Comparison of gamma ray and electron beam irradiation on antioxidant properties of glycowithanolide extracted from Withania somnifera, Indian ginseng

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1 Comparison of gamma ray and electron beam irradiation on antioxidant properties of glycowithanolide extracted from Withania somnifera, Indian ginseng Periasamy Srinivasan, Jae Kyung Kim, Jong-il Choi, Jae-Hun Kim, Beom Seok Song, Myung-Woo Byun and Ju-Woon Lee * a Team for Radiation Food Science & Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, , Korea Submitted 30 August, 2008 Abstract Glycowithanolide is an active component in Withania somnifera (WS), known as Indian ginseng, for the treatment of tumors, inflammation, arthritis, asthma, and hypertension. The current investigation was carried out to study the effect of electron beam (EB) and gamma ray (GR) irradiations on the antioxidant characteristics of extracted glycowithanolide (GW). In this study, the WS dry root was powdered and irradiated. Hydroxyl, superoxide and DPPH radical scavenging ability were all increased as the irradiation dose increased at both EB and GR irradiated WS, but the extent of increase by EB was larger than that by GR. The content of withanolide from HPLC analysis was found to be increased by EB and GR irradiations, this may be the possible reason for increased antioxidant activity of GW. Thus EB and GR irradiation could be used as an efficient tool for extraction of GW. Keywords: Withania somnifera; Glycowithanolide; Gamma ray; Electron beam irradiation; Antioxidant activities. * Corresponding author. Tel.: , Fax: address: sjwlee@kaeri.re.kr. 1

2 1 1. Introduction 2 Ashwagandha [Withania somnifera L. Dunal (Solanaceae)] is an important 3 medicinal plant, widely used as a home remedy for several diseases in India as well as 4 other parts of the world (Patwardhan et al., 1998; Sharma and Dandiya, 1991). It is 5 described as an herbal tonic and health food in Vedas and considered as Indian 6 Ginseng in traditional Indian system of medicine. In fact, it is mentioned as an official 7 drug in the Indian Pharmacopoeia as well (Indian Pharmacopoeia, 1985). Beside its use 8 as general tonic (Agarwal et al., 1999; Dhuley, 2000; Ziauddin et al., 1996), several 9 recent reports have demonstrated immunomodulator and antitumor effect of 10 ashwagandha as well (Agarwal et al., 1999; Devi, 1999; Sharad et al., 1996; Budhiraja 11 and Sudhir, 1987; Ziauddin et al., 1996). Moreover, various parts of the plant have been 12 reported to possess antiserotogenic, anticancer and anabolic properties and has 13 beneficial effects in the treatment of arthritis, stress and geriatric problems (Asthana and 14 Raina, 1989; Grandhi et al., 1994; Davis and Kuttan, 2000; Singh et al., 2001; Prakash 15 et al., 2001). 16 Therefore, the present study was conducted to evaluate the effect of gamma 17 irradiation and electron beam irradiation on the antioxidative properties of GW and the 18 feasibility of using irradiation as a tool for the extraction of GW without any adverse 2

3 19 effect Materials and Methods Sample irradiation 23 The solutions in tightly capped tubes were irradiated by a cobalt-60 irradiator 24 (point source, AECL, IR-79, Nordion, Canada) with 5 and 10 kgy. The source strength 25 was approximately 11.1 PBq with a dose rate at the location of the sample of 26 approximately 10 kgy/hr. Irradiation was carried out at 15 ± 0.5ºC. Dosimetry was 27 performed by using alanine dosimeters (Bruker Instruments, Rheinstetten, Germany) 28 measured with a Bruker EMS 104 EPR Analyzer. The alanine pellet was attached to the 29 tube. The actual doses were within ±2% of the target dose. The containers were 30 continuously rotated during an irradiation to obtain a uniform dose. Electron beam 31 irradiation was carried out using electron acceletor (UELV-10-10S accelerator, NII EFA, 32 Moscow, Russia) at 5 and 10 kgy. The samples were kept at 4 ºC in a refrigerator prior 33 to the following experiments Extraction of glycowithanolides 36 Glycowithanolides were extracted as follows, to 1 part of withania powder 37 added 4 part of (1:1) ethanol : water mixture and cold percolated for 48 h, evaporated 3

