Malaysian Journal of Analytical Sciences Vol 20 No 5 (2016): 1191 - 1202

DOI: http://dx.doi.org/10.17576/mjas-2016-2005-26

 

 

 

IN VITRO NITRIC OXIDE SCAVENGING AND ANTI INFLAMMATORY ACTIVITIES OF DIFFERENT SOLVENT EXTRACTS OF VARIOUS PARTS OF Musa paradisiaca

 

(Aktiviti Pemerangkapan Nitrik Oksida dan Anti-Radang Secara In Vitro oleh Ekstrak Pelarut Berbeza dari Pelbagai Bahagian Musa paradisiaca)

 

U.S Mahadeva Rao¹*, Bashir Ado Ahmad¹, Khamsah Suryati Mohd^2,3

 

¹Faculty of Medicine,,

Universiti Sultan Zainal Abidin, Medical Campus, 21400 Kuala Terengganu, Terengganu, Malaysia

²Faculty of Bioresources and Food Industry

³Agriculture Production and Food Innovation Research Institute

 Universiti Sultan Zainal Abidin, Tembila Campus, 22200 Besut, Terengganu, Malaysia

 

*Corresponding author: raousm@gmail.com

 

 

Received: 14 April 2015; Accepted: 3 August 2016

 

 

Abstract

Inflammatory diseases are an important health concern, and the growing rate is on the rise. Finding a safe drug for these diseases remains an important issue. The study aimed to determine the nitric oxide (NO) scavenging as well as anti-inflammatory activities of Musa paradisiaca (banana). Musa paradisiaca plant parts used in this study namely tepal, skin (peel) and flesh (pulp) were extracted with methanol (tepal, flesh and skin), ethanol (tepal) and water (tepal) using cold maceration technique. Phytochemicals screening of the extracts was carried out. The ability of the extracts to scavenge NO radical was evaluated using Griess reagent. The anti-inflammatory activity of the extracts was assessed by evaluating the ability of the extract to inhibit RAW 264.7 macrophage cell line from generating harmful NO induced by bacterial lipopolysaccharide (LPS). In order to determine the toxicity of the extracts to the cells while exerting its anti-inflammatory activity,cytotoxicity was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.The extract was found to contain bioactive chemicals like flavonoids, saponins, phenols, etc. Maximum NO radical inhibition of 41.05% was recorded in the tepal aqueous extract, and minimum inhibition of 22.34% was recorded in the flesh extract. All others showed mild inhibition as well. Flesh extract has the highest NO (generated by LP) inhibitory activity, with the maximum inhibition of 52.21% at 250 µg/mL, followed by tepal aqueous extract with maximum inhibition of 48.16% at 62.5 μg/mL. The least inhibition was noted in the tepal methanol extract that has the maximum inhibition of 19.63% at     62.5 μg/mL. The viability of the activated macrophages was not affected by the extracts as confirmed by the MTT assay, thereby indicating that the inhibition of NO synthesis by the extracts was not due to cytotoxic effects. Overall, Musa paradisiaca plant parts have the potential as anti-inflammatory and studies in this aspect should be intensified.

 

Keywords:  anti-Inflammation, MTT, Musa paradisiaca, RAW 264.7

 

