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Evaluation of anticancer activity of ethanol extract of Sesbania grandiflora (Agati Sesban) against Ehrlich ascites carcinoma in Swiss albino mice
Journal of Ethnopharmacology 134 (2011) 984–987
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Evaluation of anticancer activity of ethanol extract of Sesbania grandiflora (Agati Sesban) against Ehrlich ascites carcinoma in Swiss albino mice S. Sreelatha a,c,∗ , P.R. Padma b , E. Umasankari c a
Faculty of Science, NUS, Singapore Department of Biochemistry, Avinashilingam University, Tamilnadu, India c Department of Chemistry and Biosciences, SASTRA University, Tamilnadu, India b
a r t i c l e
i n f o
Article history: Received 18 June 2010 Received in revised form 22 December 2010 Accepted 10 January 2011 Available online 18 January 2011 Keywords: Sesbania grandiflora Ethanol extract Ehrlich’s ascites carcinoma (EAC) Anticancer agent
a b s t r a c t Ethno pharmacological relevance: The plant Sesbania grandiflora (Fabaceae) is commonly known as “Sesbania” and “agathi” in ayurvedic system of medicine and reputed in the indigenous medicine in India. It is also known as “Agati Sesban” or “humming bird tree” in English. All parts of this unique plant are useful and have a wide spectrum of medicinal properties. The plant has various uses in folk and traditional medicines for headache, swellings, anemia, bronchitis, pains, liver disorders and tumors. Aim of the study: The objective of the present study was to explore the anticancer activity of the ethanol extract of Sesbania grandiflora against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. Materials and methods: Anticancer activity of ethanol extract of Sesbania grandiflora (EESG) of both leaves and flowers were evaluated in Swiss albino mice against Ehrlich Ascites Carcinoma (EAC) cell line at the doses of 100 and 200 mg/kg body weight intraperitoneally. The extracts were administered for 14 consecutive days. Twenty-four hours of last dose and 18 h of fasting, the mice were sacrificed and the anticancer effect of EESG was assessed by evaluating tumor volume, viable and nonviable tumor cell count, tumor weight, hematological parameters and biochemical parameters of EAC bearing host. Results: Sesbania grandiflora extracts showed significant decrease in (p < 0.01) tumor volume, viable cell count, tumor weight and elevated the life span of EAC bearing mice. Hematological profile such as RBC, hemoglobin and lymphocyte count reverted to normal level in EESG treated mice. The extracts significantly (p < 0.05) decreased the levels of lipid peroxidation and significantly (p < 0.05) increased the levels of GSH, SOD and CAT. Conclusion: The results showed that the ethanol extract of Sesbania grandiflora was effective in inhibiting the tumor growth in ascitic models and that is comparable to 5-Fluorouracil. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Plant The plant material Sesbania grandiflora L. pers (Fabaceae) was collected from Kumbakonam, Tanjore District, Tamilnadu, India and authenticated by the Botanical survey of India, Coimbatore, Tamilnadu, India, where a voucher specimen has been preserved for future reference (BSI/SC/5/23/Tech-905). The leaves and flowers of Sesbania grandiflora were shade dried, powdered, and extracted (180 g) with ethanol and water in the ratio 1:4 in a Soxhlet extractor for 18–20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature. The crude extracts obtained were a dark brown solid weighing 40 g and 35 g with the yield of the extracts as 22.01% and 18.01% respectively. The extracts
∗ Corresponding author at: Faculty of Science, NUS, Singapore-117546, Singapore. Tel.: +65 82834498. E-mail address: [email protected] (S. Sreelatha). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.01.012
were preserved in a refrigerator at 4 ◦ C until further use. Preliminary phytochemical screening indicated the presence of flavonoids, saponins, tannins and alkaloids.
2. Uses in traditional medicine and reported activities Sesbania grandiflora L. pers (Fabaceae) commonly known as “sesbania” and “agathi,” is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases including leprosy, gout, rheumatism, tumor and liver disorders (Joshi, 2000). All parts of Sesbania grandiflora are utilized for medicine in South eastern Asia and India including preparations derived from the roots, bark, gum, leaves, flowers, and fruit. In ayurvedic medicine the root is applied as a poultice (wet substance applied to inflammation or sore spots) for application to inflammation and fever and the leaves are utilized for the treatment of epileptic fits and to cure night blindness (Burbidage, 1965). The juices of the flowers have a special ability to improve vision and the crushed leaves are applied
S. Sreelatha et al. / Journal of Ethnopharmacology 134 (2011) 984–987
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to sprains and bruises of all kinds. A tea made from the leaves is believed to have antibiotic, anthelmintic, antitumor and contraceptive properties. The bark is considered as a tonic and an antipyretic, and a remedy for gastric troubles and diabetes. The principal medicinal effects are due to the tree’s astringency; hence it is used against inflammation, venom and other poisons, bacterial infections and tumors (Anon., 1986; Mannetje and Jones, 1992; Timyan, 1996). Sesbania grandiflora has been used as an important dietary nutritive source in South East Asian countries and the leaves are the richest source of aminoacids, minerals and vitamins (Govindan and Shanmugasundaram, 1987). Recently, its (antianxiety) anxiolytic (Kasture et al., 2002), hepatoprotective (Pari and Uma, 2003), cardio protective (Ramesh et al., 2008), antiurolithiatic and antioxidant activities were reported (Doddola et al., 2008). However, so far no systematic study has examined the anticancer activity in the leaves and flowers of this plant. Hence, the present study based on the ethno medical claims, was sought to evaluate the scientific validity for the anticancer activity of the ethanol extract of Sesbania grandiflora both leaves and flowers against EAC tumor model.
