ANTIOXIDANT STUDIES, ERYTHROCYTE MEMBRANE STABILIZATION AND THROMBOLYTIC POTENTIAL OF DIALLYL DISULPHIDE

ANTIOXIDANT STUDIES, ERYTHROCYTE MEMBRANE STABILIZATION AND THROMBOLYTIC POTENTIAL OF DIALLYL DISULPHIDE

N. Suchetha Kumari ^1*, T. Yogish Somayaji ², R. Tejashvi ², K. M. Damodara Gowda ³ and C. Rao ⁴

Professor, Dept. of Biochemistry ¹, Nitte University Center for Science Education and Research ^2, Department of Physiology ³, Department of Pathology ⁴ KS Hegde Medical Academy. Deralakatte, Mangalore, Karnataka, India.

ABSTRACT: Diallyl Disulphide is an essential component of garlic which has numerous medicinal applications. Thus it was evaluated for its free radical scavenging potential. The compound was assayed for its ability to stabilize the erythrocyte membrane by osmotic fragility test and heat induced hemolysis assay. The thrombolytic potential was evaluated by the ability of the compound to dissolve the clot formed. The antioxidant studies were assessed by DPPH radical scavenging assay, ferric reducing antioxidant potential, Superoxide radical scavenging potential and Nitric oxide radical scavenging potential. The total antioxidant capacity was also estimated and the values were expressed in terms of Ascorbic acid equivalents. The results show a poor free radical scavenging potential and antioxidant capacity.  However it has shown a moderate percentage in dissolution of clot implicating in thrombolytic potential. The compound has also shown a higher erythrocyte membrane stabilization by reducing the percentage of hemolysis in heat induced hemolytic assay than Ascorbic Acid. The results suggest that Diallyl Disulphide is a poor antioxidant but a potential anti-hemolytic in In-vitro studies

DADS- Diallyl Disulphide, DPPH- 1, 1-diphenyl-2-picryl hydrazyl, TPTZ- 2, 4, 6-tripyridyl–S-triazine, FRAP- Ferric Reducing Antioxidant Potential, Erythrocyte Membrane Stabilization, Thrombolytic Potential, Reactive Oxygen Species

INTRODUCTION: Diallyl Disulphide is an organosulphur compound derived form allicin, natural compound found in garlic and related plants ^{1, 2}. Diallyl Disulphide has many physiological effects, many that are cardio- and neuroprotective. Diallyl Disulphide is also a known antiatherosclerotic, antibacterial and antimutagenic ^{3, 4}. These effects are due to, atleast in part because Diallyl Disuphide is converted, in the presence of thiols, to the gaseous mediator Hydrogen Sulphide⁵.

Thus, Diallyl Disulphide serves as a thiol- dependent Hydrogen Sulphide donor in biological systems. There is limited literature available about the ability of Diallyl Disulphide in stabilizing the erythrocyte membrane at elevated temperatures. Previously aged garlic extracts have been shown to be a potential therapeutic source for sickle cell anemia ⁶. The Anticancer property of garlic has been attributed to the presence of Diallyl Disulphide and other organosulfur compounds ⁷. Thus, Diallyl disulphide could be a good source of therapeutic and other biomedical applications. The present study attempts to evaluate the antioxidant, erythrocyte membrane stabilization and thrombolytic potential of Diallyl Disulphide.

 MATERIALS AND METHODS:

The entire experiment was carried out at the Nitte University center for Science Education and Research (NUCSER), Nitte University, Mangalore, India.

The Diallyl Disulphide (>75% pure; GC graded) was obtained from TCI chemicals, Japan. The DPPH, TPTZ were purchased from Himedia pvt. Ltd., Mumbai. The Antioxidant studies were carried out by DPPH radical scavenging assay, Nitric oxide radical scavenging assay and Superoxide radical scavenging assay. The ferric reducing antioxidant power assay was done by TPTZ.

DPPH Assay:

The method of ⁸ was followed. The free radical scavenging activity of the various extracts of Carica papaya (Linn.) were measured with formation of stable 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical using a spectrophotometer at 517nm. The percentage inhibition value was calculated using the formula,

Absorbance of Control- Absorbance of Test   _×100

Absorbance of Control

FRAP-TPTZ Assay:

The method of ⁹ was used. The Ferric Reducing Antioxidant Power was determined using 2, 4, 6-tripyridyl–S-triazine (TPTZ) in a spectrophotometer at 595nm. The results were expressed in terms of EC₅₀,the concentration at a particular absorbance at which it is exactly half of the absorbance of the control.

 Nitric Oxide Radical Scavenging Assay:

The method of ¹⁰ was followed to evaluate the Nitric Oxide radical scavenging property. The Nitric Oxide Radical Scavenging Activity was measured by the formation of nitrite from sodium nitroprusside using Griess reagent (1% Sulphanilamide in 2.5% ortho-phosphoric acid, 0.1% Naphthyl ethylenediamine dihydrochloride) spectrophotometrically at 540nm.

