Isolation of hippadine from the bulbs of crinum latifolium L amaryllidaceae

Southeast-Asian J. of Sciences, Vol. 6, No. 1 (2018) pp. 73-79 ISOLATION OF HIPPADINE FROM THE BULBS OF CRINUM LATIFOLIUM L. AMARYLLIDACEAE Nguyen Thi Tuyet Nhung∗, Huynh Cong Tin∗, Phan Van Ho Nam†, and Vo Thi Bach Hue† Faculty of Pharmacy, Lac Hong University, Dong Nai, Vietnam e-mail: tuyetnhung@lhu.edu.vn † Faculty of Pharmacy Ho Chi Minh University of Medicine and Pharmacy Ho Chi Minh city , Vietnam e-mail: vothibachhue@gmail.com Abstract The purpose of study is extraction and isolation of hippadine from the bulbs of Crinum latifolium for using as a reference standard for quality control of herbs and hippadine-containing preparations. Bulbs powder of Crinum latifolium was percolated with 70% ethanol, percolate obtained was evaporated at 65 0C to give a liquid extract. The extract was frac- tionated by distribution with n-hexane. The n-hexane extract was then chromatographed on silicagel 60 (0.04 - 0.063 mm). Hippadine was iso- lated, purified and elucidated by NMR, MS, IR and UV-Vis spectrum. The purity of hippadine was determined by HPLC-PDA. About 565,3 mg of hippadine was isolated and its purity was detemined as pure as 100%. Hippadine was isolated with the purity suitable for using as ref- erence standard for quality control of herbs and hippadine-containing preparations. 1. Introduction Amaryllidaceae is a widely spread family on the world, it contains about 90 genera and 1310 species (Benson L., 1970). The genus Crinum is an important Key words: hippadine, Amaryllidaceae, Crinum latifolium, isolation. 73 74 Isolation of hippadine from the bulbs of... representative of family Amaryllidaceae with broad geographical distribution throughout the tropics, subtropics and warm moderate regions (Mabberly DJ., 2008). And Crinum latifolium L. (Amaryllidaceae) has been used in Asian folk and traditional medicine in the treatment of various illnesses like rheuma- tism, fistula, tumors, earaches, rubefacient, tubercle and whitlow, it’s also used in prostatitis adenoma, benign prostate enlargement (Ghosal S et al., 1985). In Vietnamese and Chinese traditional medicine, Crinum latifolium (CL) are used until nowadays because of its anti-inflammatory, anti-tumour and anti- microbial effects. These effects are mainly attributed to the presence of many different alkaloids in CL (Loi DT., 2000). Today, a large number of functional foods, especially drugs from CL are circulating in the Vietnamese market such as Crila, CrilaOPC, Tadimax, etc. Therefore, the management and quality control of these products is essential, the study was carried out with the aim of isolating hippadine from the bulbs of Crinum latifolium L. as a reference standard for quality control of herbs and hippadine-containing preparations. Materials and Methods Plants materials. The bulbs of CL were collected in Binh Dinh province, Vietnam, in March, 2016. The bulbs were identified and authenticated by Prof. Vo Thi Bach Hue, Analytical Department, Medicine and Pharmacy University of Ho Chi Minh city, Vietnam. After collection, the bulbs were cleaned and shade-dried in a cool place and then ground into a homogeneous powder. Instrumentation. The structure of the pure compound was characterized by spectral anal- ysis. UV-Vis spectroscopy was performed on Thermo Scientific Evolution 300. Fourier-transform infrared spectroscopy (IR) was performed on Shimadzu IRTracer-100. Mass spectroscopy (MS) was performed on Shimadzu LCMS- 8040. Nuclear magnetic resonance spectroscopy (NMR) was recorded on Bruker Avance 500 Mhz using CDCl3 solvent. The purity was determined by high per- formance liquid chromatography - photodiode array detector (HPLC - PDA) Agilent 1260. Methods. Bulbs powder of CL was percolated with 70% ethanol, percolate obtained was evaporated at 65 0C to give a liquid extract. The extract was fractionated by distribution with n-hexane. Isolation was performed by conventional column chromatography for the separation of the pure compound from n-hexane crude extract. The column was prepared using silicagel (0.40 - 0.063 mm) by wet packing method. The column prepared for the separation and isolation of the phytoconstituents should be N. T. Tuyet Nhung, H. C. Tin, P. V. Ho Nam, V. T. Bach Hue 75 properly grasped till the complete analysis. The sample was mixed uniformly and was introduced into the column using a long Pasteur pipette followed by addition of the eluent slowly from the top without disturbing the column. The eluent strength was changed from least polar solvent (n-hexane) system to polar solvent (methanol). The pure compound was isolated, purified and elucidated by NMR, MS, IR and UV-Vis spectrum. The isolated compound was determined its purity by thin layer chromatography with three different solvent systems and by high performance liquid chromatography (HPLC). Results and Discussion Extract and Isolation. Bulbs powder of CL (10 kg) was percolated with 70% ethanol. The ethanolic extract was heated to 65 0C to concentrate solution, give a 4200 g of concen- trated ethanolic extract. Then, the extract was dissolved in 