Separation and hplc quantitative analysis of murrayafoline a from the roots of glycosmis stenocarpa - Tran Quoc Toan

Journal of Science and Technology 54 (2C) (2016) 530-537 SEPARATION AND HPLC QUANTITATIVE ANALYSIS OF MURRAYAFOLINE A FROM THE ROOTS OF GLYCOSMIS STENOCARPA Tran Quoc Toan1, Le Xuan Duy1, Tran Thu Huong1, To Dao Cuong1, Tran Thi Thu Thuy1, Nguyen Quang Tung2, Bui Huu Tai3, Nguyen Manh Cuong1, * 1Institute of Natural Products Chemistry, VAST, 18 Hoang Quoc Viet road, Cau Giay, Hanoi 2Faculty of Chemical Technology, Hanoi University of Industry, km 13, 32 Road, Minh Khai commune, Bac Tu Liem, Hanoi 3Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet road, Cau Giay,Hanoi *Email: nmcuong_inpc@yahoo.com.vn Received: 15 June 2016; Accepted for publication: 29 October 2016 ABSTRACT In this study, we investigated two methods for the separation and purification of alkaloid Murrayafoline A from the roots of the Vietnamese plant Glycosmis stenocarpa and developed an HPLC method for the determination of its contents in plant materials and the methanol extract. The yield of Murrayafoline A using steam distillation method was 1.5 times higher than the separation method using column chromatography. By HPLC method, the content of Murrayafoline A was determined to be 0.38 % (w/w) in the roots and 16.69 % (w/w) in the methanol extract of G. stenocarpa. Keywords:Glycosmis stenocarpa, Rutaceae, carbazole alkaloid, Murrayafoline A. 1. INTRODUCTION Murrayafoline A is a carbazole alkaloid which has strong activity against cancer cells [1 - 3], enhances myocardial contractility, prevents myocardial ischemia and minimizes the risk of stroke [4]. In Vietnam, Murrayafoline A was found in the medicinal plant Glycosmis stenocarpa Drake. (Rutaceae), which is widely distributed in some northern provinces [5, 6, 7]. Our recent studies showed that the methanol extract from the leaves and stem of G. stenocarpa has anti-cancer, anti- fungal and anti-bacterial activities [8, 9]. In this paper, we report a new and effective method for the isolation of Murrayafoline A from G. stenocarpa using steam distillation and an HPLC method for the determination of its content in plant materials and extracts. 2. MATERIALS AND METHODS 2.1. Plant materials Separation and HPLC quantitative analysis of murrayafoline A from the roots of Glycosmis... 531 Plant samples of G. stenocarpa were collected at Hoang Hoa Tham commune, Hai Duong province, Vietnam in March 2011 and were identified by Dr. Ngo Van Trai - Department of Botany, National Institute of Medicinal Materials (NIMM). A voucher specimen (TC-036) was deposited in the Herbarium of the Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam. The roots of G. stenocarpa were collected, washed, dried and crushed to obtain the plant material as dried powder. 2.2. General experimental procedures 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were measured on a Bruker Avance 500 MHz spectrometer. Column chromatography was carried out on silica gel 60 (40 - 63 μm, Merck). Pre-coated TLC plates (Si 60 F254) were used for analytical purposes. HPLC solvents were from Burdick & Jackson, USA and other solvents were redistilled before use. 2.3. Quantification of Murrayafoline A by HPLC 2.3.1. HPLC apparatus and chromatographic conditions HPLC was carried out using a Gilson system equipped with a Gemini® 5 μm C18 100 Å column (250 x 4.6 mm, 5 μm particle size, Phenomenex) and a UV/VIS-151 detector. Data was collected and analyzed using Gilson Trilution software. Chromatographic analysis was conducted with isocratic elution using a solution of 60 % acetonitrile in water at a flow rate of 1.0 mL/min, in 25 minutes. The injection volume was 20 μL. 2.3.2. Preparation of standard solutions of Murrayafoline A Stock standard solution of Murrayafoline A (1.0 mg/mL) was correctly weighed and dissolved in methanol and then diluted to prepare seven solutions with concentrations of 10, 30, 50, 100, 150, 200 and 300 μg/mL for the establishment of calibration curve. These solutions were stored away from light at 5 oC. 2.3.3. Preparation of sample solution The dried powdered roots of G. stenocarpa (2 g) were weight accurately, added into filter bag, and extracted with 20 mL methanol by reflux for three times (each 60 mins) to give methanol extract. Methanol extract was then re-dissolved in methanol to form 3 mg/mL solution. After filtration using filter membrane 0.45 μm (Whatman), filtrate was injected into the HPLC system in triplicate. The content of Murrayafoline A was determined from the corresponding calibration. 