Microsoft Word - 73-479-1-LayoutEfinal.docx Low effects on cytotoxicity against neuroblastoma cancer cell line (SK-N-SH) and free-radical scavenging activity of Stemona alkaloids Sumet Kongkiatpaiboon, Primchanien Moongkarndi, Wandee Gritsanapan All Res. J. Biol., 2013, 4, 19-23 The publication cost of this article might be covered by external sponsors. More info for sponsors at: sponsors@arjournals.com ARTICLE                                                                                                                    Issue 3, Vol 4, 2013, 19-23 Low effects on cytotoxicity against neuroblastoma cancer cell line (SK-N- SH) and free-radical scavenging activity of Stemona alkaloids Sumet Kongkiatpaiboona, Primchanien Moongkarndib, and Wandee Gritsanapana,* a) Department of Pharmacognosy b) Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand *Corresponding author email: wandee.gri@mahidol.ac.th; wandeegrit@yahoo.co.th Abstract: Stemona plants contain alkaloids with different skeletal types. They are well-characterized and are commonly used for insecticide, killing head lice, treating skin diseases, antitussive, and anticancer treatment. Eleven Stemona alkaloids of three skeletal types, protestemonine-, croomine-, and stichoneurine-type, i.e. protostemonine-type (didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, oxystemokerrine), croomine-type (croomine), and stichoneurine-type (tuberostemonine, tuberostemonine A, tuberostemonine N, neotuberostemonine) were isolated from various Stemona roots and investigated for their cytotoxicity and free radical scavenging activity, which have not yet been reported. Cytotoxicity on neuroblastoma cancer cell (SK-N-SH) was determined by MTT assay, while free radical scavenging activity was investigated using the DPPH radical scavenging method. Protostemonine type alkaloids possessed insignificant effect on the cytotoxicity and free radical scavenging activity, while croomine and stichoneurine type alkaloids showed weak to moderate effects. Keywords: anticancer; antioxidant; MTT assay; stemofoline; Stemona; tuberostemonine Introduction Stemona spp. of the family Stemonaceae are commonly known in Thailand under the name of “Non Tai Yak.” These plants have been used as a natural insecticide against head lice, scabicide, anticancer agent, and for treatment of skin and respiratory diseases1-3. Stemona alkaloids represent a unique chemical character of the family Stemonaceae4,5. Stemona plants contain various amounts of these alkaloids6,7, which can be divided into 3 main skeletal types4, i.e. protostemonine-type, croomine-type, and stichoneurine-type (Fig. 1). The major alkaloids of the first skeletal type includes didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, oxystemokerrine, and stemokerrine, while the second type includes croomine, and the last type includes tuberostemonine, tuberostemonine A, tuberostemonine N, and neotuberostemonine. The reported biological activities of these Stemona alkaloids are insect toxicity8,9, antitussive10,11, oxytocin antagonist12, nitric oxide inhibition13, and increasing chemosensitivity via P-glycoprotein-mediated multidrug resistance14-16. Cytotoxicity of Stemona extracts or their isolated alkaloids has been reported against several human cancer cell lines such as medullary thyroid carcinoma17,18, human fibroblast18, leukemia carcinoma19, nasopharyngeal19, gastric carcinoma19, breast adenocarcinoma20. However, free radical scavenging activity and cytotoxicity of these alkaloids on neuroblastoma cancer cells has not been reported. Oxidative stress can lead to numerous pathophysiological conditions, including cancer. Antioxidants can terminate or retard the oxidation process by scavenging and sequestering the free radicals that are implicated in many chronic diseases. In the present study, eleven Stemona alkaloids (Fig. 1), which were isolated from underground parts of various Stemona plants in our previous works6, were investigated for their free radical scavenging activity and cytotoxicity against neuroblastoma cell line (SK-N-SH). Materials and methods Chemical and reagents RPMI 1640 medium and fetal calf serum (FCS) were obtained from Biochrom (Berlin, Germany). 