Abstrak

Penyakit radang merupakan gejala penyakit yang semakin mendapat perhatian dan kadar kedapatan (penyakit) semakin meningkat. Pencarian ubat yang selamat untuk penyakit ini masih menjadi isu penting. Kajian ini bertujuan menentukan aktiviti pemerangkapan nitrik oksida (NO) serta anti radang oleh Musa paradisiaca (pisang). Bahagian – bahagian Musa paradisiaca yang diguna dalam kajian ini adalah jantung, kulit (kupasan) dan isi (pulpa). Sampel – sampel telah diekstrak dengan metanol (jantung, kulit dan isi), etanol (jantung) dan air (jantung) menggunakan kaedah rendaman sejuk. Penyaringan fitokimia keatas ekstrak telah dijalankan. Keupayaan ekstrak memerangkap radikal NO dinilai menggunakan reagent Griess. Aktiviti anti radang oleh bahan terekstrak telah ditentukan melalui penilaian terhadap keupayaan menghalang sel makrofaj RAW 264.7 daripada menghasilkan NO yang diaruh oleh lipopolisakarida (LPS) bakteria. Bagi menentukan ketoksikkan ekstrak terhadap sel semasa ianya bertindak sebagai anti radang, tahap sitotiksik telah diukur menggunakan ujian MTT [3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromida]. Kajian mendapati bahawa ekstrak pisang mengandungi bahan kimia bioaktif seperti flavonoids, saponins, fenols, dan lain - lain. Perencatan maksima radikal NO sebanyak 22.34% telah direkodkan bagi ekstrak akueus jantung pisang, dan kadar perencatan minima telah direkodkan pada ekstrak isi pisang. Kesemua ekstrak yang lain menunjukkan perencatan yang rendah. Ekstrak isi pisang mempunyai aktiviti perencatan NO (yang dihasilkan oleh LP) tertinggi dengan perencatan maksima  52.21% pada kepekatan 250 µg/mL, diikuti oleh ekstrak akueus jantung pisang dengan perencatan maksima  48.16% pada kepekatan 62.5μg/mL. Perencatan terendah telah dicatatkan oleh ekstrak metanol jantung pisang, yang mempunyai perencatan maksima 19.63% pada kepekatan 62.5 μg/mL. Kebolehhidupan makrofaj yang diaktifkan tidak terjejas oleh ekstrak sepertimana yang dibuktikan oleh ujian MTT, ia menunjukkan bahawa sintesis NO oleh ektrak bukanlah dari kesan sitotoksik. Secara keseluruhan, bahagian – bahagian Musa paradisiacal mempunyai potensi sebagai anti radang dan kajian mengenainya perlu diperluaskan.

Kata kunci:  anti radang, MTT, Musa paradisiaca, RAW 264.7

 

References

1.        O’Byrne, K. J. and Dalgleish, A. G. (2001). Chronic immune activation and inflammation as the cause of malignancy. British Journal of Cancer, 85:473 – 483.

2.        O’Byrne, K. J., Dalgleish, A. G., Browning, M. J., Steward, W. P. and Harris, A. L. (2000), The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease. European Journal of Cancer, 36: 151 – 169.

3.        Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M. and Fiebich, B. L. (2000). Inflammation and Alzheimer’s disease. Neurobiology of Aging, 21: 383 – 421.

4.        Libby, P., Ridker, P. M. and Maseri, A. (2002). Inflammation and atherosclerosis. Circulation, 105: 1135 – 1143.

5.        Kulander, L., Pauksens, K. and Venge, P. (2001). Soluble adhesion molecules, cytokines and cellular markers in serum in patients with acute infections. Scandinavian Journal of Infectious Diseases, 33: 290 – 300.

6.        Bellanti, J. A. (1998). Cytokines and allergic diseases: clinical aspects. Allergy Asthma Proceeding, 19: 337 – 341.

7.        Romagnani, S. (2000). T-cell subsets (Th1 versus Th2). Ann. Allergy Asthma. Immunology, 85: 9 – 21.

8.        Silvestri, M., Sabatini, F., Defilippi, A. C., Ghiro, L., Baraldi, E. and Rossi, G. A. (2003). A marker of asthma inflammation: orally exhaled nitric oxide. Allergy and Clinical Immunology International, 15: 37 – 43.

9.        Alderton, W. K., Cooper, C. E. and Knowles, R. G. (2001). Nitric oxide synthases: structure, function and inhibition. Biochemical Journal, 357(P3): 593 – 615.

10.    Bogdan, C. (2001). Nitric oxide and the immune response. Nature Immunology, 2(10): 907 –916.

11.    Dawn, B. and Bolli, R. (2002). Role of nitric oxide in myocardial preconditioning. Annals of the New York Academy of Sciences, 962: 18 – 41.

12.    Moncada, S. and Higgs, E. A. (1991). Endogenous nitric oxide: physiology, pathology and clinical relevance. European Journal of Clinical Investigation, 21(4): 361 – 374.

13.    Aktan, F., Henness, S., Roufogalis, B. D. and Ammit, A. J. (2003). Gypenosides derived from Gynostemma pentaphyllum suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-B-mediated iNOS protein expression. Nitric Oxide: Biology and Chemistry, 8(4): 235 – 242.

