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1 1252 药学学报 Acta Pharmaceutica Sinica 2009, 44 (11): Insulin secretion stimulating effects of mogroside V and fruit extract of Luo Han Kuo (Siraitia grosvenori Swingle) fruit extract ZHOU Ying 1, ZHENG Yan 2, EBERSOLE Jeff 2, HUANG Chi-fu 2* (1. College of Life Science, Guizhou University, Guiyang , China; 2. Center for Oral Health Research, College of Dentistry, University of Kentucky, Lexington, Kentucky 40503, USA) Abstract: Luo Han Kuo fruit (Siraitia grosvenori Swingle), a fruit native to China, has been used as a natural sweetening agent for centuries and has been reported to be beneficial for diabetic population. However, limited research has been conducted to elucidate the relationship between the sweetening action and biological parameters that may be related to potential health benefits of LHK fruit (Luo Han Kuo fruit). The present study examined the effect of LHK fruit and its chemical components on insulin secretion using an in vitro cell model system. Mogroside V is the most abundant and the sweetest chemical component among the mogrosides in LHK fruit. The experimental data demonstrated that the crude LHK extract stimulated the secretion of insulin in pancreatic beta cells; furthermore, pure mogroside V isolated from LHK fruit also exhibited a significant activity in stimulating insulin secretion by the beta cells, which could partially be responsible for the insulin secretion activity of LHK fruit and fruit extract. The current study supports that LHK fruit/extract has the potential to be natural sweetener with a low glycemic index, and that mogroside V, possible other related mogrosides, can provide a positive health impact on stimulating insulin secretion. Key words: Luo Han Kuo fruit; insulin secretion; mogroside V CLC number: R284.1 Document code: A Article ID: (2009) 罗汉果提取物和罗汉果苷 V 对胰岛素分泌的调节作用周英 1, 郑艳 2, EBERSOLE Jeff 2, 黄赤夫 2* (1. 贵州大学生命科学学院, 贵州贵阳 ; 2. 美国肯塔基大学口腔学院口腔健康研究中心, 肯塔基, 列克星敦, 40503) 摘要 : 罗汉果作为一种保健食品和甜味剂已有长期的应用, 有报道认为罗汉果及其提取物对糖尿病有一定的防治作用, 然而目前还未见对其相关活性成分以及活性的报道本研究运用鼠 β 细胞系细胞 RIN-5F 研究了罗汉果提取物以及罗汉果苷 V 对于胰岛素分泌的影响本研究结果显示, 罗汉果提取物和罗汉果苷 V 对胰岛素分泌有显著的促进作用本研究从细胞水平揭示了罗汉果和罗汉果苷 V 对于糖尿病人具有血糖调节作用, 提示罗汉果提取物以及罗汉果苷对于 II 型糖尿病的防治作用关键词 : 罗汉果 ; 胰岛素分泌 ; 罗汉果苷 V Luo Han Kuo (LHK) fruit (Siraitia grosvenori Swingle) has been used in China for centuries as a natural sweetener, and also been used to treat sore throats and coughing, and treat diabetes in folk medicine [1 5]. Received Project supported by National Institute of Dental & Craniofacial Research (NIH/NIDCR (1R41DE )). * Corresponding author Tel: , chuan2@uky.edu LHK extract is a high intensive sweetener that is about 300 times sweeter than sucrose. The sweetening components of the LHK fruit were identified as mogrosides [6, 7], which are 300 to 400 times sweeter than sucrose. The mogrosides are a group of compounds primarily consisting of mogroside I to VI with variations in the position and the number of the glucose moieties attached to a mogrol group [8 10]. Mogroside