Healthy male Swiss albino mice weighing 20 ± 2 g were procured from Tamilnadu University of Veterinary and Animal Sciences (TANUVAS) were used for the study. The animals were housed in large polypropylene cages in a temperature-controlled room and provided with standardized pelleted feed (TANUVAS) and clean drinking water ad libitum. All the procedures described were reviewed and approved by the University Animals Ethical Committee. The animals were divided into seven groups. All the animals were injected with EAC cells (0.2 ml of 2 × 106 cells/mouse) intraperitoneally except the normal group, for the development of ascites tumor (Kuttan et al., 1988). After 24 h of tumor inoculation the extracts of Sesbania grandiflora in variable doses of 100 mg/kg and 200 mg/kg were administered once daily for 14 days to groups 3, 4, 5 and 6.After the last dose and 24 h fasting, six mice from each group were sacrificed. The antitumor efficacy of plant extracts was compared with the standard which served as the seventh group (5-Flourouracil, 20 mg/kg/day i.p.).
3. Previously isolated constituents
4.6. Tumor growth response
High content of quercetin, myricetin and kaempferol were identified in Sesbania grandiflora leaf extracts (Mustafa et al., 2010). A novel protein fraction was isolated from the flower of Sesbania grandiflora which showed chemo preventive efficacy (Laladhas et al., 2010).
The anticancer effect of EESG was assessed by change in tumor weight, tumor volume, viable and nonviable cell count, median survival time (MST), and percentage increased life span (%ILS). MST for each group was noted and anticancer activity of the test compounds were compared with that of control group by measuring ILS (Gupta et al., 2000).
4. Materials and methods
MST =
4.5. Transplantation of tumor and treatment schedule
day of first death + day of last death 2
4.1. Chemicals Increase in life span (%) = 5-Fluorouracil was obtained from Ranbaxy Laboratories, Ltd., India. All other chemicals used were of analytical grade. 4.2. Phytochemical analysis The leaves and flowers were subjected to preliminary phytochemical screening to identify the presence of various phytoconstituents present in the extract (Kokate et al., 1997). On estimating the levels of polyphenolic compounds in each fraction using standard quercetin the ethanol fraction was found to have the higher polyphenolic content. Therefore this extract was further used for all the study throughout. 4.3. Tumor cells Ehrlich’s ascites carcinoma cells were obtained from Amala Cancer Research Centre, Trissur, and Kerala, India. The EAC cells were maintained by intraperitoneal inoculation of 2 × 106 cells/mouse. EAC cells aspirated from the peritoneal activity of mice were washed with saline and were given intraperitoneally to develop ascitic tumor. 4.4. Toxicity study An acute toxicity study relating to the determination of LD50 was performed (Litchfield and Wilcoxon, 1949). Different groups of mice were treated with graded doses of the EESG of leaves and flowers (100 mg, 200 mg, 500 mg and 750 mg/kg) orally. One group was maintained as control and was given distilled water. The animals were observed continuously for 2 h, and then intermittently and after 24 h for 14 days. The animals were observed for behavioral, neurological and autonomic profiles.
MST of treated group × 100 − 100. MST of control group
The viability and nonviability of the cells were checked by trypan blue assay. The cells were stained with trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were viable and those that took the dye were nonviable. These viable and nonviable cells were counted. 4.7. Hematological parameters At the end of the experimental period, the next day after an over night fasting blood was collected from freely flowing tail vein and used for the estimation of hemoglobin (Hb) content, red blood cell (RBC) count, white blood cell (WBC) count and differential count of WBC by standard procedures. 4.8. Biochemical parameters After the collection of blood samples, the mice were sacrificed. Then the liver was excised, rinsed in ice cold normal saline followed by ice-cold 10% KCl solution, blotted, dried and weighed. A 10% w/v homogenate was prepared in ice-cold KCl solution and centrifuged at 1500 rpm for 15 min at 4 ◦ C. The supernatant thus obtained were used for the estimation of thio-barbituric acid substances (TBARS) (Okhawa et al., 1979) glutathione (GSH) (Ellman, 1979) superoxide dismutase (SOD) (Kakkar et al., 1984) catalase (CAT) (Luck, 1974) and total protein (TP) (Lowry et al., 1951). 5. Statistical analysis All the parameters studied were subjected to statistical treatment using Sigma Stat statistical package (Version 3.1). The data were expressed as mean ± S.D. (n = 6) where ‘n’ represents the no. of samples. One-way ANOVA, followed by post hoc analysis using
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Table 1 Effect of EESG on tumor growth response. Parameters
Tumor volume (ml) Tumor weight (g) MST (days) %ILS Viable tumor cell count (×107 cells/ml) Non viable cell count (×107 cells/ml)
EAC control
Treated
II
III
IV
V
VI
VII
4.8 ± 0.86 5.5 ± 0.42 23.5 ± 1.90 00.00 10.40 ± 0.56 0.50 ± 0.03
3.4* ± 0.76 3.5* ± 0.67 38.2 ± 2.90 68.51 3.70* ± 0.15 0.90 ± 0.03
2.1** ± 0.12 2.6** ± 0.21 42.5 ± 3.90 97.42 3.60** ± 0.18 1.91 ± 0.04
3.7* ± 0.52 3.6* ± 0.25 31.7 ± 2.90 32.52 6.10 ± 0.23 0.80 ± 0.21
2.5** ± 0.33 2.3** ± 0.23 39.6 ± 3.40 64.61 4.30* ± 0.09 1.71 ± 0.02
1.8 ± 0.63 1.7 ± 0.36 48.1 ± 3.40 104.61 2.10 ± 0.17 2.60 ± 0.04
Values are expressed as mean ± S.D., n = 6. III – leaf extract (100 mg/kg) + EAC; IV – leaf extract (200 mg/kg) + EAC; V – flower extract (100 mg/kg) + EAC; VI – flower extract (200 mg/kg) + EAC; VII – Standard 5-FU (20 mg/kg) + EAC. * P < 0.001: between extract treated groups and EAC. ** P < 0.01: between extract treated groups and EAC.
