5-hydroxymetylfurfural (1), bisdehydroneotuberostemonine (2) and a mixture containing neotuberostemonine (3) were isolated from Stemona tuberosa''s climbing plant. The chemical structure of these compounds is determined by 1-dimensional and 2- dimensional magnetic resonance spectroscopy methods as well as comparison with references.
Scientific Journal No35/2019 111 STUDY ON ISOLATING AND DETERMINING THE STRUCTURE OF SOME ORGANIC COMPOUNDS IN TUBERS OF STEMONA TUBEROSA IN CENTRAL HIGHLANDS OF VIETNAM Pham Van Cong Tay Bac University Abstract: 5-hydroxymetylfurfural (1), bisdehydroneotuberostemonine (2) and a mixture containing neotuberostemonine (3) were isolated from Stemona tuberosa's climbing plant The chemical structure of these compounds is determined by 1-dimensional and 2dimensional magnetic resonance spectroscopy methods as well as comparison with references Keywords: Stemona tuberosa, fural, ankaloids, stemonine, Central Highlands of Vietnam Email: phamvancongdhtb@gmail.com Received 19 October 2019 Accepted for publication 25 November 2019 PREAMBLE Stemona is one of the precious medicinal plants used to cure many folk diseases such as cough, skin diseases [1-3] With such effects, cypress in Vietnam has been studied and researched for a long time by scientists and has found many valuable compounds, especially alkaloids [4] Cypress tree is a shrub, grows wild in the plaints of Southern of Vietnam, Central Highlands of Vietnam, Northeast of Thailand According to the folk experience in the South and Central Highlands of Vietnam, people have used the roots of the cypress tree to treat many effective diseases such as skin diseases, liver cancer [1] In addition, the bulbs have the ability to make insecticides, termites The research on the trees in the Central Highlands of Vietnam is very limited, mostly only at the level of researching plant subspecies There have not been any researches on the chemical composition and biological activity of this tree in the Central Highlands 112 Ha Noi Metroplolitan University EXPERIMENTAL AND RESEARCH METHODS 2.1 Plant samples Stemona tuberosa tubers of climbing trees are collected from Central Highlands in October, 2013 Sample issued by PhD Do Huu Thu (Institute of Natural Resources and Environment Ecology, VAST) identifies the scientific name 2.2 Chemicals and equipment - Thin layer chromatography (TLC): Performed on a thin, pre-coated sheet of DCAlufolien 60 F254 (Merck-Germany) Detect the substance with ultraviolet light in two wavelengths 254 and 368 nm, sulfuric acid reagent (H2SO4 + methanol + vanillin) dried at a temperature of > 100 oC until the color appears - Column chromatography (CC) is carried out with normal phase adsorbents (Silica gel 60-160 and 240-430 mesh, Merck) - Nuclear magnetic resonance spectrum (NMR) was measured on the Bruker AM500 FT-NMR Spectrometer - MS, NMR spectra were measured at the Institute of Chemistry, Vietnam Academy of Science and Technology 2.3 Extraction and isolation of substances Cypress roots have a weight of 1.0 kilograms (dried, chopped), this amount of sample is soaked in solvent H2O: MeOH = 80:20 (5 liters) The sample was soaked for week, ultrasonic extraction several times Carry out the separation of the yellow solution and the residue The residue is further soaked in H2O: MeOH solvent Repeat several times until the filtered water remains only a pale yellow color The portion of solution used for distillation of the evaporator is high yellow, liquid, gelatinous (PVC) After that, the total alkaloid extract was treated in the direction of the alkaloids extraction yielding 5.2 grams of the total alkaloid The chromatographic column of silica gel column and crystallized