14.    Kroncke, K. D., Fehsel, K. and Kolb-Bachofen, V. (1998). Inducible nitric oxide synthase in human diseases. Clinical Experimental Immunology, 113(2): 147 – 156.

15.    Kim, Y. S., Young, M. R., Bobe, G., Colburn, N. H. and Milner, J. A., (2009). Bioactive food components, inflammatory targets, and cancer prevention. Cancer Prevention Research, 2: 200 – 208.

16.    Fabricant, D. S. and Farnsworth, N. R. (2001). The value of plants used in traditional medicine for drug discovery. Environmental Health Perspective, 109: 69 – 75.

17.    Jachak, S. M. and Saklani, A., (2007). Challenges and opportunities in drug discovery from plants. Current Science, 92: 1251 – 1257.

18.    Sharma, P. C., Yelne, M. B., Dennis and Kadali J. J. (2002). In: Data base on medical plants used in Ayurveda and Siddha. New Delhi: Public Printing, 5: 78 – 93.

19.    Trease, G. E. and Evans, W. C. (1989). Trease and Evan’s Textbook of Pharmacognosy. 13^th Ed. London: Cambridge University Press: pp. 546

20.    Sreejayan, N. and Rao, M. N. A. (1997). Nitric oxide scavenging by curcuminoids. Journal of Pharmacy and Pharmacology, 49(1): 105 – 107.

21.    Yoon, W. J., Kim, S. S., Oh, T. H., Lee, N. H. and Hyun, C. G. (2009). Abies koreana essential oil inhibits drug-resistant skin pathogen growth and LPS-induced inflammatory effects of murine macrophage. Lipids, 44: 471 – 476.

22.    Sheeba, M. S. and Asha, V. V. (2006). Effect of Cardiospermum halicacabum on ethanol-induced gastric ulcers in rats. Journal of Ethnopharmacology, 106(1): 105 – 110.

23.    Bohlin, L., Goransson, U., Alsmark, C., Weden, C. and Backlund, A. (2010). Natural products in modern life science. Phytochemistry Reviews, 9(2): 279 – 301.

24.    Doss, A. and Anand, S. P. (2012). Preliminary phytochemical screening of Asteracanthalongifolia and Pergularia daemia. World Applied Science Journal, 18(2): 233 – 235.

25.    Kubmarawa, D., Khan, M. E., Punah, A. M. and Hassan, M. (2008). Phytochemical screening and antibacterial activity of extracts from Parkia clappertoniana keay against human pathogenic bacteria. Journal of Medical Plants Research; 2(12): 352 – 355.

26.    Savithramma, N., Linga Rao, M. and Suhrulatha, D. (2011). Screening of medicinal plants for secondary metabolites. Middle-East Journal of Science Research; 8: 579 – 584.

27.    Rupasinghe, H. P., Jackson, C. J., Poysa, V., Berado, C. D., Bewley, J. D. and Jenkinson, J. (2003).  Soyasapogenol A and B distribution in Soybean (Glycine max (L.) Merr.) in relation to seed physiology, genetic variability and growing location. Journal of Agricultural and Food Chemistry, 51: 5888 – 5894.

28.    Jini, J., David, P., Kavitha, M. P., Dineshkumar, B., Jalaja S. Menon, Bhat, A. R. and Krishnakumar, K. (2014). Preliminary phytochemical screening and in vitro antioxidant activity of Banana flower (Musa paradisiaca AAB Nendran variety). Journal of Pharmacy Research, 8(2): 144 – 147.

29.    Sunil Jawla, Kumar, Y. and Khan, M. S. Y. (2012). Antimicrobial and antihyperglycemic activities of Musa paradisiaca flowers. Asian Pacific Journal of Tropical Biomedicine, 2(2): 914 – 918.

30.    Anhwange, B. A. (2008). Chemical composition of Musa sapientum (Banana) peels. Journal of Food Technology, 6(6): 263 – 268.

31.    Archibald, J. G. (1949). Nutrient composition of banana skins. Journal of Dairy Science; 32: 969 –971.

32.    Alisi, C. S., Nwanyanwu, C. E., Akujobi, C. O. and Ibegbulem, C. O. (2008). Inhibition of dehydrogenase activity in pathogenic bacteria isolates by aqueous extracts of Musa paradisiaca (var Sapientum). African Journal of Biotechnology, 7(12): 1821 – 1825.

