ASSESMENT OF CYTOTOXIC EFFECT OF METHANOLIC CRUDE EXTRACTS OF AZADIRACTA INDICAHTML Full Text
ASSESMENT OF CYTOTOXIC EFFECT OF METHANOLIC CRUDE EXTRACTS OF AZADIRACTA INDICA
Md. Mahabubul Alam Rakib 1 and Mohammad Musarraf Hussain*2
Department of Pharmacy, Noakhali Science and Technology University 1, Sonapur, Noakhali-3802, Bangladesh
Department of Pharmacy, Jagannath University 2, Dhaka-1100, Bangladesh
ABSTRACT:The aim of the research is to observe the cytotoxic activity of methanolic crude extracts of Azadirachta indica, a plant belonging to the family Meliaceae. The root of Azadirachta indica was extracted with organic solvent and the extracts were fractionated by using three fractions (n-hexane, ethyl acetate and chloroform). The crude extracts were screened for antitumor properties using brine shrimp lethality bioassay. A reputed cytotoxic agent, vincristine sulphate was used as a positive control. From the results of the brine shrimp lethality bioassay it can be well predicted that n-hexane, ethyl acetate, chloroform soluble fractions of Azadirachta indica possess cytotoxic principles (LC50 1.429 mg/mL, 2.368mg/mL and 1.479 mg/mL respectively) comparison with positive control vincristine sulphate (LC50 0.563 mg/mL).
Azadirachta indica, Meliaceae, Methanolic Crude Extracts, Cytotoxic Activity
INTRODUCTION: Azadirachta indica is a native tree of Bangladesh, found in every part in Bangladesh especially in semi-arid conditions. It is especially suited to semi-arid conditions and thrives even in the poorest soil with rainfalls as little as 18 inches (450 mm) per year and temperatures up to 50°C (120°F). It may grow up to 50 feet (15 m) tall and live for 200 years. The lifespan of the Azadirachta indica tree is described to be anywhere between 150 to 300 years. Its blossoms are small, white flowers with a very sweet, jasmine-like scent. Its edible fruit is about 3/4 of an inch (2 cm) long with white kernels.
Azadirachta indica tree generally begins bearing fruits at three to five years of age, and can produce up to 50 kg of fruit annually when mature. The pinnate leaves have a very bitter taste and a garlic-like smell.
Azadiracta indica had been used as a potencial medicinal agent like ani-inflammatory 1, 2, ani-neoplastic activity 5 melanogenesis inhibitory 2, immunostimulatory agent 3, larvae of filaria 4, tumor cell killing 6, chemotactic activity 7, larvae of Boophilus decolotatus 9, acaricidal acitivity 10, larvicidal activity 11, 37, anti-tumor activity 13, 15, 17, 33, leukocyte adopsis 14, propylactic activity16, anthelmintic activity 18, 19, hepatotoxicity 21, anti-cancer effects 22, transmission blocking activity 23, insecticidal activity 24, anti-oxidant activity 25, anti-trypanosomal 26, anti-hypertensive activity 31, immunological function 32, nutritional activity 34, free radical scavenging activity 35, antiviral activity 36, antibacterial activity 38, antimalarial activity 39, anti-diabetic 40 and anti-bovine activity 41. Some potential medicinal compounds had been isolated from A. indica like anti-plasmodial triterpenoids 8 and snake venom phospholipage A2 30.
METHODS AND MATERIALS:
Collection and identification of the plant: Plant sample of Azadirachta indica root was collected from near the Noakhali Science and Technology University area in March 2010.
Plant material preparation: The root of the plant was collected in fresh condition. It was sun-dried and then dried in an oven at reduced temperature to make it suitable for grinding purpose. The coarse powder was then stored in air-tight container with marking for identification and kept in cool, dark and dry place for future use.
Extraction of the plant material: The powdered roots (660 g) were soaked with methanol (2.5 L) in May, 2010 in a desicator through occasional shaking and stirring for 25days.