Fischer’s LSD was adopted to all the parameters under study to test the level of statistical significance. The difference was considered significant if p < 0.05. 6. Results and discussion Recently, the use of some herbs has attracted a great deal of attention as one of the alternative cancer therapies from the point of less toxicity and cost benefits. Therefore, an attempt has been made to evaluate the anticancer activity of the leaf and flower extract of Sesbania grandiflora, which is commonly used in ayurvedic system of medicine for various purposes. The extracts revealed the presence of various phytoconstituents like reducing and non-reducing sugars, polyphenolics, alkaloids, saponins, tannins and terpenoids. In acute toxicity, no gross behavioral changes and mortality was observed up to a dose level of 400 mg/kg body weight. The LD50 value of EESG leaves and flowers were found to be >2 g/kg body weight of mice indicating that it is very less toxic to the animal. Treatment with EESG both leaves and flowers at the dose of 100 and 200 mg/kg body weight increased the life span (ILS) and nonviable cell count and reduced the tumor volume, tumor weight and viable tumor cell count significantly when compared to that of EAC control group (Table 1). As shown in Table 2, hemoglobin content and RBC count decreased significantly and the total WBC count increased significantly in EAC group as compared to normal group. Treatment with EESG restored the hematological parameters to more or less normal values. The number of RBC count and hemoglobin content also increased, while the WBC and the differential count decreased as compared to that of EAC control. The reliable criteria for judging the value of any anticancer drug is the prolongation of life span of the animals and the decrease of leukemic cells from blood (Gupta et al., 2004). However with the
treatment of EESG the percent increase in tumor cell volume, and number of viable tumor cells were found to be significantly less when compared to the EAC control. Hence, it may be concluded that the extracts by a direct cytotoxic effect and by arresting the tumor growth, increased the life span of EAC-bearing mice. The percentage increase in life span at the 200 mg/kg body weight dose of the leaf extract was found to be the highest than the flower extracts indicating its potent anticancer nature. In acute toxicity studies, the administration of EESG at the dose of 100 mg and 200 mg/kg for 14 days did not exhibit any adverse effect which may be due its composite nature where the presence of phytoconstituents could counteract its toxicity. In cancer chemotherapy the major problems are myelosuppression and anemia (Maseki et al., 1981). The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or hemoglobin percentage and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions (Fenninger and Mider, 1954). Similar results were observed in the present study in animals of the EAC tumor control group. The reversal of hematological parameters indicates that the extracts may possess protective action on the hematopoietic system. This reinstates that use of herbs might be a more effective strategy in the treatment of cancer. As shown in Table 2, the level of TBARS was significantly increased in the EAC treated animals when compared to the normal group. Treatment with EESG of both leaves and flowers at 200 mg/kg body weight reversed these changes towards normal levels. Significant decrease in the levels of GSH, SOD and CAT was observed in EAC control group which was reversed significantly towards normal in the EESG treated group. Almost similar results were observed with 5-FU treatment. It was observed that tumor cells produced more peroxides when they proliferate actively after inoculation of tumor and also known to affect many functions of the vital organs, which indicated the intensification of oxygen free rad-
Table 2 Effect of EESG on hematological and biochemical parameters. Parameters
Normal I
Total RBC (×106 cells/ml) Total WBC (×104 cells/ml) Hemoglobin (%) Lymphocyte (%) Neutrophil (%) Monocyte (%) LPO (nmol MDA/mg protein) GSH (mg/g) SOD (U/mg protein) CAT (U/mg protein)
5.34 6.40 16.10 66.10 31.10 3.30 0.96 2.31 4.34 27.4
± ± ± ± ± ± ± ± ± ±
0.02 0.10 0.70 5.40 2.70 0.10 0.70 0.21 0.05 0.21
EAC control
Treated
II
III
1.22 14.60 7.30 24.10 74.20 2.20 1.40 1.39 2.22 11.6
± ± ± ± ± ± ± ± ± ±
0.06* 0.90 0.50* 1.80* 6.50* 0.50* 0.50 0.32 0.07 0.92
4.29 11.50 14.80 9.20 38.30 3.20 1.02 2.72 3.29 25.5
IV ± ± ± ± ± ± ± ± ± ±
0.03 0.30 0.40 3.60 1.90 0.70** 0.20 0.15 0.06 0.32
4.6 9 6.20 15.90 61.10 37.10 2.40 0.97 2.49 4.62 26.2
V ± ± ± ± ± ± ± ± ± ±
0.06** 0.40** 0.80** 4.70** 2.60** 0.30 0.80** 0.32** 0.06** 0.42**
4.27 11.90 13.90 57.20 41.20 2.60 1.03 2.61 3.27 24.6
VI ± ± ± ± ± ± ± ± ± ±
0.05 0.30 0.40 5.3** 3.20** 0.40 0.40 0.12 0.06 0.32
4.51 5.80 15.0 60.30 8.10 2.30 0.98 2.51 4.31 25.9
VII ± ± ± ± ± ± ± ± ± ±
0.07** 0.20** 0.80** 5.80** 1.20** 0.10 0.70** 0.23** 0.09** 0.25**
5.36 6.70 15.80 65.10 33.10 4.20 0.97 2.23 4.34 26.7
± ± ± ± ± ± ± ± ± ±
0.40** 0.50** 1.60** 4.40** 1.90** 0.090** 1.60** 0.51** 0.09** 0.52**
Values are expressed as mean ± S.D, n = 6. III – leaf extract (100 mg/kg) + EAC; IV – leaf extract (200 mg/kg) + EAC; V – flower extract (100 mg/kg) + EAC; VI – flower extract (200 mg/kg) + EAC; VII – Standard 5-FU (20 mg/kg) + EAC. * P < 0.001: between normal and EAC. ** P < 0.01: between extract treated groups and EAC.