33.    Middleton, E. J. and Kandaswanmi, C. (1992). Effects of flavonoids on immune and inflammatory cell function. Biochemical Pharmacology, 43(6): 1167 –1179.

34.    Lata, H. and Ahuja, G. K. (2003). Role of free radicals in health and disease. Indian Journal of Physiology Allied Science, 57: 124 – 128.

35.    Lalenti, S., Moncada, M. and Di Rosa (1993). Modulation of adjuvant arthritis by endogenous nitric oxide. British Journal of Pharmacology, 110: 701 – 705.

36.    Ross, R. (1993). The pathogenesis of atherosclerosis: A perspective for the 1990’s. Nature, 362: 801- 809.

37.    Palash, M., Tarun, K. M. and Mitali, G. (2009). Free-radical scavenging activity and phytochemical analysis in the leaf and stem of Drymaria diandra Blume. International Journal Integrative Biology, 7(2): 81 – 83.

38.    Ames, B. N., Shigenaga, M. K. and Hagen, T. M. (1993). Oxidants, antioxidants and the degenerative diseases of aging. Proceeding National Academy Science, 90: 7915 – 7922.

39.    Chu, Y. F., Sun J., Wu X. and Liu, R. H. (2002). Antioxidant and antiproliferative activities of common vegetables. Journal Agricultural and Food Chemistry, 50: 6910 – 6916.

40.    Choi, C. W., Kim, S. C., Hwang, S. S., Choi, B. K., Ahn, H. J., Lee, M. Y., Park, S. H. and Kim, S. K. (2002). Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison. Plant Science, 163: 1161 – 1168.

41.    Mennen, L. I., Sapinho, D., de Bree, A., Arnault, N., Ber Trais, S., Galan, P. and Hercberg, S. (2004). Consumption of foods rich in flavonoids is related to a decreased cardiovascular risk in apparently healthy French women. Journal of Nutrition, 134(4): 923 – 926.

42.    Zedler, S. and Faist, E. (2006). The impact of endogenous triggers on trauma-associated inflammation. Current Opinion in Critical Care, 12: 595 – 601.

43.    Mariathasan, S. and Monack, D. M., (2007). Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nature Review of Immunology, 7: 31 – 40.

44.    Choy, C. S., Hu, C. M., Chiu, W. T., Lam, C. S., Ting, Y., Tsai, S. H. and Wang, T. C. (2008). Suppression of lipopolysaccharide-induced of inducible nitric oxide synthase and cyclooxygenase-2 by Sanguisdraconis, a dragon's blood resin, in RAW 264.7 cells. Journal of Ethnopharmacology, 115: 455 – 462.

45.    Vane, J. R., Mitchell, J. A., Appleton, I., Tomlinson, A., Bishop-Bailey, D., Croxtall, J. and Willoughby, D. A. (1994). Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proceedings of the National Academy of Sciences, 91: 2046 – 2050.

46.    Kasama, T., Miwa, Y., Isozaki, T., Odai, T., Adachi, M. and Kunkel, S. L. (2005). Neutrophil-derived cytokines: potential therapeutic targets in inflammation. Current Drug Targets - Inflammation and Allergy, 4: 273 – 279.

47.    Wolf, A. M., Wolf, D., Rumpold, H., Ludwiczek, S., Enrich, B., Gastl, G., Weiss, G. and Tilg, H., (2005). The kinase inhibitor imatinib mesylate inhibits TNF-{alpha} production in vitro and prevents TNF-dependent acute hepatic inflammation. Proceedings of the National Academy of Sciences, 102: 13622 – 13627.

48.    Cheon, H., Rho, Y. H., Choi, S. J., Lee, Y. H., Song, G. G., Sohn, J., Won, N. H. and Ji, J. D. (2006). Prostaglandin E2 augments IL-10 signaling and function. Journal of Immunology, 177: 1092 – 2100.

49.    Esposito, E. and Cuzzocrea, S., (2007). The role of nitric oxide synthases in lung inflammation. Current Opinion in Investigational Drugs, 8: 899 – 909.