The extract was then filtered through filter-cloth. The filtrate was kept to dry in fresh and clean air to afford a greenish mass of biological investigation. The methanolic crude extract was fractionate according to Modified Kupchan partitioning method (figure 2) into n-hexane (1.0 g), ethyl acetate (1.5 g) and chloroform (0.7 g) soluble fractions (figure 1).
FIGURE 1: SCHEMATIC DIAGRAM OF THE CRUDE EXTRACTS OF AZADIRACHTA INDICA
FIGURE 2: SCHEMATIC DIAGRAM OF A MODIFIED KUPCHAN PARTITIONING METHOD
RESULT AND DISCUSSION: The brine shrimp test represents a rapid, inexpensive and simple bioassay for testing plant extract lethality which in most cases correlates reasonably well with cytotoxic and anti-tumour properties 27. Following the procedure of Mayer 28, the lethality of the methanolic crude extract (n-hexane, ethyl acetate and chloroform fractions) were determined and the LC50 values of n-hexane, ethyl acetate and chloroform soluble fraction found to be 1.429mg/mL 2.368mg/mL and 1.479 mg/mL respectively compared with the positive control vincristine sulphate (0.563mg/mL) (Table 1).From the results of the brine shrimp lethality bioassay it can be well predicted that the methanolic crude extracts (n-hexane, ethyl acetate and chloroform soluble fractions) possess cytotoxic properties. The effect of vincristine sulphate, n-hexane, ethyl acetate, and chloroform soluble fraction and are shown in table 2 and figure 3, 4, 5 and 6.
TABLE 1: RESULTS OF THE TEST SAMPLES OF AZADIRACHTA INDICA
|Methanolic crude extracts||LC50 (mg/mL)||Regression equation||R2|
|n-Hexane||1.429||y = 44.27x - 13.23||0.930|
|Ethyl acetate||2.368||y = 23.94x - 6.867||0.690|
|Chloroform||1.479||y = 43.06x - 13.71||0.925|
|Vincristine sulphate (positive control)||0.563||y = 30.056x + 56.016||0.9168|
TABLE 2: EFFECT OF n-HEXANE, ETHYL ACETATE AND CHLOROFORM SOLUBLE FRACTION ON SHRIMP NAUPLII
|Conc. (C)(mg/mL)||Log C||% Mortality||LC50 (mg/mL)||Vincristine Sulfate|
|Conc. (C)(mg/mL)||Log C||%Mortality||LC50(mg/mL)|
|n-Hexane||Ethyl acetate||Chloroform||n-Hexane||Ethyl acetate||CF|
FIGURE 3: EFFECT OF VINCRISTINE SULPHATE ON BRINE SHRIMP NAUPLII
FIGURE 4: EFFECT OF N-HEXANE SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII
FIGURE 5: EFFECT OF ETHYL ACETATE SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII
FIGURE 6: EFFECT OF CHLOROFORM SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII
CONCLUSION: Azadirachta indica leaves and roots are good for blood circulation and blood purification. Azadirachta indica has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern medicine. Azadirachta indica elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex.
More than 140 compounds have been isolated from different parts of Azadirachta indica. All parts of the Azadirachta indica tree- leaves, flowers, seeds, fruits, roots and bark have been used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders. Azadirachta indica leaf and its constituents have been demonstrated to exhibit immunomodulatory, anti-inflammatory, anti-hyperglycaemic, antiulcer, anti-malarial, antifungal, antibacterial, antiviral, anti-oxidant, antimutagenic and anticarcinogenic properties.
It comes out of my own personal experiences and study of the effects and properties of Azadirachta indica. It is also my conviction that Azadirachta indica is a wonderful cure for diseases and gives much to humans including shade, good air, health and overall well-being. I also think, it is important that people become aware of the values of Azadirachta indica. Among all the plants on this planet that have proved useful for humanity, a few are distinguished by their astonishing versatility.
Among these, the Azadirachta indica tree is one of the most important one. Azadirachta indica is deemed very effective in the treatment of scabies although only preliminary scientific proof exists which still has to be corroborated, and is recommended for those who are sensitive to permethrin, a known insecticide which might be irritant. Also, the scabies mite has yet to become resistant to Azadirachta indica, so in persistent cases Azadirachta indica has been shown to be very effective.