were obtained with two pure substances, compound [PVC2E2 (10 mgrs)], compound [PVC2F2 (84 mgrs), and an alkaloid-rich fraction [PVC2E1 (48 mgrs)] RESULTS 3.1 Compound (PVC2E2) H NMR spectrum of compound (figure 3.1) has protons, including: - A proton of the -CHO group has a chemical shift of 9.61 ppm Scientific Journal No35/2019 113 Fig 3.1: 1H NMR spectrum of PVC2E2 compound - The two protons have a chemical shift at 7.22 and 6.52 ppm with a spin-spin separation constant J = 3.5 Hz, which is predicted to be two carbon-linked protons adjacent to each other Compared to reference [6] we predict contains furan rings - Two protons equivalent, with a chemical shift of 4.73 ppm are expected to be protons of the –CH2-O- group Fig 3.2: 13C NMR spectrum of PVC2E2 compound 114 Ha Noi Metroplolitan University - 13C NMR spectrum of compound (Figure 3.2) has a signal at 177.6 ppm which is attributed to carbon of –CHO group - A signal at 57.7 ppm is attributed to the carbon of the CH2-O group - Also there are sp2 hybrid carbon with a chemical shift of 160.4; 152.5; 122.4 and 110.0 ppm To determine the structure of this compound, we conducted 2D NMR spectroscopy which is HSQC and HMBC spectra - In the HSQC spectrum (Figure 3.3) there are the following correlations: + The 7.22 ppm signal is delivered with C at 122.4 ppm + The proton signal at 6.52 ppm intersects with the C at 110.0 ppm + The signal at 4.73 ppm intersects with the C at 57.7 ppm Finally, the structure of compound is determined by the HMBC spectrum (Fig 3.4) Among them are the following interactions between proton and carbon: Fig 3.3: HSQC spectrum of compound - Two protons at 4.73 ppm have a far interaction with C-4 (110.0 ppm) and C-5 (160.4 ppm), the –CH2OH group attaches to C-5 furan ring - Proton H-3 and H-4 have a long interaction with C-2 and C-5, thus deducing the group -CHO attached to C-2 Therefore, this compound contains furfural framework 115 Scientific Journal No35/2019 Fig 3.4: HMBC spectrum of compound So, compound is 5-hydroxymethylfurfural with the following structure: O CHO HO Compound structure Table 3.1: The NMR spectrum data of and compared with reference Order number Prussia 1H NMR Prussia 13C NMR Document [5] 152,5 7,22, d, 3,5 Hz 122,4 7,24, d, 3,4 Hz 6,52, d, 3,5 Hz 110,0 6,54, d, 3,4 Hz 160,4 2-CHO 9,61, s 177,6 9,62, s 5-CH2OH 4,73, s 57,7 4,75, s 3.2 Compound (PVC2F2) Compound No is purified by the method of recrystallization in ethanol solvent for white, needle-shaped crystals H NMR spectrum of compound (Figure 3.5) has the following signals: 116 Ha Noi Metroplolitan University Fig 3.1: 1H NMR spectrum of compound - There is a proton with a chemical shift of 5.93 ppm which is attributed as H attached to sp2 hybridized carbon - The two methyl groups, which are double bonded with secondary carbon, have a chemical shift of 1.32 and 1.35 ppm - A methyl group has the shape of a triplet, deducing that with the –CH2-CH3 group Analysis of 13C NMR spectrum of compound shows: - There are two groups of C = O with chemical shift at 178.7 and 178.8 ppm - There are carbon hybrid sp2 - There are two carbon bonded to oxygen at 80.8 and 71.7 ppm Comparing NMR spectrum data of compound with bisdehydroneotuberostemonine, we found a good match So compound is bisdehydroneotuberostemonine [5] Compound structure 117 Scientific Journal No35/2019 Table 3.2: The NMR spectrum data of and compared with reference Order number H NMR C NMR 5,93, s 108.6 4,21, dd, 6.0, 14.5 44.8 2.00, m 2,03, m 28.6 1,18, m 29.1 107.1 5,95, s 2,70, m 9a 108.7 137.1 4,20, ddd, 15.5, 5.0, 1.0 44.8 1.93, m 28.6 2,03, m 29.2 1,45, m 35.0 1,30, m C NMR [6] 1.30, m 1,52, m 13 3,73, dd, 15.0, 11.4 1,30, m H NMR [6] 137.5 