2 ZHOU Ying, et al: Insulin secretion stimulating effects of mogroside V and fruit extract of Luo Han Kuo (Siraitia grosvenori Swingle) fruit extract 1253 V has five glucose moieties and is the sweetest among the mogrosides, about 500 times sweeter than sucrose [8 10]. Although the LHK fruit has been anecdotally reported to have beneficial effects for individuals with diabetes, limited research has been conducted to elucidate biological activities that could account for these observations. A recent study reported an antidiabetic effect of long-term dietary supplementation with LHK fruit extract on the spontaneously type 2 diabetes model, Goto-Kakizaki (GK) rat [11]. In this study, 7-week-old diabetic GK rats were fed a diet supplemented with 0.4% of the LHK extract for 13 weeks with no effect on overall food intake or body weight. However, with oral glucose tolerance tests (OGTT), LHK extract supplementation improved the insulin response, reduced the plasma glucose level, and elevated the total amount of insulin in whole pancreas taken from the fasting rats. This composite could explain the greater capacity to secrete insulin during the OGTT. Their data also supported that LHK extract supplementation may prevent complications and attenuate pathological conditions for type 2 diabetes. In a separate study, Suzuki et al [12] were able to use triterpene glycosides of Siraitia grosvenori to inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats. However, there have been no reports describing the effect of LHK extracts and mogrosides on the function of pancreatic beta cells. In this study, purified mogroside V has been used to investigate its biological action on beta cells. Our experimental data demonstrated that mogroside V had a significant effect on insulin secretion and confirmed that the natural sweetener LHK could be beneficial for use by diabetic populations, because of the no-caloric, low glycemic index of LHK extract/mogroside V would help control blood sugar levels and contribute in management of diabetes and obesity. Materials and methods Instruments Column chromatography (CC): silica gel ( mesh, EMD Chemicals Inc., Germany; mesh, Fisher Scientific, USA); silica gel 60 RP-18 ( mesh, EMD Chemicals Inc., Germany); Amberlite XAD-16 (Fisherscientific, USA). Thin-layer chromatography (TLC): silica gel GF 254 plates (Merck KGaA, Germany); silica gel 60 RP-18 F254 plates (Merck KGaA, Germany). TLC spots were detected by visualization under UV light or by spraying with 98% H 2 SO 4 /EtOH 10 90, followed by heating at 110. PTLC: silica gel 60 (500 μm thickness, Whatman, UK; 1000 μm thickness, Merck KGaA, Germany). Analysis high-performance liquid chromatography (HPLC): Waters 1525 binary HPLC pump (Waters, USA), Waters 2487 dual λ absorbance detector (Waters, USA) and a XTerra RP18 column (250 mm 4.6 mm i.d., particle size 5 μm; Waters, USA). Preparative high-performance liquid chromatography (HPLC): Waters 510 pump (Waters, USA), Waters 484 tunable absorbance detector (Waters, USA) and a Phenomenex Gemini C 18 column (250 mm 10.0 mm i.d., particle size 5 μm; Phenomenex, USA). The Nuclear Magnetic Resonance spectra data was obtained on Varian INOVA H NMR data was recorded at 400 MHz and 13 C NMR at 100 MHz, separately. Norell 4 mm 178 mm NMR sample tube (Norell INC., USA) was used with pyridine-d 5 as solvent. All the chemical shifts were given in value δ (ppm) downfield relative to tetramethylsilane (δ = 0 ppm) and coupling constants J were given in Hz. ESI-MS was performed on a ThermoFinnigan LTQ spectrometer in m/z. LHK extract purification Dry LHK fruit from Guilin and LHK fruit extract were commercially obtained from China Natural Products, Inc. (China, and voucher samples (No and No ) were deposited in Oral Health Research and Development Center, University of Kentucky. The LHK fruit was identified and samples were kept by Prof. Chifu Huang in University of Kentucky. LHK extract was prepared from the fruit by crushing 100 grams of dry LHK fruit using a mortar and pestle, followed by adding 