500 ml of HCl 1%. The obtained acidic ethanolic solution was filtered through paper and then was fractionated by distribution with n-hexane. The n-hexane crude extract was chromatographed by conventional column chromatography. About 60,2 g of n-hexane extract was loaded into the column (85 × 7.5 cm) with 900 g of silica gel (40 - 63 μm) and mobile phases were n-hexane, chloroform, ethyl acetate and methanol. After the implementation of column chromatography, 10 fractions were obtained, denoted by F1-F10. From the F3 fraction (n-hexane-chloroform (60:40)), an amorphous solid called CL-1 was achieved. CL-1 was crystallized in n-hexane, washed several times with cold n-hexane. Finally, we obtained 565,3mg of a colorless crystal CL-1. Structure elucidation. CL-1 was isolated as a white amorphous solid. It was a poor polarized compound, soluble in chloroform, slightly soluble in methanol and reacted with Dragendorff reagent. UV-Vis spectrum in methanol for CL-1: λmax(nm) at 248, 299 and 348 nm FT-IR spectrum for the CL-1: IR ν(cm−1) 1668,43 (C=O); 1618,28 (C=C); 1024,2 (- O-CH2-O-); and 929,69 - 632 (C=C). MS spectrum for the CL-1: It had the chemical formula C16H9NO3 as de- termined by ESI-MS (+) ( 286,0405 m/z [M+Na]+, calcd for [C16H9NO3Na]+). NMR spectrum for the CL-1: 1H-NMR (125 MHz, CDCl3): δH (ppm) 6,14 (2H; s; H-12); 6,87 (1H; d; J = 3,5 Hz; H-4); 7,44 (1H; t; J = 7,5 Hz; H-2); 7,59 (1H; s; H-11); 7,71 (1H; d; J = 7,5 Hz; H-3); 7,86 (1H; d; J = 7,5 Hz; H-1); 7,94 (1H; s; H-8); 8,01 (1H; d; J = 3,5 Hz; H-5). 76 Isolation of hippadine from the bulbs of... The 1H-NMR spectrum displayed signals for five aromatic protons, two olefinic doublets, and a methylenedioxy group. Three of the five aromatic protons were determined to be in the same spin system due to the observed coupling constants from δH 7,86 (1H; d; J = 7,5 Hz), ?H 7,71 (1H; d; J = 7,5 Hz), and δH 7,44 (1H; t; J = 7,5 Hz). The doublet at δH 7,86 is most likely the result of unresolved meta coupling. The remaining two aromatic signals are both isolated singlets at δH 7,94 (1H; s), and δH 7,59 (1H; s). There was also a characteristic methylenedioxy resonance at δH 6,14 (2H, s). Lastly, the two olefinic protons were both coupled to one another with resonances at δH 6,87 (1H; d; J = 3,5 Hz) and δH 8,01 (1H; d; J = 3,5 Hz). 13C-NMR (500 MHz, CDCl3): δC (ppm) 101,7 (C-11); 102,3 (C-12); 108,0 (C- 8); 110,6 (C-4); 116,6 (C-11a); 118,3 (C-1); 122,5 (C-3); 122,6 (C-11b); 123,5 (C-5); 123,9 (C-2); 128,4 (C-3a); 130,9 (C-11c); 131,6 (C-7a); 148,5 (C-10); 152,5 (C-9); 158,1 (C-7). Comparison of the 1H-NMR and 13C-NMR literature data for hippadine (Thuy NHL., 2014) with the observed spectroscopic data confirmed the identity of CL-1 as the known compound hippadine. (Figure 1) Determination of the purity Thin layer chromatography was applied to test the purity of CL-1, with three different solvent systems: chloroform-n-hexane (1:4); toluene - acetone (3:1); n-hexane - acetone (7:3), detected by UV 365-254 and Dragendorff reagent, hippadine gave a single spot on the chromatograms. The isolated compound - hippadine was determined its purity by HPLC- PDA with chromatographic conditions: - HPLC system of Agilent 1260. - Column: Eclipse RP C18 (100 x 4.6 mm; 3.5 μm). - Mobile phase: Acetonitrile - acid H3PO4 pH 3 (46:54). - Flow rate: 1 ml / min. - Volume of injection: 10 μl. N. T. Tuyet Nhung, H. C. Tin, P. V. Ho Nam, V. T. Bach Hue 77 Table 2. NMR spectral data of isolated compound CL-1 and Hippadine Figure 1. Structure of hippadine 78 Isolation of hippadine from the bulbs of... - Column temperature: 30 0C. - Wavelength of detector: 299 nm. - Sample concentration: 10 μg / ml. Figure 2. HPLC chromatogram of hippadine The HPLC chromatogram of hippadine showed only one peak at 9,073 min (except the peak of solvent) with 100% purity. The result is shown in Figure 2. Conclusions In conclusion, 565,3 mg of hippadine was isolated and its purity was detemined as pure as 100%. Hippadine was isolated with the purity suitable for using as reference standard for quality control of herbs and hippadine-containing preparations. Hippadine is a very weak base, a poor polarized alcaloid of the indole frame- work. In the acidic environment, the hippadine in the form of salt is easily return to the form of alglycon, therefore, hippadine was isolated from the n- hexane extract in the acidic environment. N. T. Tuyet Nhung, H. C. Tin, P. V. Ho Nam, V. T. Bach Hue 79 References [1] L. Benson, “Handbook of Plant Classification”, 1970. [2] S. Ghosal, K.S. Saini, and S. Razdan, Crinum alkaloids: their chemistry and biology, Phytochemistry, 24(10) (1985), 2141-2156. [3] D.T. Loi, “Traditional Plants of Vietnam” (in Vietnamese), Ha Noi Medicinal Publisher, 2000, 515-516. [4] D.J. Mabberley, “The plant-book: a portable dictionary of the vascular plants”, Cam- bridge University press, 2008. [5] N. H. L. Thuy, “Research on chemical constituents, establish standard references and procedures for determination alkaloid and flavonoid of Crinum latifolium L., Amarylli- dacceae” (Doctoral dissertation, Ho Chi Minh University of Medicine and Pharmacy), 2014.