Chemical constituents from ethyl acetate extract of scoparia dulcis linn - Ton Nu Lien Huong

Journal of Science and Technology 54 (2C) (2016) 278-283 CHEMICAL CONSTITUENTS FROM ETHYL ACETATE EXTRACT OF SCOPARIA DULCIS LINN. Ton Nu Lien Huong *, Le Thi Diem Trinh, Van Nguyen Anh Duy, Le Thanh Cong, Nguyen Tan Qui, Nguyen Duy Thanh Department of Chemistry, College of Natural Science, Can Tho University, Campus 2, 3/2 Street, Ninh Kieu, Can Tho City, Vietnam *Email: tnlhuong@ctu.edu.vn Received: 30 May 2016; Accepted for publication: 23 October 2016 ABSTRACT This research focuses on the isolation and characterization of eight natural compounds from the ethyl acetate extract of Scoparia dulcis L. collected in An Giang province, Vietnam. Six flavonoid compounds: apigenin (1), luteolin (2), baicalein (3), oroxylin A (4), oroxylin A 7-O-β- D-glucoside (5) and vitexin (6) along with two nitrogen-containing compounds: 2,4-dihydroxy- 1,4-benzoxazin-3-one (7) and uracil (8) were successfully isolated via the methods of natural product extraction. The structures of these compounds were elucidated base on 1D-NMR and 2D-NMR spectral data as well as MS and compared with those in the authentic reports. This study contributed new results to the phytochemical examination of Scoparia dulcis Linn. growing in Vietnam with the presence of the first isolated compounds as (3), (4), (5), (7) and (8). Keywords: Scoparia dulcis Linn., ethyl acetate extract, apigenin, luteolin, baicalein, oroxylin A, vitexin, oroxylin A 7-O-β-D-glucoside, DIBOA, uracil. 1. INTRODUCTION The medicinal plant Scoparia dulcis L., commonly known as sweet broomweed or goatweed, is a species belonging to the Scrophulariaceae family. It is an edible herb mainly found in many tropical regions around the world such as Mexico, India, Thailand, Malaysia, etc. In Vietnam, Scoparia dulcis is naturally distributed from north to south; due to the valuable medicinal properties of this herb so that it has been cultivated throughout the country. Traditionally, all parts of the plant are used to treat various diseases. The roots are used by the people in Antilles to treat menorrhagia and gonorrhea [1]. Vietnamese and Chinese use this plant as an antidote. In folk medicine of China and Taiwan, some diseases like hypertension, hepatitis, bronchitis and gastric ulcers could also be cured with Scoparia dulcis Linn. [2]. Much research has been carried out to evaluate the bioactivities of Scoparia dulcis L. including cytotoxic, β-glucuronidase inhibitory, radical scavenging, anti-inflammatory, antiviral, antimicrobial, antifungal and diuretic activities [2 - 6]. Over 50 compounds, including flavonoids, terpenoids, benzoxazinoids, etc., have been isolated from Scoparia dulcis L. around the world Chemical constituents from ethyl acetate extract of Scoparia dulcis Linn. 279 [2], yet there is few researches focusing on this plant in Vietnam. Therefore, studying about the chemical composition as well as the bioactivities of this medicinal herb in Vietnam is a necessity. 2. MATERIAL AND METHOD 2.1. Plant material Scoparia dulcis Linn was collected in An Giang province, Vietnam in July 2015. The specimen was identified the scientific name and stored with the No 02-2015 at the Organic Chemistry lab, College of Natural Sciences, CTU. After washing, the air dried parts (leaves, flower and stem), except the root, were ground into fine powder. 