3. RESULTS AND DISCUSSION 3.1. Separation of Murrayafoline A by column chromatography and steam distillation The dried root powder of G. stenocarpa (10 kg) was extracted by sonication with methanol (25 L × 3 times) for 2 hours at room temperature and concentrated under reduced pressure to yield a black crude methanol extract. The crude methanol extract was then suspended in MeOH: H2O (1:1, v/v) and partitioned with n-hexane, ethyl acetate and n-butanol. The resulting fractions Tran Quoc Toan, Le Xuan Duy, Tran Thu Huong 532 were concentrated under reduced pressure to give the corresponding n-hexane (508 g), ethyl acetate (196 g) and n-butanol residues (322 g). Half of the n-hexane residue (254 g) was subjected to silica gel column chromatography (40- 63 mesh, Merck), eluted with gradient solutions of n-hexane / ethyl acetate (100:0 to 1:1, v/v) to afford 8 sub-fractions (H1-H8). The fraction M1 (64.2 g) was obtained by combination of sub- fractions H2, H3 and H4 according to their thin layer chromatography (TLC) profiles. The fraction M1 (64.2 g) was further subjected to silica gel column chromatography (40-63 mesh, Merck), eluted with n-hexane/ethylacetate (50:1, v/v) to afford 3 sub-fractions (M1A-M1C). Murrayafoline A (20.1 g) was obtained by re-crystallization of sub-fraction M1B (30 g) in cold n-hexane/ethyl acetate (10:1, v/v). The confirmed structure of Murrayafoline A agreed well with spectral data as described in references [10, 11]. Figure 1. Chemical structure of Murrayafoline A. Murrayafoline A: white crystal, C14H13NO; EI-MS m/z: 211 (100 %) (M+), 196 (M−CH3+); UV λmax (CHCl3) nm: 209, 222, 243, 291, 327, 340; 1H-NMR (500 MHz, CDCl3), δH (ppm): 8.12 (1H, br s, NH), 8.00 (1H, d, J = 7.4 Hz, H-5), 7.46 (1H, br s, H-4), 7.39 (1H, d, J = 7.4 Hz, H-8), 7.36 (1H, t, J = 7.4 Hz, H-7), 7.18 (1H, t, J = 7.4 Hz, H-6), 6.71 (1H, br s, H-2), 3.97 (3H, s, 1- OCH3), 2.52 (3H, s, 3-CH3); 13C-NMR (125 MHz, CDCl3), δC (ppm): 145.4 (C-1), 139.5 (C-8a), 129.5 (C-3), 128.0 (C-9a), 125.5 (C-7), 124.4 (C-4a), 123.6 (C-4b), 120.4 (C-5), 119.2 (C-6), 112.5 (C-4), 110.9 (C-8), 107.7 (C-2), 55.5 (1-OCH3), 21.9 (3-CH3). The remaining n-hexane residue (254 g) was subjected to steam distillation for 2 hours. The distillate was extracted with n-hexane/ethyl acetate (10:1, v/v). The organic layer was dried over Na2SO4, filtered and then concentrated under reduced pressure to afford fraction A (38.1 g). Murrayafoline A (28.6 g) was obtained by re-crystallization of fraction A in cold n-hexane/ethyl acetate (10:1, v/v). Thus, the yield of Murrayafoline A by separation using steam distillation is 1.5 times higher than column chromatography method. 3.2. Determination of content of Murrayafoline A by HPLC 3.2.1. Selection of detection wavelength Murrayafoline A shows UV absorption in the range from 200 to 400 nm (Figure 2). The maximum wavelength of 243 nm was set for HPLC monitoring of Murrayafoline A. Separation and HPLC quantitative analysis of murrayafoline A from the roots of Glycosmis... 533 Figure 2. UV absorption spectrum of Murrayafoline A. 3.2.2. Construction of calibration curve Table 1. Analysis of Murrayafoline A at different concentrations. Concentration (μg/mL) Peak height Peak area 10 6.3330 154875.8333 30 16.9631 439035.0000 50 30.0313 763078.3274 100 63.4159 1637177.0 30 150 94.3660 2423289.5790 200 127.9864 3297047.4810 300 179.9900 4822432.0833 Figure 3. The calibration curve of Murrayafoline A. Stock solution of Murrayafoline A was diluted with methanol to prepare seven samples with different concentrations. HPLC analysis of each sample was performed in triplicates. Calibration curve was established by linear regression analysis. The results are summarized in Table 1 and Figure 3. The calibration curve of Murrayafoline A was determined as y = 16285x – 20370, R2 = 0.9995. 