4 38 under reduced pressure at 55±5 ºC to 1/6th volume and exhaustively extracted with 39 chloroform. Chloroform insoluble fraction was spray dried and used for further studies Hydroxyl radical scavenging activity 42 To 0.2 ml of sample added 10 mm of 0.2 ml of FeSO 4 and EDTA, 1 ml of 0.1 M 43 of phosphate buffer ph 7.4 to that 0.2 ml of 10 mm hydrogen peroxide was added and 44 then incubated at 37ºC for 4 h, added 1 ml of 2.8 % TCA and 1 ml of TBA and the tubes 45 were heated at boiling water bath for 10 min and read at 532 nm Superoxide radical scavenging activity 48 To 0.3 ml of sample added 2.16 ml of 50 mm of phosphate buffer ph 8.24, then 49 added 90 microlitre of 3 mm pyrogallol. Zero minute and 10 min readings were taken 50 and the difference in the O.D at 325 nm was calculated for the superoxide radical 51 scavenging capacity was estimated from the difference in the absorbance for the 52 samples or sample blank and expressed as a percentage of the superoxide radical 53 scavenging activity , 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity 4

5 56 57 The free radical scavenging effect was estimated according to the method of 58 Blois (1958) with some modifications. One milliliter of glycowithanolide (4 mg/ml) 59 were added into the 0.2 mm DPPH radical (Sigma Aldrich Co., St. Louis, MO, USA, 1 60 ml) and 95% ethanol (1 ml) for a sample blank. The mixture was shaken and left to 61 stand for 30 min at room temperature and measured at 517 nm with a spectrophotometer 62 (model UV-1601PC, Shimadzu Co.) HPLC analysis 65 The glycowithanolide was dissolved in methanol: water (1:1) and filtered 66 through 0.45 μm Millipore filter before injection into the HPLC system. Withanolides 67 were separated and quantified at 30 C employing Shimadzu HPLC system consisting 68 of a Diode Array detector and phenomenex C18 column (5 μm, mm I.D.) by 69 UV detection at 237 nm. The mobile phase consisted of methanol: water (60:40) and 70 acetonitrile:water gradient Statistical analysis 73 The data were analyzed by Statistical Package for the Social Science (SPSS 5

6 74 Inc., 10.0, 2000). Differences among mean values were obtained by LSD multiple 75 comparison tests at p < Results and discussion 78 In the present study a significant (p <0.05) increase in the hydroxyl radical 79 scavenging capacity (Fig.1) was observed in all irradiated GW when compared to that 80 of non irradiated. On comparing GR and EB, significant increase was observed in the 81 EB treated withania powder than that of GR. 10 kgy EB irradiated showed the highest 82 hydroxyl radical scavenging capacity than that of all irradiated samples. 83 The results of the current study shows that the super oxide scavenging capacity 84 (Fig.2) of all irradiated GW was significantly (p <0.05) increased when compared to 85 that of non irradaiated GW. On comparing GR and EB, significant (p <0.05) increase in 86 super oxide scavenging capacity was observed in the EB treated withania powder than 87 that of GR. 10 kgy EB irradiated showed the highest super oxide scavenging capacity 88 than that of all irradiated samples. 89 Fig. 3 shows the changes of DPPH-scavenging activity of irradiated GW. 90 Irradiation resulted in a significant increase in DPPH radical-scavenging activity on 91 irradiation dose dependent manner of GW. The maximum DPPH radical scavenging 6

7 92 activity of GW was observed in irradiated GW at 10 kgy EB irradiation. FTIR 93 spectroscopy showed no significant change in the peaks. 94 To define the reason for the increase of antioxidative activity by the irradiation, 95 HPLC analysis of GW was carried out. The results showed that there was significant 96 increase in the major peak of 10 kgygr irradiated than all irradiated samples. 10 kgy 97 EB irradiated samples was found to be increased more than that of all irradiated 98 samples except 10 kgygr irradiated withania powder Conclusions 101 To conclude, the content of withanolide from HPLC analysis was found to be 102 increased by GR than that of EB irradiation, this may be the possible reason for 103 increased antioxidant activity of GW. Thus EB and GR irradiation could be used as an 104 efficient tool for extraction of GW Acknowledgement 107 This study was cosupported by the Basic Research Support Program, Korea 108 Atomic Energy Research Institute, and by the Korea Science and Engineering 109 Foundation, Ministry of Education, Science and Technology, Korean government, 110 through its National Nuclear Technology Program. 7