2.2. Method 2.2.1. Extraction and isolation The plant powder (8 kg) was exhaustively extracted with methanol. The crude methanol extract (1.0 kg) was diluted with distilled water then partitioned in turn with petroleum ether, dichloromethane and ethyl acetate. The partitioned ethyl acetate solution was concentrated to afford ethyl acetate extract (75.0 g). This extract (60.0 g) was subjected on dry-column flash, eluted with the solvent system: n-hexane:ethyl acetate (30:70) to ethyl acetate:methanol (80:20), to afford 10 fractions (F1F10). Fraction F1 (2.4 g) was then subjected to an open column, eluted with n-hexane:ethyl acetate (5:5) to yield 1 (5 mg), 2 (6 mg) and 7 (5 mg). Fraction F2 (7 g) was also analysed with silica gel, using n-hexane:ethyl acetate (1:9) solvent system to afford 6 (4 mg) and 8 (5 mg). The rest amount of the extract (15.0 g) was subjected to another column, eluted with CH2Cl2:MeOH gradients to yield 4 fractions (P1  P4). Fraction P1 was concentrated to give crystals; 5 (3 mg) was afforded after recrystallizing. Fraction P4 was purified with open-column chromatography, using ethyl acetate: methanol gradients to yield 3 (2 mg) and 4 (3 mg). 2.2.2. Spectroscopic methods 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopy were recorded by a 500 MHz Bruker Avance spectrometer using tetramethylsilane (TMS) as the internal standard; ESI-MS was recorded with a VG 7070 Mass spectrometer operating at 70 eV. All spectroscopic methods were carried out at Institute of Chemistry, Vietnam Academy of Science and Technology. 3. RESULTS AND DISCUSSION 3.1. Compound 1 to 6 The 1H NMR and 13C NMR data of compound 1 to 6 suggest that all these six compounds have the flavone skeleton with six specific carbon signals (C-2, C-3, C-4, C-9, C-10 and C-1') as well as the proton signals of H-3 (1H; s) and aromatic protons. With the number of protons and carbons displayed in the spectral data, the coupling constants calculated and the ESI-MS results, compound 1, 2, 3 and 4 were identified as apigenin, luteolin, baicalein and oroxylin A, respectively (Fig.1). Because of the presence of five oxymethine and one oxymethylene carbon Ton Nu Lien Huong, Le Thi Diem Trinh, Van Nguyen Anh Duy 280 signals as well as the proton signals from δH (ppm) 3.00-4.00, compound 5 and 6 were determined as glucosides. However, both the anomeric carbon (73.3 ppm; C-1'') and the anomeric proton signal (4.71 ppm; H-1'') of 6 are upfield shifted, compared to those of 5 (100.2 ppm; C-1'') and (5.13 ppm; H-1''); thus, 6 was recommended as a C-glucoside, and 5 was identified as a O-glucoside. The aglycone spectra of compound 5 are very similar to those of oroxylin A, while the genin part spectra of compound 6 are analogous with those of apigenin; moreover, the HMBC spectroscopy of 6 shows the correlation between H-1'' and C-8. Therefore, compound 5 was recognized as oroxylin A 7-O-β-D-glucoside, and apigenin 8-C-β-D-glucoside (vitexin) was assigned to compound 6 (Fig.1). The spectral data of all these six compounds were also in agreement with those in published documents [7 - 10]. Figure 1. Chemical structures of compound 1 to 6. Apigenin (1): Yellow powder. ESI-MS: m/z 268.9 [M  H] (calcd. for C15H10O5 269.9). 1H NMR (DMSO-d6) δ ppm: 12.96 (1H; s; 5-OH); 6.77 (1H; s; H-3); 6.20 (1H; s; H-6); 6.49 (1H; s; H-8); 7.93 (2H; d; J = 8.5 Hz; H-2', 6'); 6.94 (2H; d; J = 8.0 Hz; H-3', 5'). 13C NMR (DMSO- d6) δ ppm: 163.7 (C-2); 102.8 (C-3); 181.7 (C-4); 161.4 (C-5); 98.9 (C-6); 164.5 (C-7); 94.0 (C- 8); 157.3 (C-9); 103.5 (C-10); 121.1 (C-1'); 128.4 (C-2', 6'); 116.0 (C-3', 5'); 161.2 (C-4'). Luteolin (2): Yellow powder. ESI-MS: m/z 284.9 [M  H] (calcd. for C15H10O6 285.9). 1H NMR (DMSO-d6) δ ppm: 12.96 (1H; s; 5-OH); 6.66 (1H; s; H-3); 6.18 (1H; s; H-6); 6.94 (1H; s; H-8); 7.39 (1H; brs; H-2'); 6.88 (1H; d; J = 8.0 Hz; H-5'); 7.40 (1H; dd; J1 = 8.0 Hz, J2 = 1.5 Hz; H-6'). 