.067 9.177*± 0.079 9.067*± 0.056 9.133*± 0.091 P latelet s (105/mm3) 6.300± 0.057 4.767± 0.088 5.100**± 0.068 5.300**± 0.186 5.117**± 0.083 5.417**± 0.087 WBC (103/mm3) 4.800± 0.096 2.267± 0.244 3.367**± 0.141 3.633**± 0.267 3.300**± 0.086 3.350**± 0.182 Bone marro w is a source of cells involved in immune activity and is the sensitive organ most affected during any immunosuppression therapy with this class of cytotoxic drugs. Stem cells degeneration and inability of bone marrow to regenerate new blood cells will g ive rise to thrombocytopenia and leucopenia[17]. Immuno modulatory activity of methanolic ext ract of leaves and flowers of Pongamia glabra Vent. was exp lored on cyclophosphamide induced myelosuppression in mice at the doses of 250mg/ kg and 500mg/kg p.o. Results of the e xperimental study proved the dose dependant counteracting effect of the ext racts to the cyclophosphamide induced myelosuppression, as indicated by increase in RBC, total WBC platelet counts, Hb % and DLC in the extract treated groups (Group III, IV, V and VI), co mpared to cyclophosphamide treated group (Group II). However the significance of counteracting activity to reduction in blood cell counts of the flowers extract was more than that of the leaves extract at the mentioned doses. The potentiated activity of flowers extract may be due to p resence of excess number of flavonoids and steroids in the extract. The results clearly indicated modulation of bone marrow act ivity suppression i.e. myelosuppression when used cyclophosphamide alone and stimu lation to counteract the cyclophosphamide induced myelosuppression in pretreated methanolic ext ract groups of leaves and flowers of Pongamia glabra Vent.. Table 3. Effect of methanolic extracts of Pongamia glabra Vent. leaves and flowers on cyclophosphamide induced myelosuppression in mice Animal gr. n=6 Neutro -phils (%) Lympho -cytes (%) Eosino -phils (%) Baso -phils (%) Mono -cytes (%) I 22.00± 71.83± (Control) 0.577 0.654 2.667± 1.500± 1.667± 0.210 0.232 0.210 II (Cyclopho sphamide) III(Leaf Ex.250mg /kg ) IV(Leaf Ex.500mg /kg) V(Flower Ex.250mg /kg) VI(Flower Ex.500mg /kg) 12.17± 0.703 15.33* ±0.882 18.00* ±0.816 15.83* ±0.703 16.33* ±0.494 62.17± 0.477 62.00** ±0.966 68.67* ±0.843 64.67* ±1.116 67.67*± 0.918 0.666± 0.166 0.210 ±0.166 1.167*± 0.333* 0.307 ±0.211 1.500* 0.500* ±0.223 ±0.223 1.337* 0.500* ±0.333 ±0.224 1.667* ±0.210 0.500* ±0.223 0.167± 0.167 0.333* ±0.211 0.666* ±0.210 0.500* ±0.224 0.500* ±0.223 *= p<0.01, **= p<0.001 Test drug treated groups were compared with control group (Group I) 4. Conclusions The phytochemical investigation of the methanolic extracts of leaves and flowers of Pongamia glabra Vent. revealed the presence of saponins, steroids, proteins, tannins and flavonoids. Saponins are steroidal glycosides proven as important phytoconstituents to have immuno modulating property. The isolated diosgenyl saponins of Paris polyphylla were reported for immunostimulating act ivity[18]. Tannins are also reported to possess immunostimu lating activites. The ingredients of well known Ayurvedic formulat ion, Chyawanprash was used traditionally as a rejuvenator includes Terminalia chebula, Terminalia 173 Advances in Life Sciences 2012, 2(6): 170-173 belerica, Emblica officialis and many other tannin containing drugs, which have been reported for their immunostimu lating activ ity[19]. Hence the collective presence of steroids, saponins, tannins and flavonoids in the methanolic ext racts would be attributed for immunostimu lating activity. However the flo wer methanolic extract of Pongamia glabra Vent. exhib ited mo re immune-potentiating activity than that of methanolic extract of leaves. However this is a preliminary research work and the precise mechanis m of immunomodulatory action exerted by the bio-active constituents of methanolic extracts of leaves and flowers of Pongamia glabra Vent. against cyclophosphamide induced myelosuppression needs to be investigated. [7] Goodman JW , In : Stites DP, Terr. AI, Parslow TG. (Editors). The Immune response in Basic and Clinical Immunology, 8th ed. Prentice-Hall, Engle wood Cliffs, NJ, pp. 40-47, 1994. [8] Angulo I, Jimenez M B, Garcia Bustos JF and Gargallo D, “Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of sup pressive myeloid progenitors”, cell Immunology, vol. 218, pp .46-58,2002. [9] Kirtikar KR and Basu BD, Indian M edicinal Plants, vol.1, 2nd ed. Oriental Enterprises. Dehradun, India, pp. 830-32, 1984. [10] Nadkarni KM , Indian M ateria M edica, vol.1, Popular Prakashan, M umbai, India, pp. 1001 – 1004, 1996. [11] Hartwell JL , Plants used against cancer. A Survey, Lloydia, pp. 30-34, 1967-1971. ACKNOWLEDGEMENTS Authors are thankful to authorities of HKE society and MTR Institute of Pharmaceutical Sciences, Gu lbarga, Karnataka, India, for provid ing the necessary facilities to carry out the study. REFERENCES [12] Khandelwal KR , Practical Pharmocognosy Techniques and Experiments, 10th ed., Nirali Prakashan, Pune, India, pp. 149 – 158, 2003. [13] Hildebert Wagner and Sabine Bladt , Plant Drug Analysis-A Thin layer Chomoatography Atlas, 2nd ed., Springer-verlag. Berlin Heidelberg, New York, pp. 1-3, 195-197, 305-206, 2001. 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[2] Bafna AR and M ishra SH, “Immunostimulatory effect of [16] M anjarekar PN, Jolly CL and Narayan S, “Comparative methanol extract Of Curculigo orchioides on immunosup studies of immunomodulatory activity of Tinospora pressed mice”, Journal of Ethnopharmacology, vol. 104, cordifolia and Tinopora sinensis”, Fitoterpia, vol. 71, pp. pp.1-4, 2006. 254-257, 2001. [3] Patwardhan B, Kalbag D, Patki PS and Nagasampagi BA, [17] Pelczar MJ, Chan ECS and Krieg NR, M icrobiology, 5th ed., “Search of Immunomodulatory agents : a review”, Indian Tata M cgraw Hill, New Delhi, India, pp.703-715, 1990. Drugs, vol. 28, no. 2,pp. 56- 63,1990. [18] Xiu-feng Z, Yan C, Jiajun H, Ya-Zhou Z, Zhou N and Ya-Lin [4] Wagner H, “ Immunomodulatory agents” in proceedings of T, Immunostimulating properties of diosgenyl saponins iso the Alfred Benzon Symposium, 20, p. 559, 1983. lated from Paris polyphylla. Bioorganic and M edicine [5] M akare N, Bodhankar S and Rangari V, “Immunomodulatory Chemical Letters, 2007. activity of alcoholic extracts of Mangifera indica Linn. in [19] R. Srikumar, Parthasarathy NJ and Sheeladevi R, “Immuno mice”, Journal of Ethnopharmacology, vol.78, pp.133-137, modulatory activity of Triphala on Neutrophil Functions”, 2001. Biology and Pharma Bulletin, vol.28, no.8, pp.1398 – 1403, [6] Baumann F and Preiss R, “ Cyclophosphamide and related 2005. anticancer drugs”, Biomedical Science Applications, vol. 764, pp .173-192,2000.

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