500 ml of 50% EtOH. This LHK mixture was allowed to shake for 24 h in a flask and was then filtered with 3M paper. The LHK extract powder was prepared by lyophilization. The LHK extract powder (10 g) was further treated by chromatography on an Amberlite column and eluted with an EtOH solution of increasing polarity by 10% increments. Ethanol fractions at 50% 90% (ca. 100 ml each) were collected and combined on the basis of TLC analysis. The Amberlite fractions and commercial LHK fruit extract were tested for insulin secretion activity (see below). The purchased commercial LHK extract has 80% mogrosides, of

3 1254 药学学报 Acta Pharmaceutica Sinica 2009, 44 (11): which 40% are mogroside V according to the COA of manufacturer. Mogroside V purifcation The LHK extract powder (10 g) was also chromatographed on a silica gel column and eluted with CHCl 3 -MeOH-H 2 O mixure of increasing polarity. A total of 51 fractions (ca. 100 ml each) were collected and combined on the basis of TLC analysis. Fractions were further separated by Amberlite eluting with MeOH-H 2 O gradient mixtures to give 32 fractions (ca. 75 ml). Sub-fractions was purified by silica gel RP-18 column, and the fractions eluted with MeOH-H 2 O (6 4), which mostly composed of mogroside V were chromatographed by preparative HPLC with MeOH-H 2 O (2 1) as the mobile phase at the flow rate of 6 ml min 1. Pure mogroside V was obtained from this purification step. Structure elucidation Mogroside V: Colorless powder. C 60 H 102 O 29, ESI-MS: [M+H] +. 1 H NMR (400 MHz, C 5 D 5 N-d 5 ) δ: 5.49 (1H, d, J = 8.0 Hz, H-6), 4.15 (1H, d, J = 8.5 Hz, H-11β), 3.78 (1H, d, J = 8.0 Hz, H-24), 3.70 (1H, br s, H-3), 1.53 (3H, s, Me-29β), 1.47 (3H, s, Me-26), 1.36 (3H, s, Me-19), 1.35 (3H, s, Me-27), 1.10 (3H, d, J = 5.6 Hz, Me-21β), 1.09 (3H, s, Me-28α), 0.92 (3H, s, Me-18β), 0.92 (3H, s, Me-30α), glucose: A 5.49 (1H, d, J = 8.0 Hz, H-1), B 5.19 (1H, d, J = 8.0 Hz, H-1), C 4.91 (1 H, d, J = 5.6 Hz, H-1), D 4.83 (1H, d, J = 8.0 Hz, H-1), E 4.81 (1H, d, J = 7.2 Hz, H-1); 13 C NMR (100 MHz, C 5 D 5 N-d 5 ) δ: 27.2 (C-1), 29.8 (C-2), 87.8 (C-3), 42.6 (C-4), (C-5), (C-6), 25.0 (C-7), 43.8 (C-8), 40.5 (C-9), 37.0 (C-10), 78.2 (C-11), 41.4 (C-12), 47.8 (C-13), 50.0 (C-14), 34.9 (C-15), 28.9 (C-16), 51.4 (C-17), 17.5 (C-18), 26.6 (C-19), 36.7 (C-20), 19.4 (C-21), 33.6 (C-22), 29.8 (C-23), 92.2 (C-24), 73.1 (C-25), 25.0 (C-26), 27.5 (C-27), 28.0 (C-28), 26.6 (C-29), 19.8 (C-30); Glc (A): (C-1 ), 75.8 (C-2 ), 79.0 (C-3 ), 72.0 (C-4 ), 78.4 (C-5 ), 70.7 (C-6 ); Glc (B): (C-1 ), 75.7 (C-2 ), 78.9 (C-3 ), 71.8 (C-4 ), 78.2 (C-5 ), 63.0 (C-6 ); Glc (C): (C-1 ), 82.9 (C-2 ), 77.7 (C-3 ), 72.9 (C-4 ), 76.8 (C-5 ), 70.5 (C-6 ); Glc (D): (C-1 ), 75.6 (C-2 ), 78.7 (C-3 ), 71.8 (C-4 ), 79.0 (C-5 ), 62.9 (C-6 ); Glc (E): (C-1 ), 76.3 (C-2 ), 78.7 (C-3 ), 72.1 (C-4 ), 78.8 (C-5 ), 64.0 (C-6 ). The MS and NMR spectral data were in agreement with those reported for mogroside V [10, 13]. Bioassay screening of mogroside V RIN-5F cells (rat insulinoma) were obtained from ATCC (CRL-2058) and cultured in RPMI 1640 (10 mmol L 1 glucose) supplemented with 10% (v/v) fetal calf serum, 2 mmol L 1 L-glutamine, 1 mmol L 1 sodium pyruvate, 100 u ml 1 penicillin, 100 µg ml 1 streptomycin at 37, and 5% CO 2. RIN-5F cells ( ) were cultured in 75-cm 2 tissue culture flasks and fed every 3 d. Cells were subcultured and seeded into 96-well plates with each well having RIN-5F cell density of To determine the effect of mogroside V on insulin secretion, rat insulinoma cells were incubated with