50.    Murakami, A. and Ohigashi, H. (2007). Targeting NOX, INOS and COX-2 in inflammatory cells: Chemoprevention using food phytochemicals. International Journal of Cancer; 121: 2357 – 2363.

51.    Zeilhofer, H. U. and Brune, K., (2006). Analgesic strategies beyond the inhibition of cyclooxygenases. Trends Pharmacology Science, 27: 467 – 474.

52.    Jachak, S. M. (2007). PGE synthase inhibitors as an alternative to COX-2 inhibitors. Current Opinion in Investigational Drugs, 8: 411 – 415.

53.    Xie, Q. W., Kashiwabara, Y. and Nathan, C. (1994). Role of transcription factor NFkappa B/Rel in induction of nitric oxide synthase. Journal of Biology Chemistry, 269: 4705 – 4708.

54.    Henkel, T., Machleidt, T., Alkalay, I., Konke, M., Ben-Neriah, Y. and Baeuerle, P. A. (1993).  Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature, 365: 182 – 185.

55.    Arya, V. and Arya, M. L. (2011). A review on anti-inflammatory plant barks. International Journal of Pharmtech Research, 3: 899 – 908.

56.    Zheng, W. and Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. Journal of Agricultural and Food Chemistry, 49(11): 5165 – 5170.

57.    Cai, Y. Z., Sun, M. and Corke, H. (2003). Antioxidant activity of betalains from plants of the Amaranthaceae. Journal of Agricultural Food Chemistry, 51(8): 2288 – 2294.

58.    Sala, A., Recio, M. D., Giner, R. M., Manez, S., Tournier, H., Schinella, G. and Rios, J. L. (2002). Antiinflammatory and antioxidant properties of Helichrysum italicum. Journal Pharmacy and Pharmacology, 54(3): 365 – 371.

59.    Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M. and Pridham, J. B. (1995). The relative activities of Plant-derived polyphenolic flavonoid. Free Radical Research, 22: 375 – 383.

60.    Kang, J. H., Sung, M. K., Kawada, T., Yoo, H., Kim, Y. K., Kim, J. S. and Yu, R. (2005). Soybean saponins suppress the release of proinflammatory mediators by LPS-stimulated peritoneal macrophages. Cancer Letters, 230: 219 – 227.

61.    Yao, Y., Yang, X., Shi, Z. and Ren., G. (2014). Anti-inflammatory activity of saponins from quinoa (chenopodium quinoa willd.) seeds in lipopolysaccharide-stimulated RAW 264.7 macrophages cells. Journal of Food Science, 79(5): 1021 – 1022.

62.    Reddy, A. M., Lee, J. Y., Seo, J. H., Kim, B. H., Chung, E. Y., Ryu, S. Y., Kim, Y. S., Lee, C. K., Min, K. R. and Kim, Y. (2006). Artemisolide from Artemisia asiatica: nuclear factor-kappaB (NF-kappaB) inhibitor suppressing prostaglandin E2 and nitric oxide production in macrophages. Archives Pharmacal Research, 29: 591 – 597.

63.    Choi, Y., Lee, M. K., Lim, S. Y., Sung, S. H. and Kim, Y. C. (2009). Inhibition of inducible NO synthase, cyclooxygenase-2 and interleukin-1beta by torilin is mediated by mitogen-activated protein kinases in microglial BV2 cells. British Journal of Pharmacology, 156: 933 – 940.

64.    Kim, J. M., Lee, P., Son, D., Kim, H. and Kim, S. Y. (2003). Falcarindiol inhibits nitric oxidemediated neuronal death in lipopolysaccharide-treated organotypic hippocampal cultures. Neuroreport, 14: 1941 –1944.

65.    Kim, B. H., Hong, S. S., Kwon, S. W., Lee, H. Y., Sung, H., Lee, I. J., Hwang, B. Y., Song, S., Lee, C. K., Chung, D., Ahn, B., Nam, S. Y., Han, S. B. and Kim, Y. (2008). Diarctigenin, a lignan constituent from Arctium lappa, down-regulated zymosan-induced transcription of inflammatory genes through suppression of DNA binding ability of nuclear factor-kappaB in macrophages. Journal Pharmacology and Experimental Therapeutics, 327: 93 – 401.