 Superoxide Radical Scavenging Assay:

The method of ¹¹ was followed with slight modification. The superoxide Radical generated by the photo-oxidation of methionine-riboflavin and their scavenging property was determined spectrophotometrically at 560nm using Nitroblue tetrazolium (NBT) as the chromogenic substrate.

Total Antioxidant Assay:

The estimation of total antioxidants was done by the method of ¹². The formation of blue- green phosphomolybdenum complex was measured at 695nm.

1.  
2. Estimation of Total Phenolic Content:

The method of ¹³ was followed to estimate the total phenolic content using Folin- Ciocalteau reagent. The absorbance was measured at 760nm.

 In-vitro Erythrocyte Membrane Stabilization and Thrombolytic Potential:

Heat Induced Hemolysis assay:

The method of ¹⁴ was followed. The red blood cell suspension was pre-incubated with different extracts of Carica papaya (Linn.) for 20minutes at 55⁰C in a water bath. Then the absorbance of the suspension was measured at 540nm. Aspirin was used as positive control.

 Osmotic Fragility of Erythrocytes:

The method of ¹⁵ was followed with slight modifications. The erythrocyte suspension with the extracts was added to serially diluted saline from 0.9% to 0.48% and distilled water. The absorbance of the suspension was measured at 540nm. The absorbance at 0.44% was taken against the absorbance with erythrocyte suspension in distilled water as control.

 Thrombolytic Potential:

The thrombolytic potential was evaluated by the method of ¹⁶. The extracts were incubated with pre-weighed clot for 90 minutes at 37⁰C.  The clots were centrifuged, the supernatant discarded and clot weighed again. The percentage of clot dissolved was calculated.

RESULTS:

In vitro Antioxidant study on Diallyl Disulphide:

• DPPH assay:

TABLE 1 SHOWING THE DPPH RADICAL SCAVENGING POTENTIAL OF DIALLYL DISULPHIDE COMPARED WITH STANDARD ASCORBIC ACID

FIG.1 SHOWING THE COMPARISON BETWEEN DPPH RADICAL SCAVENGING POTENTIAL OF DIALLYL DISULPHIDE AND ASCORBIC ACID

 FRAP-TPTZ assay:

The Ferric Reducing Power of Diallyl Disulphide was found to be 566.83±231.5mg compared to that of Ascorbic Acid standard with a FRAP value of 25.34±1.55 mg.

Superoxide radical scavenging assay:

TABLE 2 SHOWING THE SUPEROXIDE RADICAL SCAVENGING ACTIVITY OF DIALLYL DISULPHIDE COMPARED WITH STANDARD ASCORBIC ACID

FIG. 2: SUPEROXIDE RADICAL SCAVENGING ASSAY OF DIALLYL DISULPHIDE AND ASCORBIC ACID

Nitric Oxide radical scavenging assay:

The Nitric oxide scavenging potential of Diallyl Disulphide was found to be 5.30% compared to that of Ascorbic Acid with 27% of Scavenging potential.

 Estimation of Total Antioxidants:

The total antioxidants present in Diallyl Disulphide were about 41.33±13.32 µM Ascorbic Acid Equivalents.

In-vitro Erythrocyte Membrane Stabilization and Thrombolytic Potential:

Heat Induced Hemolysis assay: The results of heat induced hemolytic assay are shown in Figure 3. A concentration of 100mg of the compounds was selected for the experiment.

FIG. 3: HEAT INDUCED HEMOLYSIS ASSAY OF DIALLYL DISULPHIDE AND ASCORBIC ACID

Osmotic Fragility of Erythrocytes: Diallyl Disulphide showed 0% inhibition in hypotonicity induced hemolysis as compared to that of Ascorbic Acid with 29.6% inhibition of hemolysis.

 Thrombolytic Potential: The thrombolytic potential of Diallyl Disulphide is displayed in Figure 4.

FIG. 4: PERCENTAGE OF CLOT DISSOLVED BY DIALLYL DISULPHIDE AND ASCORBIC ACID

DISCUSSION: The antioxidant results from DPPH, TPTZ indicate a poor hydrogen donating ability and thus diminished ability to reduce the free radicals. The ability to donate electrons and scavenge the free radicals by Diallyl Disulphide is not significant compared to that of Ascorbic Acid which shows a five time higher capacity. Its thrombolytic potential when compared to streptokinase ¹⁶ was not significant (p value >0.05). But Diallyl Disulphide has shown a higher erythrocyte membrane stabilization by reducing the percentage of hemolysis in heat induced hemolytic assay than Ascorbic Acid. Thus Diallyl disulphide, a component of garlic oil might also have an important role in inhibiting the hemolysis and enhancing the membrane integrity at elevated temperatures.

CONCLUSIONS: The results suggest that Diallyl Disulphide is a poor antioxidant but a potential anti-hemolytic in In-vitro studies. Hence further In-vivo studies can be undertaken to evaluate the mechanism of the various physiological changes caused by Diallyl Disulphide.

ACKNOWLEDGEMENTS: The authors would like to thank the Board of Research in Nuclear Science (BRNS) for providing the funding for the project and Nitte University for providing the laboratory facilities to carry out the work. The authors also thank all the colleagues of Nitte University Center for Science Education and Research (NUCSER) for their support.