various concentrations of 1, 10, and 30 mmol L 1 mogroside V in the absence of D-glucose as a negative control. Briefly, RIN-5F cells were grown for 14 h on 1 mmol L 1 D-glucose and then pre-incubated with KRB (Krebs-Ringer Bicarbonate) buffer for 2 h, followed by treatment with 1 mmol L 1 or 30 mmol L 1 D-glucose as a positive control and with the various concentrations of commercial LHK extract or mogroside V for 1 h. Aliquots of the media were collected and used to measure secreted insulin levels with a rat insulin EIA assay (ALPCO, Salem NH). Data were presented as the means of insulin concentration of two independent experiments, each with duplicate determinations. Statistical determinations were done by using a Student t test compared to control cultures. Results and discussion Mogroside V was successfully purified from LHK fruit following chromatography and HPLC purification. Pure mogroside V was obtained with the characteristics of colorless powder (data not shown). The ESI-MS spectra analysis confirmed the identity and the molecular weight of the obtained mogroside V (Figure 1). NMR analysis further confirmed the structure of mogroside V as its spectral data is identical to that of the published literature [10, 13]. The structure of mogroside V is shown in Figure 2. The crude LHK extract exhibited insulin-stimulating activity on the RIN-5F cells. As compared Figure 1 MS spectra and HPLC of isolated mogroside V

4 ZHOU Ying, et al: Insulin secretion stimulating effects of mogroside V and fruit extract of Luo Han Kuo (Siraitia grosvenori Swingle) fruit extract 1255 to the D-glucose control, crude LHK extract induced significantly greater insulin secretion than the D-glucose control in beta cells (Figure 3). The crude LHK extract was further purified with Amberlite chromatography and 10 fractions were collected ranging from 10% to 100% ethanol elution. The ten LHK extract fractions were tested for insulin secretion inducing bioactivity. Insulin secretion bioactivity was observed on fractions between 40% and 70% EtOH (Figure 4). Further separation of the LHK extract yielded 20 mg of mogroside V. To determine the effect of the mogroside V on insulin secretion, the RIN-5 insulinoma cell line was incubated with 1, 5 mmol L 1 mogroside V solution in the absence of glucose. As shown in Figure 5 treatment of these cells with mogroside V and LHK extract stimulated insulin secretion. Mogroside V produced significant more insulin than the crude LHK extract, indicating that mogroside V is one of the key factors responsible for inducing insulin secretion in the LHK extracts. Also, the mogroside V induced significantly greater insulin secretion than D-glucose (Figure 6). This study demonstrated that the LHK extract increased insulin production in vitro from a pancreatic beta cells culture. This data suggest that the LHK extract can function as a potent insulin secretagogue, Figure 2 Structure of mogroside V Figure 4 LHK extract fractions stimulate insulin secretion in the rat insulinoma RIN-5 cell line. RIN-5 cells ( ) were grown for 14 h on 1 mmol L 1 D-glucose in per well of 96-well plate and then pre-incubated with KRB (Krebs-Ringer Bicarbonate) buffer for 2 h and treated with 10 µl of LHK extract fraction for 1 h. Data are the average value of two independent experiments with duplicate measures. The vertical bracket encloses 1 SD Figure 3 LHK extract stimulates insulin secretion in the RIN-5 cell line. RIN-5 cells were grown for 14 h on 1 mmol L 1 D-glucose and then pre-incubated with KRB (Krebs-Ringer Bicarbonate) buffer for 2 h and treated with 1, 10 or 50 mmol L 1 D-glucose as control and with 0.32, 1.6 mmol L 1 of mogroside V/LHK extract (LHK extract was normalized with mogroside V, as the commercial obtained LHK extract has 80% mogrosides, of which 40% are mogroside V) for 1 h. Aliquots of the media (containing secreted insulin) were collected and used to measure insulin levels with an insulin ELISA assay from ALPCO. Data are the averages of two independent experiments measured in duplicates Figure 5 Effects of mogroside V on insulin secretion in the rat insulinoma cell line. RIN-5 cells were grown for 14 h on 1 mmol L 1 D-glucose and then pre-incubated with KRB (Krebs- Ringer Bicarbonate) buffer for 2 h and then treated with 1 mmol L 1 or 5 mmol L 1 pure mogroside V and with 0.32, 1.6 mmol L 1 of mogroside V/LHK extract for 1 h. Data were the means of two independent experiments with duplicate measures. The vertical bracket encloses 1 SD

5 1256 药学学报 Acta Pharmaceutica Sinica 2009, 44 (11): Figure 6 Dosage effect of mogroside V on insulin secretion. Mogroside V stimulates insulin secretion in the rat insulinoma RIN cell line. RIN cells were grown for 14 h on 1 mmol L 1 D-glucose, then pre-incubated with KRB (Krebs-Ringer Bicarbonate) buffer for 2 h, and treated with 5, 10 and 50 mmol L 1 D-glucose as control, or with various concentrations (1.5, 7.5 and 15 µmol L 1 ) of pure mogroside V for 1 h. Data were the averages of two independent experiments with duplicate measures. The vertical bracket encloses 1 SD which may be beneficial to patients with type 2 diabetes using this material as a zero-calorie natural sweetener. Mogroside V has five glucoses and is the sweetest among mogrosides, about 500 times sweeter than sucrose. The ability of mogroside V to stimulate insulin secretion suggests that mogroside V may have a very positive health value. The glucose transporter plays a critical role for insulin secretion [14], although, it is unlikely that mogroside V would work through the glucose transporter, since the mogroside V is significantly bigger than glucose molecule. It is possible that the glucoses of the mogroside V could be cleaved off the mogrol moiety and the free glucoses could bind to the glucose transporters, such as GLUT4 or sweet taste receptors thus triggering the beta cells [14, 15]. However, alternative mechanisms including specific intracellular signaling [16, 17] pathways upregulating insulin gene transcription or translation [18], or enhanced secretion of insulin [19] could account for these findings. Future studies will address the mechanism(s) by which LHK extract components, particularly mogroside V, stimulate the insulin secretion in these beta cells. Defining these biological parameters of LHK and mogrosides may support that this material represents an ideal natural sweetener. Acknowledgments: We would like to thank Dr. Sabire Ozcan, Department of Biochemistry, University of Kentucky, for her kind contribution in providing intellectual and technical help on this project. Supported by the grant 1R41DE from the NIH/NIDCR. References [1] Qi XY, Chen WJ, Song YF, et al. Efficacy study on Siraitia grosvenori powder and its extracts on reducing blood glucose in diabetic rabbits [J]. Food Sci ( 食品科学 ), 2003, 24: [2] Nabors LO. A review of various other alternative sweeteners [J]. Food Sci Technol, 1986, 17: [3] Takeo E, Yoshida H, Tada N, et al. Sweet elements of Siraitia grosvenori inhibit oxidative modification of low-density lipoprotein [J]. J Atheroscler