While Azadirachta indica products have some shortcomings as a conventional alternative, they fit in well as a tool to be used in integrated pest management systems. As more and more synthetic chemicals are being pulled from the market, Azadirachta indica is an environmentally benign alternative. It has significant effect on pests without harming beneficial organisms. Toxicology studies have indicated it to be quite safe to mammal’s also. Researchers, however, still have much work ahead of them to characterize the responses of sensitive insects in the field.
- Akihisa T, Noto T, Takahashi A, Fujita Y, Banno N, Tokuda H, Koike K, Suzuki T, Yasukawa K, Kimura Y., Melanogenesis inhibitory, anti-inflammatory, and chemopreventive effects of limonoids from the seeds of Azadirachta indicia A. Juss ,J Oleo Sci. 2009, 58(11):581-94.
- Akihisa T, Takahashi A, Kikuchi T, Takagi M, Watanabe K, Fukatsu M, Fujita Y, Banno N, Tokuda H, Yasukawa K, The melanogenesis-inhibitory, anti-inflammatory, and chemo preventive effects of limonoids in n-Hexane extract of Azadirachta indica , J Oleo Sci. 2011, 60 (2):53-9.
- Baral R, Mandal I, Chattopadhyay U, Immunostimulatory Azadirachta indica leaf preparation acts as an adjuvant to enhance the efficacy of poorly immunogenic B16 melanoma surface antigen vaccine., Int Immunopharmacol., 2005, 5(7-8), : 1343-52.
- Batabyal L, Sharma P, Mohan L, Maurya P, Srivastava CN, Relative toxicity of Azadirachta indica fruit, bitter gourd, and castor seed extracts against the larvae of filaria vector, Culex quinquefasciatus, Parasitol Res. 2009, 105(5), 1205-10.
- Beuth J, Schneider H, Ko HL, Enhancement of immune responses to Azadirachta indica leaf extract correlates with antineoplastic activity in BALB/c-mice. In Vivo, 2006, 20(2): 247-51.
- Bose A, Chakraborty K, Sarkar K, Goswami S, Chakraborty T, Pal S, Baral R., Azadirachta indica leaf glycoprotein induces perforin-mediated tumor cell killing by T and NK cells through differential regulation of IFNgamma signaling., J Immunother. 2009, 32(1):42-53.
- Chakraborty K, Bose A, Pal S, Sarkar K, Goswami S, Ghosh D, Laskar S, Chattopadhyay U, Baral R, Azadirachta indica leaf glycoprotein restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance, Int Immunopharmacol. 2008, 8(2):330-40.
- Chianese G, Yerbanga SR, Lucantoni L, Habluetzel A, Basilico N, Taramelli D, Fattorusso E, Taglialatela-Scafati O, Antiplasmodial triterpenoids from the fruits of Azadirachta indica, J Nat Prod. 2010, 73(8):1448-52.
- Choudhury MK, Toxicity of Azadirachta indica Seed Oil against the Larvae of Boophilus decoloratus, A One-Host Tick In Cattle., Indian J Pharm Sci. 2009, 71(5):562-3.
- Du YH, Li JL, Jia RY, Yin ZQ, Li XT, Lv C, Ye G, Zhang L, Zhang YQ, Acaricidal activity of four fractions and octadecanoic acid-tetrahydrofuran-3,4-diyl ester isolated from chloroform extracts of Azadirachta indica oil against Sarcoptes scabiei var. cuniculi larvae in vitro. Vet Parasitol. 2009, 163(1-2):175-8.
- Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash AP., Larvicidal activity of Azadirachta indica oil formulation against mosquitoes, Malar J., 2009, 8:124.
- Dulcie et al., 2000, Ghani, A 1998. Medicinal Plants of Bangladesh: Chemical Constituents and Uses, 1st edition, Asiatic Society of Bangladesh.