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ical production. The elevation of lipid per oxidation is also known to be associated with cancer (Navarro et al., 1997). Decrease in SOD, GSH and CAT activities described in tumors is regarded as markers of malignant transformation (Kavitha and Manoharan, 2006). Therefore the significant elevation of GSH, SOD and CAT, and significant reduction in LPO by the extract treatment confirms the potent antioxidant activity and free radical quenching property of EESG. The preliminary phytochemical studies indicated the presence of flavonoids, saponins, triterpenoids, glycosides, and tannins in the extracts of leaf and flower. A number of scientific reports indicate certain terpenoids; steroids and phenolic compounds such as tannins, coumarins and flavonoids have a chemo preventive role in cancer through their effects on signal transduction in cell proliferation and angiogenesis (Blois, 2002). Many such compounds are known to possess potent anticancer properties (Weber et al., 1996). The potent anticancer properties of the Sesbania grandiflora extracts may be attributed to the antioxidant and anticancer principles present in the extract. The results from the present study are consistent with the earlier reports, which support the antioxidant potential of Sesbania grandiflora under various oxidative stress conditions (Pari and Uma, 2003; Ramesh et al., 2008). In conclusion, the present study demonstrates the potent anticancer properties of Sesbania grandiflora extracts. The Sesbania grandiflora leaf extracts were more potent than the flower extracts where the maximum benefit of anticancer effect can be derived. Further studies on the elucidation of its mechanism of action and on the isolation of its active constituents may prove rewarding in cancer treatment. Acknowledgement Authors express sincere gratitude to Dr. K. Jaswanth, Department of Pharmacology, Periyar University for his keen interest and expert guidance. References Anon., 1986. The Useful Plants of India. Publications & Information Directorate, CSIR, New Delhi, India, pp. 465–466. Blois, M.S., 2002. Antioxidant determination by the use of a stable free radical. Nature 26, 1199–1200. Burbidage, N.T., 1965. Australian species of sesbania scop (leguminosae). Australian Journal of Botany 13, 103–141. Doddola, S., Pasupulati, H., Koganti, B., Prasad, K.V., 2008. Evaluation of Sesbania grandiflora for antiurolithiatic and antioxidant properties. Journal of Natural Medicines 62, 300–307. Ellman, G.L., 1979. Tissue sulphydryl groups. Archives in Biochemistry & Biophysics 82, 70–77. Fenninger, L.D., Mider, G.B., 1954. Energy and nitrogen metabolism in cancer. Advances in Cancer Research 9, 95–102.
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Govindan, S., Shanmugasundaram, E.R.B., 1987. Evaluation of the nutritive value of agathi (Sesbania grandiflora) leaf protein concentrates. The Indian Journal of Nutrition & Dietetics 24, 370–375. Gupta, M., Mazumder, U.K., Rath, N., Mukhopadhyay, D.K., 2000. Antitumor activity of methanol extract of Cassia fistula L. seed against Ehrlich ascites carcinoma. Journal of Ethnopharmacology 72, 151–156. Gupta, M., Mazumder, U.K., Sambath Kumar, R., Siva Kumar, T., Vamsi, M.L.M., 2004. Antitumor activity and antioxidant status of Caesalpinia bonducella against Ehrlich ascites carcinoma in Swiss albino mice. Journal of Pharmacological Sciences 94, 77–184. Joshi, S.G., 2000. Leguminoceae: Text book of medicinal plants. Oxford Publishing, New Delhi, pp. 130-132. Kasture, V.S., Deshmukh, V.K., Chopde, C.T., 2002. Anxiolytic and anticonvulsive activity of Sesbania grandiflora leaves in experimental animals. Phytotherapy Research 16, 455–460. Kakkar, P., Das, B., Vishwanathan, P.N., 1984. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemistry & Biophysics 21, 130–132. Kavitha, K., Manoharan, S., 2006. Anticarcinogenic and antilipidperoxidative effects of Tephrosia purpurea (Linn) pers. In 7, 12-dimethyl benz (a) anthracene (DMBA) induced hamster buccal pouch carcinoma. Indian Journal of Pharmacology 38, 185–189. Kokate, C.K., Purohit, A.P., Gokhale, S.B., 1997. Analytical pharmacognosy: phytochemical investigations. In: Prakashan, N. (Ed.), Pharmacognosy. , fifth ed. Nirali Prakashan, India, pp. 119–137. Kuttan, G., Vasudevan, D.M., Kuttan, R., 1988. Isolation and identification of a tumor reducing component from mistletoe extract (Iscador). Cancer Letters 41, 307–315. Laladhas, K.P., Cheriyan, V.T., Puliappadamba, V.T., Bava, S.V., Unnithan, R.G., Vijayammal, P.L., Anto, R.J., 2010. A Novel protein fraction from Sesbania grandiflora shows potential anticancer and chemo preventive efficacy, in vitro and in vivo. Journal of Cellular & Molecular Medicine 14, 636–646. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose effect experiments. Journal of Pharmacology & Experimental Therapeutics 96, 99–133. Lowry, O.H., Rosebrough, N., Farr, L.A., Randal, R.J., 1951. Protein measurement with Folin’s phenol reagent. Journal of Biological Chemistry 193, 265–275. Luck, H., 1974. Methods in Enzymatic Analysis, II. Ed., Berg Mayer, Academic Press, New York, pp. 885–886. Mannetje, L., Jones, R.M., 1992. Plant Resources of South-East Asia. No. 4: Forages. Pudoc Scientific Publishers, Wageningen. Maseki, M., Nishiagaki, I., Hagishara, M., Tomoda, Y., Yagi, K., 1981. Lipid per oxidation levels and lipid content of serum lipoprotein fractions of pregnant subjects with or without preeclampsia. Journal of Clinica Chemica Acta 41, 424–426. Mustafa, R.A., Hamid, A.A., Mohammed, S., Bakar, F.A., 2010. Total phenolic compounds flavonoids and radical scavenging activity of 21 selected Tropical plants. Journal of Food Science 75, C28–C35. Navarro, J., Obrador, E., Pellicer, J.A., Asensi, M., Vina, J., Esrela, J.M., 1997. Blood glutathione as an index of radiation-induced oxidative stress in mice and humans. Free Radical Biology & Medicine 22, 1203–1209. Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pari, L., Uma, A., 2003. Protective effect of Sesbania grandiflora against erythromycin estolate-induced hepatotoxicity. Journal of Medicinal Food 58, 439–443. Ramesh, T., Mahesh, R., Sureka, C., Begum, V.H., 2008. Cardio protective effects of Sesbania grandiflora in cigarette smoke-exposed rats. Journal of Cardiovascular Pharmacology 52, 338–343. Timyan, J., 1996. Bwa Yo: Important Trees of Haiti. South-East Consortium for International Development, Washington, DC. Weber, G., Shen, F., Prajda, N., Yeh, Y.A., Yang, H., Herenyiova, et al., 1996. Increased signal transduction activity and down regulation in human cancer cells. Anticancer Research 16, 3271–3282.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Evaluation of anticancer activity of ethanol extract of Sesbania grandiflora (Agati Sesban) against Ehrlich ascites carcinoma in Swiss albino mice S. Sreelatha a,c,∗ , P.R. Padma b , E. Umasankari c a