3.2.3. Repeatability of method Table 2. Repeatability data of determination of Murrayafoline A. No Concentration (μg/mL) Peak area Average peak area RSD (%) 1 60 907308.3 909935.4 0.27 2 60 909506.6 3 60 913791.6 4 60 908381.5 5 60 910689.2 Abs nm 200 220 240 260 280 Tran Quoc Toan, Le Xuan Duy, Tran Thu Huong 534 The repeatability of the method was evaluated by assaying five replicate injections of Murrayafoline A at the same concentration (60 μg/mL), during the same day, under the same experimental conditions. The RSD values of the retention time, area, and height of Murrayafoline A peak were found to be < 0.3 %. The results are summarized in Table 2. 3.2.4. Accuracy Recovery test was used to evaluate the accuracy of the analysis. Dried powdered roots of G. stenocarpa were spiked with the known amount of standard before extraction. The mixtures were extracted and analyzed under the above-established method. Sample was analyzed in triplicate. For comparison, a blank sample (not spiked with standard compound) was prepared and analyzed. The average percentage recoveries were evaluated by calculating the ratio of detected amount vs added amount. As shown in Table 3, the recovery rates were in the range 99.7 - 100.1 %, and their RSD values were less than 2 %. Table 3. Recovery of Murrayafoline A from the methanol extract of G. stenocarpa. No Amount added (μg/mL) Average recovery (n = 3) (μg/mL) Recovery (%) RSD (%) 1 10.00 9.98 ± 0.16 99.8 1.63 2 20.00 19.94 ± 0.11 99.7 0.57 3 40.00 40.03 ± 0.19 100.1 0.48 3.2.5. Sample analysis Figure 4. Chromatogram of Murrayafoline A mixture with methanol extract from the roots of G. stenocarpa. The developed HPLC method was applied to determine the content of Murrayafoline A in G. Stenocarpa plant material and extracts. HPLC chromatograms are shown in Figure 4. Based on the regression equation, the content of Murrayafoline A was determined to be 0.38 % (w/w) in the roots and 16.69 % (w/w) in the methanol extract of G. stenocarpa. Murrayafoline A Separation and HPLC quantitative analysis of murrayafoline A from the roots of Glycosmis... 535 4. CONCLUSION In this study, we investigated two separation methods for the isolation of the bioactive compound Murrayafoline A from the roots of the Vietnamese plant Glycosmis stenocarpa and developed an HPLC method for the determination of its contents in plant material and methanol extracts. The yield of Murrayafoline A using steam distillation method was 1.5 times higher than that of the separate method using column chromatography. By HPLC method, the content of Murrayafoline A was determined to be 0.38 % (w/w) in the roots and 16.69 % (w/w) in the methanol extract of G. stenocarpa. Acknowledgements. This study was supported by the Institute of Natural Products Chemistry, VAST. REFERENCES 1. Choi H., Gwak J., Cho M., Ryu M. J., Lee J. H., Kim Y. H., Lee G. W., Yun M. Y., Cuong N. M., Shin J. K., Song G. Y., Oh S. T. - Murrayafoline A attenuates the wnt/beta- catenin pathway by promoting the degradation of intracellular beta-catenin protein, Biochem. Biophys. Res. 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Tran Quoc Toan, Le Xuan Duy, Tran Thu Huong 536 TÓM TẮT PHÂN LẬP VÀ PHÂN TÍCH ĐỊNH LƯỢNG BẰNG HPLC HỢP CHẤT MURRAYAFOLINE A TỪ RỄ CÂY CƠM RƯỢU TRÁI HẸP (GLYCOSMIS STENOCARPA) Trần Quốc Toàn1, Lê Xuân Duy1, Trần Thu Hường1, Tô Đạo Cường1, Trần Thị Thu Thủy1, Nguyễn Quang Tùng2, Bùi Hữu Tài3, Nguyễn Mạnh Cường , * 1Viện Hóa học các hợp chất thiên nhiên, Viện HLKHCNVN, 18 Hoàng Quốc Việt, Hà Nội 2Khoa Công nghệ Hóa, Trường Đại học Công nghiệp Hà Nội, Km 13, Đường 32, Minh Khai, Bắc Từ Liêm, Hà Nội 3Viện Hóa sinh biển, Viện HLKHCNVN 18 Hoàng Quốc Việt, Cầu Giấy, Hà Nội *Email: nmcuong_inpc@yahoo.com.vn Trong nghiên cứu này chúng tôi thực hiện 2 phương pháp phân lập và tinh chế hợp chất có hoạt tính sinh học Murrayafoline A từ rễ cây Cơm rượu trái hẹp (Glycosmis stenocarpa) của Việt Nam và xây dựng phương pháp định lượng hợp chất này bằng HPLC. Hiệu suất phân lập Murrayafoline A bằng phương pháp cất lôi cuốn hơi nước cao gấp 1,5 lần so với phương pháp sắc ký cột. Định lượng bằng HPLC, hàm lượng Murrayafoline A trong rễ khô cây Cơm rượu trái hẹp là 0,38 % và trong cao chiết methanol là 16,69 %. Từ khóa: Glycosmis stenocarpa Drake., Rutaceae, carbazole alkaloid, Murrayafoline A.