- Ghosh D, Bose A, Haque E, Baral R , Pretreatment with Azadirachta indica (Azadirachta indica) leaf preparation in Swiss mice diminishes leukopenia and enhances the antitumor activity of cyclophosphamide, Phytother Res. 2006, 20(9):814-8.
- Ghosh D, Bose A, Haque E, Baral R ,Azadirachta indica leaf preparation prevents leukocyte apoptosis mediated by cisplatin plus 5-fluorouracil treatment in Swiss mice, Chemotherapy, 2009, 55(3):137-44.
- Goswami S, Bose A, Sarkar K, Roy S, Chakraborty T, Sanyal U, Baral R, Azadirachta indica leaf glycoprotein matures myeloid derived dendritic cells and optimizes anti-tumor T cell functions, Vaccine, 2010, 28(5):1241-52.
- Haque E, Baral R , Azadirachta indica leaf preparation induces prophylactic growth inhibition of murine Ehrlich carcinoma in Swiss and C57BL/6 mice by activation of NK cells and NK-T cells, Immunobiology, 2006, 211(9):721-31.
- Haque E, Mandal I, Pal S, Baral R, Prophylactic dose of Azadirachta indica leaf preparation restricting murine tumor growth is nontoxic, hematostimulatory and immuno stimulatory, Immunopharmacol Immunotoxicol. 2006, 28(1):33-50.
- Iqbal Z, Lateef M, Jabbar A, Gilani A. In vivo anthelmintic activity of Azadirachta indica A. Juss seeds against gastrointestinal nematodes of sheep, Vet Parasitol. 2010, 68 (3-4), 342-5.
- Iqbal Z, Lateef M, Jabbar A, Gilani AH , In vivo anthelmintic activity of Azadirachta indica A. Juss seeds against gastrointestinal nematodes of sheep. Vet Parasitol. 2010, 168(3-4):342-5.
- Kashman et al., Gani, A. Medicinal Plants of Bangladesh: Chemical Constituents and Uses, 1998, 1st edition, Asiatic Society of Bangladesh.
- Koul A, Binepal G, Gangar SC, Impediment of diethylnitrosamine induced hepatotoxicity in male Balb/c mice by pretreatment with aqueous Azadirachta indica leaf extract, Indian J Exp Biol. 2007, 45(4):359-66.
- Kumar S, Suresh PK, Vijayababu MR, Arunkumar A, Arunakaran J, Anticancer effects of ethanolic Azadirachta indica leaf extract on prostate cancer cell line (PC-3), J Ethnopharmacol. 2006, 105(1-2):246-50.
- Lucantoni L, Yerbanga RS, Lupidi G, Pasqualini L, Esposito F, Habluetzel A., Transmission blocking activity of a standardized Azadirachta indica seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi, Malar J. 2010, 9:66.
- Maciel MV, Morais SM, Bevilaqua CM, Silva RA, Barros RS, Sousa RN, Sousa LC, Machado LK, Brito ES, Souza-Neto MA , In vitro insecticidal activity of seed Azadirachta indica oil on Lutzomyia longipalpis (Diptera: Psychodidae)., Rev Bras Parasitol Vet. 2010, 19(1):7-11.
- Manikandan P, Anandan R, Nagini S., Evaluation of Azadirachta indica leaf fractions for in vitro antioxidant potential and protective effects against H2O2-induced oxidative damage to pBR322 DNA and red blood cells. J Agric Food Chem. 2009, 57(15):6990-6.
- Mbaya AW, Ibrahim UI, God OT, Ladi S, Toxicity and potential anti-trypanosomal activity of ethanolic extract of Azadirachta indica (Maliacea) stem bark: an in vivo and in vitro approach using Trypanosoma brucei, J Ethno pharmacol. 2010, 128(2):495-500.
- Mclaughlin Jerry L., Anderson Jon E., Rogers, Lingling L. Drug Information Journal, 1998, 32, 513–524.