Faculty of Science, NUS, Singapore Department of Biochemistry, Avinashilingam University, Tamilnadu, India c Department of Chemistry and Biosciences, SASTRA University, Tamilnadu, India b
a r t i c l e
i n f o
Article history: Received 18 June 2010 Received in revised form 22 December 2010 Accepted 10 January 2011 Available online 18 January 2011 Keywords: Sesbania grandiflora Ethanol extract Ehrlich’s ascites carcinoma (EAC) Anticancer agent
a b s t r a c t Ethno pharmacological relevance: The plant Sesbania grandiflora (Fabaceae) is commonly known as “Sesbania” and “agathi” in ayurvedic system of medicine and reputed in the indigenous medicine in India. It is also known as “Agati Sesban” or “humming bird tree” in English. All parts of this unique plant are useful and have a wide spectrum of medicinal properties. The plant has various uses in folk and traditional medicines for headache, swellings, anemia, bronchitis, pains, liver disorders and tumors. Aim of the study: The objective of the present study was to explore the anticancer activity of the ethanol extract of Sesbania grandiflora against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. Materials and methods: Anticancer activity of ethanol extract of Sesbania grandiflora (EESG) of both leaves and flowers were evaluated in Swiss albino mice against Ehrlich Ascites Carcinoma (EAC) cell line at the doses of 100 and 200 mg/kg body weight intraperitoneally. The extracts were administered for 14 consecutive days. Twenty-four hours of last dose and 18 h of fasting, the mice were sacrificed and the anticancer effect of EESG was assessed by evaluating tumor volume, viable and nonviable tumor cell count, tumor weight, hematological parameters and biochemical parameters of EAC bearing host. Results: Sesbania grandiflora extracts showed significant decrease in (p < 0.01) tumor volume, viable cell count, tumor weight and elevated the life span of EAC bearing mice. Hematological profile such as RBC, hemoglobin and lymphocyte count reverted to normal level in EESG treated mice. The extracts significantly (p < 0.05) decreased the levels of lipid peroxidation and significantly (p < 0.05) increased the levels of GSH, SOD and CAT. Conclusion: The results showed that the ethanol extract of Sesbania grandiflora was effective in inhibiting the tumor growth in ascitic models and that is comparable to 5-Fluorouracil. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Plant The plant material Sesbania grandiflora L. pers (Fabaceae) was collected from Kumbakonam, Tanjore District, Tamilnadu, India and authenticated by the Botanical survey of India, Coimbatore, Tamilnadu, India, where a voucher specimen has been preserved for future reference (BSI/SC/5/23/Tech-905). The leaves and flowers of Sesbania grandiflora were shade dried, powdered, and extracted (180 g) with ethanol and water in the ratio 1:4 in a Soxhlet extractor for 18–20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature. The crude extracts obtained were a dark brown solid weighing 40 g and 35 g with the yield of the extracts as 22.01% and 18.01% respectively. The extracts
∗ Corresponding author at: Faculty of Science, NUS, Singapore-117546, Singapore. Tel.: +65 82834498. E-mail address: [email protected] (S. Sreelatha). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.01.012
were preserved in a refrigerator at 4 ◦ C until further use. Preliminary phytochemical screening indicated the presence of flavonoids, saponins, tannins and alkaloids.
2. Uses in traditional medicine and reported activities Sesbania grandiflora L. pers (Fabaceae) commonly known as “sesbania” and “agathi,” is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases including leprosy, gout, rheumatism, tumor and liver disorders (Joshi, 2000). All parts of Sesbania grandiflora are utilized for medicine in South eastern Asia and India including preparations derived from the roots, bark, gum, leaves, flowers, and fruit. In ayurvedic medicine the root is applied as a poultice (wet substance applied to inflammation or sore spots) for application to inflammation and fever and the leaves are utilized for the treatment of epileptic fits and to cure night blindness (Burbidage, 1965). The juices of the flowers have a special ability to improve vision and the crushed leaves are applied
S. Sreelatha et al. / Journal of Ethnopharmacology 134 (2011) 984–987
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to sprains and bruises of all kinds. A tea made from the leaves is believed to have antibiotic, anthelmintic, antitumor and contraceptive properties. The bark is considered as a tonic and an antipyretic, and a remedy for gastric troubles and diabetes. The principal medicinal effects are due to the tree’s astringency; hence it is used against inflammation, venom and other poisons, bacterial infections and tumors (Anon., 1986; Mannetje and Jones, 1992; Timyan, 1996). Sesbania grandiflora has been used as an important dietary nutritive source in South East Asian countries and the leaves are the richest source of aminoacids, minerals and vitamins (Govindan and Shanmugasundaram, 1987). Recently, its (antianxiety) anxiolytic (Kasture et al., 2002), hepatoprotective (Pari and Uma, 2003), cardio protective (Ramesh et al., 2008), antiurolithiatic and antioxidant activities were reported (Doddola et al., 2008). However, so far no systematic study has examined the anticancer activity in the leaves and flowers of this plant. Hence, the present study based on the ethno medical claims, was sought to evaluate the scientific validity for the anticancer activity of the ethanol extract of Sesbania grandiflora both leaves and flowers against EAC tumor model.
Healthy male Swiss albino mice weighing 20 ± 2 g were procured from Tamilnadu University of Veterinary and Animal Sciences (TANUVAS) were used for the study. The animals were housed in large polypropylene cages in a temperature-controlled room and provided with standardized pelleted feed (TANUVAS) and clean drinking water ad libitum. All the procedures described were reviewed and approved by the University Animals Ethical Committee. The animals were divided into seven groups. All the animals were injected with EAC cells (0.2 ml of 2 × 106 cells/mouse) intraperitoneally except the normal group, for the development of ascites tumor (Kuttan et al., 1988). After 24 h of tumor inoculation the extracts of Sesbania grandiflora in variable doses of 100 mg/kg and 200 mg/kg were administered once daily for 14 days to groups 3, 4, 5 and 6.After the last dose and 24 h fasting, six mice from each group were sacrificed. The antitumor efficacy of plant extracts was compared with the standard which served as the seventh group (5-Flourouracil, 20 mg/kg/day i.p.).