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) were purchased from Sigma (St. Louis, MO). Eleven Stemona alkaloids of three skeletal types, i.e. didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, oxystemokerrine of protostemonine-type; croomine of croomine-type; and tuberostemonine, tuberostemonine A, tuberostemonine N, neotuberostemonine of stichoneurine-type were isolated from the roots of various Stemona plants and identified in our previous work6. 19 All Res. J.Biol, 2013, 4, 19-23     Cell culture SK-N-SH cell line was obtained from the American Type Culture Collection (Rockville, MD) was cultured in RPMI 1640 medium supplemented with 5% (v/v) FSC, 100 mg/l of streptomycin and 100,000 U/l of penicillin G, at 37°C in 5% CO2 incubator. Cell viability evaluation by MTT assay MTT assay was a conventional method to assess the number of viable cells growing in microtiter plate after treatment with various substances. Serial dilutions of samples (50 µl) were added into each of 96-well plates. The cells were plated at a density of 1 × 104 cells/well and incubated for 48 hrs. After incubation, the medium was removed and the cells in each well were incubated with phosphate buffered saline (PBS) containing 1 mg/ml MTT for 2 hrs at 37°C in 5% CO2 incubator. MTT solution was then discarded and 50 µl of isopropanol was added into each well to dissolve insoluble formazan crystals. Plates were then agitated for 5 min at room temperature to complete the solubilization. The color of formazan derivative was analyzed on a microplate reader (Molecular Devices, CA) at a wavelength of 590 nm. Each concentration was done in triplicate. The percentage of cell viability was calculated according to the following equation: cell viability (%) = (OD of treated cells / OD of control cells) × 100%. Determination of free radical scavenging activity The free radical scavenging activity of Stemona alkaloids was investigated using DPPH radical scavenging assay. The solution of DPPH• was prepared at concentration of 60 mg/l (152 µM) in methanol, while stock solution of the sample (1 mg/ml) was serially diluted between 12.5 and 800 µg/ml. The DPPH• scavenging reaction was performed by adding DPPH• solution (100 µl) to the sample solution of the same volume. The mixture was incubated for 30 min at room temperature and measured for absorbance at 517 nm by a microplate reader. The corresponding blank sample was also taken and percent inhibition was then calculated as follows: Scavenging activity (%) = [1 - (A1 - A2) / A0] × 100% where A0 was the absorbance of control (DPPH• solution without sample), A1 was the absorbance of DPPH• solution in the presence of the sample and A2 was the absorbance of the sample without DPPH• solution. Statistical analysis The experiments were repeated three times and the results were expressed as mean + S.D. Statistical analysis was done using two-tailed Student’s t test and P values at a level of 95% confidence limit. Results Cytotoxicity on neuroblastoma cell line SK-N-SH The cytotoxic effect of Stemona alkaloids on neuroblastoma cell line SK-N-SH was investigated by MTT assay. Cells were treated with Stemona alkaloids at concentrations ranging from 0 to 200 µg/ml for 48 hours and the percentage of cell viability was analysed. The EC50 was then calculated as shown in Table 1. Croomine and stichoneurine derivatives, i.e. tuberostemonine, tuberostemonine A, tuberostemonine N, and neotuberostemonine inhibited the proliferation of SK-N- SH cells in a dose-dependent manner. A similar result was observed when quercetin was used as a positive control. Free radical scavenging activity The free radical scavenging activity of Stemona alkaloids was determined by the DPPH• free radical scavenging assay. At a concentration of 400 µg/ml, protostemonine alkaloid derivatives - didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, and oxystemokerrine - increased free radical scavenging activity by only 8.81%, 9.92%, 45.03%, 8.55%, 8.52%, and 59.27%, respectively, whereas