- Meyer, B. N, Ferringni, N, R., Puam, J, E., Lacobsen, L, B., Nichols, D.E. and McLaughlin, J. L. ‘Brine shrimp: a convenient general bioassay for active constituents, 1982, Planta Medica, 45, 31-32.
- Mia, 1990, Gani, A. Medicinal Plants of Bangladesh: Chemical Constituents and Uses, 1st edition, 1998, Asiatic Society of Bangladesh.
- Mukherjee AK, Doley R, Saikia D, Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica, mechanism of PLA2 inhibition by AIPLAI in vitro condition, Toxicon, 2008, 51(8):1548-53.
- Obiefuna I, Young R, Concurrent administration of aqueous Azadirachta indica leaf extract with DOCA-salt prevents the development of hypertension and accompanying electro cardiogram changes in the rat, Phytother Res. 2005, 19(9):792-5.
- Punzo F, Azadirachta indica seed extract containing azadirachtin affects mortality, growth, and immunological function in the whipscorpion Mastigoproctus giganteus (Lucas), Bull Environ Contam Toxicol. 2005, 75(4):684-90.
- Sarkar K, Bose A, Chakraborty K, Haque E, Ghosh D, Goswami S, Chakraborty T, Laskar S, Baral R., Azadirachta indica leaf glycoprotein helps to generate carcinoembryonic antigen specific anti-tumor immune responses utilizing macrophage-mediated antigen presentation, Vaccine, 2008, 26(34):4352-62.
- Saxena M, Ravikanth K, Kumar A, Gupta A, Singh B, Sharma A., Purification of Azadirachta indica seed cake and its impact on nutritional and antinutritional factors., J Agric Food Chem. 2010, 58(8):4939-44.
- Sithisarn P, Supabphol R, Gritsanapan W, Comparison of free radical scavenging activity of Siamese Azadirachta indica tree leaf extracts prepared by different methods of extraction, Med Princ Pract. 2006, 15(3):219-22.
- Tiwari V, Darmani NA, Yue BY, Shukla D , In vitro antiviral activity of Azadirachta indica (Azardirachta indica L.) bark extract against herpes simplex virus type-1 infection, Phytother Res. 2010, 24(8):1132-40.
- Tonk S, Bartarya R, Maharaj Kumari K, Bhatnagar VP, Srivastava SS, Effective method for extraction of larvicidal component from leaves of Azadirachta indica and Artemisia annua Linn, J Environ Biol. 2006, 27(1):103-5.
- Tripathi A, Chandrasekaran N, Raichur AM, Mukherjee A , Antibacterial applications of silver nanoparticles synthesized by aqueous extract of Azadirachta indica (Azadirachta indica) leaves, J Biomed Nanotechnol. 2009, 5(1):93-8.
- Udeinya IJ, Brown N, Shu EN, Udeinya FI, Quakeyie I , Fractions of an antimalarial Azadirachta indica-leaf extract have activities superior to chloroquine, and are gameto cytocidal, Ann Trop Med Parasitol. 2006, 100(1):17-22.
- Waheed A, Miana GA, Ahmad SI ,Clinical investigation of hypoglycemic effect of seeds of Azadirachta-indica in type-2 (NIDDM) diabetes mellitus, Pak J Pharm Sci. 2006, 19(4):322-5.
- Water-extracted polysaccharides from Azadirachta indica leaves: Structural features, chemical modification and anti-bovine herpesvirus type 1 (BoHV-1) activity. J Environ Biol. 2010, 31(4):409-12.
- Yusuf, M. Chowdhury, J.U, Wahab, M.A. and Begum, J., Medicinal Plants of Bangladesh 1994. Bangladesh Council of Scientific & Industrial Research (BCSIR), Dhaka, Bangladesh.
How to cite this article:
Rakib MMA and Hussain MM: Assessment of cytotoxic effect of Methanolic crude extracts of Azadirachta indica. Int J Pharm Sci Res 2013; 4(4); 1585-1590.
Md. Mahabubul Alam Rakib and Mohammad Musarraf Hussain*
Assistant Professor, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
25 November, 2012
21 February, 2013
29 March, 2013
01 April, 2013