3. Previously isolated constituents
4.6. Tumor growth response
High content of quercetin, myricetin and kaempferol were identified in Sesbania grandiflora leaf extracts (Mustafa et al., 2010). A novel protein fraction was isolated from the flower of Sesbania grandiflora which showed chemo preventive efficacy (Laladhas et al., 2010).
The anticancer effect of EESG was assessed by change in tumor weight, tumor volume, viable and nonviable cell count, median survival time (MST), and percentage increased life span (%ILS). MST for each group was noted and anticancer activity of the test compounds were compared with that of control group by measuring ILS (Gupta et al., 2000).
4. Materials and methods
MST =
4.5. Transplantation of tumor and treatment schedule
day of first death + day of last death 2
4.1. Chemicals Increase in life span (%) = 5-Fluorouracil was obtained from Ranbaxy Laboratories, Ltd., India. All other chemicals used were of analytical grade. 4.2. Phytochemical analysis The leaves and flowers were subjected to preliminary phytochemical screening to identify the presence of various phytoconstituents present in the extract (Kokate et al., 1997). On estimating the levels of polyphenolic compounds in each fraction using standard quercetin the ethanol fraction was found to have the higher polyphenolic content. Therefore this extract was further used for all the study throughout. 4.3. Tumor cells Ehrlich’s ascites carcinoma cells were obtained from Amala Cancer Research Centre, Trissur, and Kerala, India. The EAC cells were maintained by intraperitoneal inoculation of 2 × 106 cells/mouse. EAC cells aspirated from the peritoneal activity of mice were washed with saline and were given intraperitoneally to develop ascitic tumor. 4.4. Toxicity study An acute toxicity study relating to the determination of LD50 was performed (Litchfield and Wilcoxon, 1949). Different groups of mice were treated with graded doses of the EESG of leaves and flowers (100 mg, 200 mg, 500 mg and 750 mg/kg) orally. One group was maintained as control and was given distilled water. The animals were observed continuously for 2 h, and then intermittently and after 24 h for 14 days. The animals were observed for behavioral, neurological and autonomic profiles.
MST of treated group × 100 − 100. MST of control group
The viability and nonviability of the cells were checked by trypan blue assay. The cells were stained with trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were viable and those that took the dye were nonviable. These viable and nonviable cells were counted. 4.7. Hematological parameters At the end of the experimental period, the next day after an over night fasting blood was collected from freely flowing tail vein and used for the estimation of hemoglobin (Hb) content, red blood cell (RBC) count, white blood cell (WBC) count and differential count of WBC by standard procedures. 4.8. Biochemical parameters After the collection of blood samples, the mice were sacrificed. Then the liver was excised, rinsed in ice cold normal saline followed by ice-cold 10% KCl solution, blotted, dried and weighed. A 10% w/v homogenate was prepared in ice-cold KCl solution and centrifuged at 1500 rpm for 15 min at 4 ◦ C. The supernatant thus obtained were used for the estimation of thio-barbituric acid substances (TBARS) (Okhawa et al., 1979) glutathione (GSH) (Ellman, 1979) superoxide dismutase (SOD) (Kakkar et al., 1984) catalase (CAT) (Luck, 1974) and total protein (TP) (Lowry et al., 1951). 5. Statistical analysis All the parameters studied were subjected to statistical treatment using Sigma Stat statistical package (Version 3.1). The data were expressed as mean ± S.D. (n = 6) where ‘n’ represents the no. of samples. One-way ANOVA, followed by post hoc analysis using
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Table 1 Effect of EESG on tumor growth response. Parameters
Tumor volume (ml) Tumor weight (g) MST (days) %ILS Viable tumor cell count (×107 cells/ml) Non viable cell count (×107 cells/ml)
EAC control
Treated
II
III
IV
V
VI
VII
4.8 ± 0.86 5.5 ± 0.42 23.5 ± 1.90 00.00 10.40 ± 0.56 0.50 ± 0.03
3.4* ± 0.76 3.5* ± 0.67 38.2 ± 2.90 68.51 3.70* ± 0.15 0.90 ± 0.03
2.1** ± 0.12 2.6** ± 0.21 42.5 ± 3.90 97.42 3.60** ± 0.18 1.91 ± 0.04
3.7* ± 0.52 3.6* ± 0.25 31.7 ± 2.90 32.52 6.10 ± 0.23 0.80 ± 0.21
2.5** ± 0.33 2.3** ± 0.23 39.6 ± 3.40 64.61 4.30* ± 0.09 1.71 ± 0.02
1.8 ± 0.63 1.7 ± 0.36 48.1 ± 3.40 104.61 2.10 ± 0.17 2.60 ± 0.04
Values are expressed as mean ± S.D., n = 6. III – leaf extract (100 mg/kg) + EAC; IV – leaf extract (200 mg/kg) + EAC; V – flower extract (100 mg/kg) + EAC; VI – flower extract (200 mg/kg) + EAC; VII – Standard 5-FU (20 mg/kg) + EAC. * P < 0.001: between extract treated groups and EAC. ** P < 0.01: between extract treated groups and EAC.