REFERENCES:

1. Lawson, L.D., Wang, Z.J. and Hughes, B.G. Identification and HPLC quantitation of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products. Planta Med., 1991 57, 363-370.
2. Yan,X., Wang,Z. and Barlow,P. Quantitative estimation of garlic content in garlic oil based health products. Food Chem., 1992 45, 135-139.
3. Dong Yeok Shin, Gi-Young Kim, Jun Hyuk Lee, Byung Tae Choi, Young Hyun Yoo and Yung Hyun Choi, Apoptosis Induction of Human Prostate Carcinoma DU145 Cells by Diallyl Disulfide via Modulation of JNK and PI3K/AKT Signaling Pathways, Int. J. Mol. Sci. 2012, (13), pp.14158-14171.
4. Gupta N, Porter TD, Garlic and garlic-derived compounds inhibit human squalene monooxygenase, J. Nutr. 2001, 131, 1662-1667.
5. Hye-Sook Chang, Daiji Endoh, Yushi Ishida, Hiroyuki Takahashi, Shuji Ozawa, Masanobu Hayashi, Akira Yabuki, and Osamu Yamato, Radioprotective Effect of Alk(en)yl Thiosulfates Derived from Allium Vegetables against DNA Damage Caused by X-Ray Irradiation in Cultured Cells: Antiradiation Potential of Onions and Garlic, Sci. Wld. J. 2012,  846750, 5pg.
6. Junichiro Takasu, Rolando Uykimpang, Maria Alenor Sunga, Harunobu Amagase, and Yutaka Niihara, Aged Garlic Extract Is a Potential Therapy for Sickle-Cell Anemia,  J. Nutr. (2006) 136: 803S–805S.
7. Reddy, B.S., Rao, C.V., Rivenson,A. and Kelloff, G. (1993) Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res., 53, 3493-3498.
8. R. Senthil Kumar, B. Rajkapoor, P. Perumal, Antioxidant Potential of Indigofera linnaei Al I. An In-vitro Study, Pharmacologyonline, 2011, (1), pp. 710-720.
9. Atul Kaushik, Chanderesh Jijta, Jeevan J Kaushik, Robel Zeray, Anghesom Ambesajir, Lwam Beyene, FRAP (Ferric reducing ability of Plasma) Assay and effect of Diplazium esculentum (Retz) Sw. (a green vegetable of North India) on Central Nervous System, Ind. J. Nat. Pr. Res. 2012, (3) 2, 228-231.
10. Suchetha Kumari, N., Yogish Somayaji, T., Tejashvi, R., Damodara Gowda, K. M. and Chandrika Rao, Antioxidant, Erythrocyte Membrane Stabilization and Thrombolytic potential of Carica papaya (Linn.), Int. J. Curr. Res. 2014, (6) 5, pp.6474-6480.
11. Ganga Rao B, P. Rajeswararao, P. Prayaga Murty, E. Sambasiva Rao, P. Madhukiran, T. Mallikarjuna Rao and V. S. Praneeth D, Investigation on Regional Variation in Total Phenolic, Alkaloid Content and In-vitro Antioxidant Activity of Leucas aspera , IJPSR, 2011; 2(10): 2699-2703.
12. Reşat Apak, Shela Gorinstein, Volker Böhm, Karen M. Schaich, Mustafa Özyürek, and Kubilay Güçlü, Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report), Pure Appl. Chem., 2013, Vol.85, No.5, pp. 957–998.
13. Harsha SN, Latha BV, In vitro Antioxidant and In vitro Anti inflammatory Activity of Ruta graveolens Methanol Extract, asian J Pharm Clin Res, 2011 (5), 1, 32-35
14. Priyanga Ranasinghe, Pathmasiri Ranasinghe, WP Kaushalya M. Abeysekera, GA Sirimal Premakumara, Yashasvi S Perera, Padmalal Gurugama, Saman B Gunatilake. In vitro erythrocyte membrane stabilization properties of Carica papaya L. leaf extracts Phcog Res 2012, Vol. 4 (4), pp. 196-202.
15. Umapathy E, Ndebia EJ, Meeme A, Adam B, Menziwa P, Nkeh- Chungag BN, et al. An experimental evaluation of Albuca setosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation. J Med Plants Res 2010; Vol. 4, pp. 789-95.
16. Prasad,S., Kashyap, S.R., Deopujari, J. Y., Purohit, H.J., Taori,G.M. and Daginawala, H.F. Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis, BMC. Complement. Altern Med. (2007), vol. 7(36), pp. 1-6.
    

    ---

    How to cite this article:

    SuchethaKumari N, Somayaji TY, Tejashvi R, Damodara Gowda KMand Rao C:Antioxidant Studies, Erythrocyte Membrane Stabilization and Thrombolytic Potential of Diallyl Disulphide^. Int J Pharm Sci Res2014; 5(11): 4929-32.doi: 10.13040/IJPSR.0975-8232.5 (11).4929-32

    ---

    All © 2014 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---