8 References 113 Agarwal, R., Diwanay, S., Patki, P., Patwardhan, B., (1999), Studies on 114 immunomodulatory activity of Withania somnifera (ashwagandha) extracts in 115 experimental immune inflammation. J. Ethanopharmocol. 67 (1), Asthana, R., Raina, M.K., (1989), Pharmacology of Withania somnifera. Indian Drugs , Budhiraja, R.D., Sudhir, S., (1987), Review of biological activity of withanolides. J. 119 Scientific Ind. Res. 46, Davis, L., Kuttan, G., (2000), Effect of Withania somnifera on cyclophosphamide 121 induced urotoxicity. Cancer Lett. 148(1), Devi, P.U., (1999), Withania somnifera dunal (ashwagandha): Potential plant source of 123 a promising drug for cancer chemotherapy and radiosensitisation. Indian J. Exp. 124 Biol. 34 (10), Dhuley, J.N., (2000), Adaptogenic and cardioprotective action of ashwagandha in rats 126 and frogs. J. Ethanopharmocol. 70 (1), Grandhi, A., Majumdar, A.M., Patwardhan, B., (1994), A comparative pharmacological 128 investigation of ashwagandha and ginseng. J. Ethnopharmacol. 44 (3),

9 129 Indian Pharmacopoeia, (1985), Ashwagandha. In: IP 1985, appendix 3.3: 10:69, 130 Ministry of Health and Family welfare, Government of India, Controller of 131 Publications, Delhi. 132 Patwardhan, B., Panse, G.T., Kulkarni, P.H., (1998), Ashwagandha a review. J. Natl. 133 Integrated Med. Assoc. 30, Prakash, J., Gupta, S.K., Kochupillai, V., Gupta, Y.K., Joshi, S., (2001), 135 Chemopreventive activity of Withania somnifera in experimentally induced 136 fibrosarcoma tumors in Swiss albino mice. Phytother. Res. 15 (3), Sharad, A.C., Soloman, F.E., Devi, P.U., Udupa, N., Srinivasan, K.K., (1996), 138 Antitumor and radio sensitizing effect of withaferin A on mouse Ehrlich ascites 139 carcinoma in vivo. Acta Oncol. 35 (1), Sharma, K., Dandiya, P.C., (1991), Withania somnifera (Dunal) Present Status. 141 Indian Drugs 29 (6), Singh, B., Saxena, A.K., Chandan, B.K., Gupta, D.K., Bhutani, K.K., Anand, K.K., 143 (2001), Adaptogenic activity of a novel, withanolide-free aqueous fraction from the 144 roots of Withania somnifera Dun. Phytother. Res. 15 (4), Ziauddin, M., Phansalkar, N., Patki, P., Patwardhan, B., (1996), Studies on 146 immunomodulatory activity of ashwagandha. J. Ethanopharmocol. 50 (2),

10 List of Figure Captions Fig. 1. Hydroxy radical scavenging ablity of gamma and electron beam irradiaited glycowithanolides. Results are expressed as mean ± SD, Multiple comparison test by LSD significance at *, UIGW Vs 5 kgygigw; 10 kgygigw; 5 kgyebigw; 10 kgyebigw. UIGW, GIGW, and EBIGW mean unirradiated GW, gamma ray irradiated GW, and electron beam irradiated GW, respectively. Fig. 2. Superoxide radical scavenging ablity of extracted glycowithanolides. Results are expressed as mean ± SD, Multiple comparison test by LSD significance at *, UIGW Vs 5 kgygigw; 10 kgygigw; 5 kgyebigw; 10 kgyebigw. Fig. 3. DPPH radical scavenging ablity of gamma and electron beam irradiated glycowithanolides. Results are expressed as mean ± SD, Multiple comparison test by LSD significance at *, UIGW Vs 5 kgygigw; 10 kgygigw; 5 kgyebigw; 10 kgyebigw. 10