Fischer’s LSD was adopted to all the parameters under study to test the level of statistical significance. The difference was considered significant if p < 0.05. 6. Results and discussion Recently, the use of some herbs has attracted a great deal of attention as one of the alternative cancer therapies from the point of less toxicity and cost benefits. Therefore, an attempt has been made to evaluate the anticancer activity of the leaf and flower extract of Sesbania grandiflora, which is commonly used in ayurvedic system of medicine for various purposes. The extracts revealed the presence of various phytoconstituents like reducing and non-reducing sugars, polyphenolics, alkaloids, saponins, tannins and terpenoids. In acute toxicity, no gross behavioral changes and mortality was observed up to a dose level of 400 mg/kg body weight. The LD50 value of EESG leaves and flowers were found to be >2 g/kg body weight of mice indicating that it is very less toxic to the animal. Treatment with EESG both leaves and flowers at the dose of 100 and 200 mg/kg body weight increased the life span (ILS) and nonviable cell count and reduced the tumor volume, tumor weight and viable tumor cell count significantly when compared to that of EAC control group (Table 1). As shown in Table 2, hemoglobin content and RBC count decreased significantly and the total WBC count increased significantly in EAC group as compared to normal group. Treatment with EESG restored the hematological parameters to more or less normal values. The number of RBC count and hemoglobin content also increased, while the WBC and the differential count decreased as compared to that of EAC control. The reliable criteria for judging the value of any anticancer drug is the prolongation of life span of the animals and the decrease of leukemic cells from blood (Gupta et al., 2004). However with the
treatment of EESG the percent increase in tumor cell volume, and number of viable tumor cells were found to be significantly less when compared to the EAC control. Hence, it may be concluded that the extracts by a direct cytotoxic effect and by arresting the tumor growth, increased the life span of EAC-bearing mice. The percentage increase in life span at the 200 mg/kg body weight dose of the leaf extract was found to be the highest than the flower extracts indicating its potent anticancer nature. In acute toxicity studies, the administration of EESG at the dose of 100 mg and 200 mg/kg for 14 days did not exhibit any adverse effect which may be due its composite nature where the presence of phytoconstituents could counteract its toxicity. In cancer chemotherapy the major problems are myelosuppression and anemia (Maseki et al., 1981). The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or hemoglobin percentage and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions (Fenninger and Mider, 1954). Similar results were observed in the present study in animals of the EAC tumor control group. The reversal of hematological parameters indicates that the extracts may possess protective action on the hematopoietic system. This reinstates that use of herbs might be a more effective strategy in the treatment of cancer. As shown in Table 2, the level of TBARS was significantly increased in the EAC treated animals when compared to the normal group. Treatment with EESG of both leaves and flowers at 200 mg/kg body weight reversed these changes towards normal levels. Significant decrease in the levels of GSH, SOD and CAT was observed in EAC control group which was reversed significantly towards normal in the EESG treated group. Almost similar results were observed with 5-FU treatment. It was observed that tumor cells produced more peroxides when they proliferate actively after inoculation of tumor and also known to affect many functions of the vital organs, which indicated the intensification of oxygen free rad-
Table 2 Effect of EESG on hematological and biochemical parameters. Parameters
Normal I
Total RBC (×106 cells/ml) Total WBC (×104 cells/ml) Hemoglobin (%) Lymphocyte (%) Neutrophil (%) Monocyte (%) LPO (nmol MDA/mg protein) GSH (mg/g) SOD (U/mg protein) CAT (U/mg protein)
5.34 6.40 16.10 66.10 31.10 3.30 0.96 2.31 4.34 27.4
± ± ± ± ± ± ± ± ± ±
0.02 0.10 0.70 5.40 2.70 0.10 0.70 0.21 0.05 0.21
EAC control
Treated
II
III
1.22 14.60 7.30 24.10 74.20 2.20 1.40 1.39 2.22 11.6
± ± ± ± ± ± ± ± ± ±
0.06* 0.90 0.50* 1.80* 6.50* 0.50* 0.50 0.32 0.07 0.92
4.29 11.50 14.80 9.20 38.30 3.20 1.02 2.72 3.29 25.5
IV ± ± ± ± ± ± ± ± ± ±
0.03 0.30 0.40 3.60 1.90 0.70** 0.20 0.15 0.06 0.32
4.6 9 6.20 15.90 61.10 37.10 2.40 0.97 2.49 4.62 26.2
V ± ± ± ± ± ± ± ± ± ±
0.06** 0.40** 0.80** 4.70** 2.60** 0.30 0.80** 0.32** 0.06** 0.42**
4.27 11.90 13.90 57.20 41.20 2.60 1.03 2.61 3.27 24.6
VI ± ± ± ± ± ± ± ± ± ±
0.05 0.30 0.40 5.3** 3.20** 0.40 0.40 0.12 0.06 0.32
4.51 5.80 15.0 60.30 8.10 2.30 0.98 2.51 4.31 25.9
VII ± ± ± ± ± ± ± ± ± ±
0.07** 0.20** 0.80** 5.80** 1.20** 0.10 0.70** 0.23** 0.09** 0.25**
5.36 6.70 15.80 65.10 33.10 4.20 0.97 2.23 4.34 26.7
± ± ± ± ± ± ± ± ± ±
0.40** 0.50** 1.60** 4.40** 1.90** 0.090** 1.60** 0.51** 0.09** 0.52**
Values are expressed as mean ± S.D, n = 6. III – leaf extract (100 mg/kg) + EAC; IV – leaf extract (200 mg/kg) + EAC; V – flower extract (100 mg/kg) + EAC; VI – flower extract (200 mg/kg) + EAC; VII – Standard 5-FU (20 mg/kg) + EAC. * P < 0.001: between normal and EAC. ** P < 0.01: between extract treated groups and EAC.