croomine and the stichoneurine derivatives - tuberostemonine, tuberostemonine A, tuberostemonine N, and neotuberostemonine - increased scavenging activity by 88.84%, 88.76%, 79.98%, 84.81%, and 84.73%, respectively. Serial dilutions of each sample were also measured for their free radical scavenging activity. The EC50 of each alkaloid was then calculated (Table 1). Croomine and stichoneurine derivatives possessed free radical scavenging activity in a dose-dependent manner. Similar results were observed when quercetin and trolox were used as positive control. Discussion Stemona alkaloids could be classified, based on biosynthetic hypothesis, into three skeletal types: protostemonine-, stichoneurine-, and croomine-type. Protostemonine type alkaloids, which include didehydrostemofoline, stemofoline, stemocurtisine, stemocurtisinol, stemokerrine, and oxystemokerrine, possessed insignificant cytotoxicity on SK- N-SH cells and low free radical scavenging activity, while croomine and the stichoneurine-type alkaloids, which are tuberostemonine, tuberostemonine A, tuberostemonine N, and neotuberostemonine, showed weak to moderate activities compared to the positive control (Table 1). Although Stemona roots have been traditionally used as an ingredient in several anti-cancer preparations, only a few scientific studies on their anti-cancer properties were reported. Only medullary thyroid carcinoma cell lines gave positive results when treated with Stemona extracts17,18 whereas other cell lines showed weak responses or no cytotoxicity activity15,19,20. Although those compounds do not seem to exhibit a strong cytotoxicity, they might still be acting to potentiate the effect of anti-neoplastics. For example, stemofoline, stemocurtisine, and stemocurtisinol were reported to sensitize the multidrug resistant cancer cell to putative chemotherapeutic agents including vinblastine, paclitaxel and colchicine via P-glycoprotein mediated pathway14-16. 20 All Res. J.Biol, 2013, 4, 19-23 Figure 1 Structure of Stemona alkaloids used in this study. 21 All Res. J.Biol, 2013, 4, 19-23     Table 1 Cytotoxicity and free radical scavenging activity of Stemona alkaloids Typea Compounds EC50 (µg/ml) Cytotoxicityb Free radical scavenging activityb I Didehydrostemofoline > 200 >> 400 Stemofoline > 200 >> 400 Stemocurtisine > 200 > 400 Stemocurtisinol > 200 >> 400 Stemokerrine > 200 > 400 Oxystemokerrine > 200 156.68 + 2.72 II Tuberostemonine 140.49 + 7.92 34.88 + 1.92 Tuberostemonine A 144.58 + 10.39 218.33 + 2.00 Tuberostemonine N 193.44 + 10.57 57.03 + 0.68 Neotuberostemonine 71.27 + 2.52 66.74 + 1.56 III Croomine 198.42 + 5.48 107.74 + 0.33 Postive control Trolox - 2.62 + 0.05 Quercetin 36.67 + 4.39 2.29 + 0.02 a I = protostemonine-type alkaloid, II = stichoneurine-type alkaloid, III = croomine-type alkaloid b Results expressed as mean + SD (n = 3) Conclusion Protostemonine alkaloid derivatives showed an insignificant effect on both cytotoxicity of SK-N-SH cells and free radical scavenging activity, while stichoneurine alkaloid derivatives and croomine alkaloid demonstrated these activities at low to moderate levels. The highest cytotoxic effect was observed in neotuberostemonine with an EC50 of 71.27 µg/ml, which was 2-fold lower activity than that of quercetin (EC50 36.67 µg/ml). The highest free radical scavenging activity was observed in tuberostemonine with EC50 of 34.88 µg/ml, which is about 15 times lower than those observed with trolox and quercetin (EC50 2.62 and 2.29 µg/ml, respectively). These results demonstrate that Stemona alkaloids, the major contents in the roots of Stemona species, may not directly possess cytotoxicity on cancer cells. When included in traditional anti-cancer preparations, Stemona possibly synergizes and enhances the activity of other active compounds through increasing chemosensitivity. Acknowledgements :    This study is a part of a Ph.D. thesis at Mahidol University, financially supported by Thailand Research Fund and Mahidol University (Royal Golden Jubilee Ph.D. Program Grant No. PHD/0139/2550). 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