Thromb, 2002, 9: [4] Ukiya M, Ahihisa T, Tokuda H, et al. Inhibitory effects of cucurbitane glycosides and other triterpenoids from the fruit of Momordica grosvenori on epstein-barr virus early antigen induced by tumor promoter 12-O-tetradecanoylphorbol-13- acetate [J]. J Agric Food Chem, 2002, 50: [5] Takasaki M, Konoshima T, Murata Y, et al. Anticarcinogenic activity of natural sweeteners, cucurbitane glycosides, from Momordica grosvenori [J]. Cancer Lett, 2003, 198: [6] Kasai R, Nie RL, Nashi K, et al. Sweet cucurbitane glycosides form fruits of Siraitiasiamensis (chi-zi Luo-han-guo), a Chinese folk medicine [J]. Agric Biol Chem, 1989, 53: [7] Lee C. Intense sweetener from Lo Han Kuo (Momordica grosvenori) [J]. Experientia, 1975, 31: [8] Takemoto T, Arihara S, Nakajima T, et al. Studies on the constituents of Fructus Momordicae. I. On the sweet principle [J]. Yakugaku Zasshi, 1983, 103: [9] Takemoto T, Arihara S, Nakajima T, et al. Studies on the constituents of Fructus Momordicae. III. Structure of mogrosides [J]. Yakugaku Zasshi, 1983, 103: [10] Takemoto T, Arihara S, Nakajima T, et al. Studies on the constituents of Fructus Momordicae. II. Structure of sapogenin [J]. Yakugaku Zasshi, 1983, 103:

6 ZHOU Ying, et al: Insulin secretion stimulating effects of mogroside V and fruit extract of Luo Han Kuo (Siraitia grosvenori Swingle) fruit extract 1257 [11] Suzuki YA, Tomoda M, Murata Y, et al. Antidiabetic effect of long-term supplementation with Siraitia grosvenori on the spontaneously diabetic goto-kakizaki rat [J]. Br J Nutr, 2007, 97: [12] Suzuki YA, Murata Y, Inui H, et al. Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration [15] Mace OJ, Affleck J, Patel N, et al. Sweet taste receptors in rat small intestine stimulate glucose absorption through apical GLUT2 [J]. J Physiol, 2007, 582: [16] Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription [J]. Diabetologia, 2002, 45: [17] Jang WG, Kim EJ, Park KG, et al. Glucocorticoid receptor mediated repression of human insulin gene expression is of maltose in rats [J]. J Agric Food Chem, 2005, 53: 2941 regulated by PGC-lalpha [J]. Biochem Biophys Res Commun, , 352: [13] Li DP, Ikeda T, Matsuoka N, et al. Cucurbitane glycosides from unripe fruits of Lo Han Kuo (Siraitia grosvenori) [J]. [18] Goodge KA, Hutton JC, et al. Translational regulation of proinsulin biosynthesis and proinsulin conversion in the Chem Pharm Bull, 2006, 54: [14] Navarro-Tableros V, Fiordelisio T, Hernández-Cruz A, et al. pancreatic beta-cell [J] Semin Cell Dev Biol, 2000, 11: 235 Physiological development of insulin secretion, calcium channels, and GLUT2 expression of pancreatic rat beta-cells [19] Johnson D, Shepherd RM, Gill D, et al. Glucokinase activators: molecular tools for studying the physiology of insulin-secreting [J]. Am J Physiol Endocrinol Metab, 2007, 292: E cells [J]. Biochem Soc Trans, 2007, 35: 欢迎订阅中国临床药学杂志中国临床药学杂志主要报道我国临床药学及相关领域的新成果新技术新方法, 有关临床用药的基础研究, 合理用药药物不良反应药物相互作用药物动力学血药浓度监测以及有关医院药学学科的研究成果与实践旨在为我国广大临床药学工作者提供一个学术交流信息传递和了解国内外临床药学及相关学科发展情况的窗口与平台作为一本专业特色明显的全国性期刊, 本刊被收录为国家科技部中国科技论文统计源期刊 ( 中国科技核心期刊 ) 国家级火炬计划项目中国科学引文数据库来源期刊中国学术期刊综合评价数据库来源期刊中国期刊网中国学术期刊 ( 光盘版 ) 和万方数据资源系统 (China info) 数字化期刊群, 并从 1998 年起被美国国际药学文摘 (IPA) 和美国化学文摘 (CA) 摘录收载本刊的主要读者对象为医院药师医师和大专院校科研单位研究人员和临床药学工作者本刊为双月刊, 大 16 开本, 64 页, 逢单月 25 日出版每期定价 8 元, 全年 48 元邮发代号 4-573, 欢迎广大读者到本地邮局订阅国外代号 4726BM ( 中国国际图书贸易公司, 北京 399 信箱 ) 国内统一刊号: CN ; 国际标准出版物编号 : ISSN 杂志社地址: 上海市医学院路 138 号 290 信箱, 邮编 : , 电话 : (021) ; 传真 : (021) ; cjcpfdu@citiz.net

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