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ical production. The elevation of lipid per oxidation is also known to be associated with cancer (Navarro et al., 1997). Decrease in SOD, GSH and CAT activities described in tumors is regarded as markers of malignant transformation (Kavitha and Manoharan, 2006). Therefore the significant elevation of GSH, SOD and CAT, and significant reduction in LPO by the extract treatment confirms the potent antioxidant activity and free radical quenching property of EESG. The preliminary phytochemical studies indicated the presence of flavonoids, saponins, triterpenoids, glycosides, and tannins in the extracts of leaf and flower. A number of scientific reports indicate certain terpenoids; steroids and phenolic compounds such as tannins, coumarins and flavonoids have a chemo preventive role in cancer through their effects on signal transduction in cell proliferation and angiogenesis (Blois, 2002). Many such compounds are known to possess potent anticancer properties (Weber et al., 1996). The potent anticancer properties of the Sesbania grandiflora extracts may be attributed to the antioxidant and anticancer principles present in the extract. The results from the present study are consistent with the earlier reports, which support the antioxidant potential of Sesbania grandiflora under various oxidative stress conditions (Pari and Uma, 2003; Ramesh et al., 2008). In conclusion, the present study demonstrates the potent anticancer properties of Sesbania grandiflora extracts. The Sesbania grandiflora leaf extracts were more potent than the flower extracts where the maximum benefit of anticancer effect can be derived. Further studies on the elucidation of its mechanism of action and on the isolation of its active constituents may prove rewarding in cancer treatment. Acknowledgement Authors express sincere gratitude to Dr. K. Jaswanth, Department of Pharmacology, Periyar University for his keen interest and expert guidance. References Anon., 1986. The Useful Plants of India. Publications & Information Directorate, CSIR, New Delhi, India, pp. 465–466. Blois, M.S., 2002. Antioxidant determination by the use of a stable free radical. Nature 26, 1199–1200. Burbidage, N.T., 1965. Australian species of sesbania scop (leguminosae). Australian Journal of Botany 13, 103–141. Doddola, S., Pasupulati, H., Koganti, B., Prasad, K.V., 2008. Evaluation of Sesbania grandiflora for antiurolithiatic and antioxidant properties. Journal of Natural Medicines 62, 300–307. Ellman, G.L., 1979. Tissue sulphydryl groups. Archives in Biochemistry & Biophysics 82, 70–77. Fenninger, L.D., Mider, G.B., 1954. Energy and nitrogen metabolism in cancer. Advances in Cancer Research 9, 95–102.
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Govindan, S., Shanmugasundaram, E.R.B., 1987. Evaluation of the nutritive value of agathi (Sesbania grandiflora) leaf protein concentrates. The Indian Journal of Nutrition & Dietetics 24, 370–375. Gupta, M., Mazumder, U.K., Rath, N., Mukhopadhyay, D.K., 2000. Antitumor activity of methanol extract of Cassia fistula L. seed against Ehrlich ascites carcinoma. Journal of Ethnopharmacology 72, 151–156. Gupta, M., Mazumder, U.K., Sambath Kumar, R., Siva Kumar, T., Vamsi, M.L.M., 2004. Antitumor activity and antioxidant status of Caesalpinia bonducella against Ehrlich ascites carcinoma in Swiss albino mice. Journal of Pharmacological Sciences 94, 77–184. Joshi, S.G., 2000. Leguminoceae: Text book of medicinal plants. Oxford Publishing, New Delhi, pp. 130-132. Kasture, V.S., Deshmukh, V.K., Chopde, C.T., 2002. Anxiolytic and anticonvulsive activity of Sesbania grandiflora leaves in experimental animals. Phytotherapy Research 16, 455–460. Kakkar, P., Das, B., Vishwanathan, P.N., 1984. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemistry & Biophysics 21, 130–132. Kavitha, K., Manoharan, S., 2006. Anticarcinogenic and antilipidperoxidative effects of Tephrosia purpurea (Linn) pers. In 7, 12-dimethyl benz (a) anthracene (DMBA) induced hamster buccal pouch carcinoma. Indian Journal of Pharmacology 38, 185–189. Kokate, C.K., Purohit, A.P., Gokhale, S.B., 1997. Analytical pharmacognosy: phytochemical investigations. In: Prakashan, N. (Ed.), Pharmacognosy. , fifth ed. Nirali Prakashan, India, pp. 119–137. Kuttan, G., Vasudevan, D.M., Kuttan, R., 1988. Isolation and identification of a tumor reducing component from mistletoe extract (Iscador). Cancer Letters 41, 307–315. Laladhas, K.P., Cheriyan, V.T., Puliappadamba, V.T., Bava, S.V., Unnithan, R.G., Vijayammal, P.L., Anto, R.J., 2010. A Novel protein fraction from Sesbania grandiflora shows potential anticancer and chemo preventive efficacy, in vitro and in vivo. Journal of Cellular & Molecular Medicine 14, 636–646. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose effect experiments. Journal of Pharmacology & Experimental Therapeutics 96, 99–133. Lowry, O.H., Rosebrough, N., Farr, L.A., Randal, R.J., 1951. Protein measurement with Folin’s phenol reagent. Journal of Biological Chemistry 193, 265–275. Luck, H., 1974. Methods in Enzymatic Analysis, II. Ed., Berg Mayer, Academic Press, New York, pp. 885–886. Mannetje, L., Jones, R.M., 1992. Plant Resources of South-East Asia. No. 4: Forages. Pudoc Scientific Publishers, Wageningen. Maseki, M., Nishiagaki, I., Hagishara, M., Tomoda, Y., Yagi, K., 1981. Lipid per oxidation levels and lipid content of serum lipoprotein fractions of pregnant subjects with or without preeclampsia. Journal of Clinica Chemica Acta 41, 424–426. Mustafa, R.A., Hamid, A.A., Mohammed, S., Bakar, F.A., 2010. Total phenolic compounds flavonoids and radical scavenging activity of 21 selected Tropical plants. Journal of Food Science 75, C28–C35. Navarro, J., Obrador, E., Pellicer, J.A., Asensi, M., Vina, J., Esrela, J.M., 1997. Blood glutathione as an index of radiation-induced oxidative stress in mice and humans. Free Radical Biology & Medicine 22, 1203–1209. Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pari, L., Uma, A., 2003. Protective effect of Sesbania grandiflora against erythromycin estolate-induced hepatotoxicity. Journal of Medicinal Food 58, 439–443. Ramesh, T., Mahesh, R., Sureka, C., Begum, V.H., 2008. Cardio protective effects of Sesbania grandiflora in cigarette smoke-exposed rats. Journal of Cardiovascular Pharmacology 52, 338–343. Timyan, J., 1996. Bwa Yo: Important Trees of Haiti. South-East Consortium for International Development, Washington, DC. Weber, G., Shen, F., Prajda, N., Yeh, Y.A., Yang, H., Herenyiova, et al., 1996. Increased signal transduction activity and down regulation in human cancer cells. Anticancer Research 16, 3271–3282.