3,70, dd, 14.0, 11.0 107.1 13 1.10, ddd, 11.7, 3.2, 8.2 35.1 1.30, m 36.0 2.73, m 127.5 36.1 127.8 10 1,99, m 34.8 1,90, m 34.9 11 4,70, ddm 2.0, 4.5 80.8 4,62 brd, 5.0 80.8 12 3,45, brt, 5.0 39.5 3,51, dd, 7.1, 5.0 39.6 13 2,97, m 41.9 2,95, dq, 7.1, 7.3 41.9 178.7 1,30, d, 7.3 178.7 14 15 1,30, d, 7.0 15.0 1,75-1,85, m 15.0 16 1,75-1,85, m 23.2 1,03, t (7.5) 23.2 17 1,06, t, 6.5 11.4 5,33, dd(5.2, 11.2) 11.4 18 5,53, dd, 5.0, 8.0 71.7 2.77, ddd (5.2, 5.4, 11.7) 71.7 19 2,70, m 30.9 2.15, ddd (11.7, 11.9, 11.2) 30.8 2,15, m 20 2,81, m 21 22 44.8 178.8 1,35, d, 7.0 11.8 2.80, ddq (5.4, 11.9, 6.9) 1.35, d (6.9) 44.8 178.7 11.8 3.3 Compound (PVC2E1) In addition, we isolated a segment of PVC2E1 that is rich in alkaloids We conducted a TLC test and column chromatography, but couldn't be purer Analysis of 1H NMR 118 Ha Noi Metroplolitan University spectrum (Figure 3.6) of this mixture showed that many signals coincide with the spectrum of neotuberostemonine (3) 15 CH3 O H H 14 13 O 11 H H 10 16 22 CH3 H N H H H 18 21 O O 17 CH3 Compound structure Thus, from the extract of the vines, S tuberosa collected from Central Highlands of Vietnam, we purified 5-hydroxymethylfurfural (1), bisdehydroneotuberostemonine (2) and a mixture containing neotuberostemonine (3) CONCLUSION By means of one-and-two-dimensional magnetic resonance spectroscopy, combined with reference, we have determined the structure of two compounds, 5hydroxymethylfurfural (1) and bisdehydroneotuberostemonine (2), recommended structure of neotuberostemonine (3) This result is completely consistent with the previous research results on alkaloids in tubers of climbing plants in Ha Nam of Vietnam [4] and Kunming of China [5] REFERENCES Do Tat Loi (1977), Vietnamese medicinal plants and herbs, - Science and Technology Publishing House, Hanoi, p.180-181 (in Vietnamese) Nguyen Tien Ban (2003), List of Vietnamese plant species 3, - Publishing house Agriculture (in Vietnamese) Scientific Journal No35/2019 119 Pham Hoang Ho (1999), Vietnamese Plants, Volume 3, - Young Publishing House (in Vietnamese) Pham Huu Dien, Pham Van Kiem, Luu Van Chinh and Chau Van Minh (2000), “Alkaloids from Stemona tuberosa Lour root tubers, - Journal of Chemistry, volume 38, pp.64-67 (in Vietnamese) Suqin Hu, Zhaofu Zhang, Jinliang Song, Yinxi Zhou and Buxing Han (2009), “Efficient conversion of glucose into 5-hydroxymethylfurfural catalyzed by a common Lewis acid SnCl4 in an ionic liquid”, - Green Chem., 2009, 11, pp.1746-1749 Yang Ye, Guo-Wei Qin and Ren-Sheng Xu (1994), “Alkaloids from Stemona tuberosa”, Phytochemistry, 37, pp.1201-1203 NGHIÊN CỨU PHÂN LẬP VÀ XÁC ĐỊNH CẤU TRÚC MỘT SỐ HỢP CHẤT HỮU CƠ TRONG CỦ CÂY BÁCH BỘ THÂN LEO (STEMONA TUBEROSA) Ở TÂY NGUYÊN Tóm tắt: 5-hydroxymetylfurfural (1), bisdehydroneotuberostemonine (2) hỗn hợp chứa neotuberostemonine (3) phân lập từ củ bách thân leo Stemona tuberosa Cấu trúc hóa học hợp chất xác định phương pháp phổ cộng hưởng từ hạt nhân chiều chiều kết hợp so sánh với tài liệu tham khảo Từ khóa: Stemona tuberosa, fural, ankaloids, stemonin, Tây Nguyên ... Thus, from the extract of the vines, S tuberosa collected from Central Highlands of Vietnam, we purified 5-hydroxymethylfurfural (1), bisdehydroneotuberostemonine (2) and a mixture containing neotuberostemonine... with the C at 110.0 ppm + The signal at 4.73 ppm intersects with the C at 57.7 ppm Finally, the structure of compound is determined by the HMBC spectrum (Fig 3.4) Among them are the following interactions... containing neotuberostemonine (3) CONCLUSION By means of one -and- two-dimensional magnetic resonance spectroscopy, combined with reference, we have determined the structure of two compounds, 5hydroxymethylfurfural