13C NMR (DMSO-d6) δ ppm: 163.9 (C-2); 102.9 (C-3); 181.6 (C-4); 161.5 (C-5); 98.8 (C-6); 164.1 (C-7); 93.8 (C-8); 157.3 (C-9); 103.7 (C-10); 121.5 (C-1'); 113.4 (C-2'); 145.7 (C- 3'); 149.7 (C-4'); 116.0 (C-5'); 119.0 (C-6'). Baicalein (3): Yellow needles. 1H NMR (DMSO-d6) δ ppm: 12.61 (1H; s; 5-OH); 6.92 (1H; s; H-3); 6.63 (1H; s; H-8); 8.05 (2H; d; J = 8.0 Hz; H-2', 6'); 7.57 (3H; m; H-3', 4', 5'). 13C NMR (DMSO-d6) δ ppm: 162.9 (C-2); 104.3 (C-3); 182.1 (C-4); 147.0 (C-5); 129.3 (C-6); 153.6 (C-7); 94.0 (C-8); 149.8 (C-9); 104.5 (C-10); 131.0 (C-1'); 126.3 (C-2', 6'); 129.1 (C-3', 5'); 131.8 (C-4') Oroxylin A (4): Yellow needles. 1H NMR (DMSO-d6) δ ppm: 12.92 (1H; s; 5-OH); 6.96 (1H; s; H-3); 6.63 (1H; s; H-8); 8.06 (2H; m; H-2', 6'); 7.59 (3H; m; H-3', 4', 5'); 3.76 (3H; s; 6 OCH3). 13C NMR (DMSO-d6) δ ppm: 163.2 (C-2); 104.6 (C-3); 182.2 (C-4); 152.6 (C-5); 130.7 (C-6); 157.8 (C-7); 94.4 (C-8); 152.7 (C-9); 104.6 (C-10); 131.5 (C-1'); 126.4 (C-2', 6'); 129.1 (C-3', 5'); 132.0 (C-4'); 59.9 (6OCH3). Oroxylin A 7-O-β-D-glucoside (5): Yellow crystal. ESI-MS: m/z 446.9 [M + H]+ (calcd. for C22H22O10 445.9). 1H NMR (DMSO-d6) δ ppm: 12.79 (1H; s; 5-OH); 7.05 (1H; s; H-3); 7.07 (1H; s; H-8); 8.10 (2H; d; J = 7.0 Hz; H-2', 6'); 7.62 (3H; m; H-3', 4', 5'); 3.78 (3H; s; 6OCH3); 5.13 (1H; d; J = 7.0 Hz; H-1''); 3.10  3.75 (6H; m; H-2''6''). 13C NMR (DMSO-d6) δ ppm: 163.7 (C-2); 105.0 (C-3); 182.5 (C-4); 152.3 (C-5); 132.2 (C-6); 156.7 (C-7); 94.5 (C-8); 152.4 (C-9); 106.0 (C-10); 130.6 (C-1'); 126.5 (C-2', 6'); 129.2 (C-3', 5'); 132.6 (C-4'); 60.3 (6OCH3); 100.2 (C-1''); 73.2 (C-2''); 77.3 (C-3''); 69.6 (C-4''); 76.7 (C-5''); 60.6 (C-6''). Chemical constituents from ethyl acetate extract of Scoparia dulcis Linn. 281 Vitexin (6): Yellow powder. ESI-MS: m/z 430.9 [M  H] (calcd. for C21H20O10 431.9). 1H NMR (DMSO-d6) δ ppm: 13.15 (1H; s; 5-OH); 8.01 (2H; s; H-2', 6'); 6.89 (2H; d; J = 8.5 Hz; H- 3', 5'); 6.75 (1H; s; H-3); 6.24 (1H; s; H-8); 4.71 (1H; d; J = 8.5 Hz; H-1''); 3.004.00 (6H; m; H-2'' 6''). 13C NMR δ ppm: 163.8 (C-2); 102.4 (C-3); 181.9 (C-4); 160.4 (C-5); 98.1 (C-6); 162.5 (C-7); 104.6 (C-8); 160.4 (C-9); 104.0 (C-10); 121.6 (C-1'); 128.9 (C-2', 6'); 115.8 (C- 3',5'); 161.1 (C-4'); 73.3 (C-1''); 70.9 (C-2''); 78.7 (C-3''); 70.5 (C-4''); 81.3 (C-5''); 61.3 (C-6''). 3.2. Compound 7 Compound 7 was obtained as brown yellow powder. The ESI-MS of 7 shows the pseudo- molecular ion peak at m/z 163.9 [M – H2O + H]+; this suggests the molecular formula would be C8H7O4N (181 amu). The 1H NMR (CD3OD) spectra displays four aromatic proton signals at δ ppm: 7.38 and 7.05 (1H; dd; J = 1.5 Hz and 8.0 Hz); 7.12 and 7.09 (1H; ddd; J = 1.5 Hz; 7.5 Hz and 8.0 Hz). The coupling patterns and coupling constants of these signals indicate the presence of a 1,2-disubstituted benzene ring. Two quaternary carbon signals at δC 142.5 and 129.7 ppm, together with the correlation between four aromatic protons above with these two carbons in HMBC show that the two atoms attaching to the benzene ring are oxygen and nitrogen. Moreover, with the degree of unsaturation calculated from the molecular formula (∆ = 6) as well as other carbon and proton signals, compound 7 was identified as 2,4-dihydroxy-1,4- benzoxazin-3-one (DIBOA). The result was enhanced by the correspondance of NMR data of compound 7 and those of 2-O-β-D-glucopyranosyl-4-hydroxy-1,4-benzoxazin-3-one (DIBOA- glc) in published data [10] (Table 1). Table 1. NMR spectral data of compound 7 in comparison with those of DIBOA-glc (D2O). Position 1H NMR δ ppm, (J, Hz) 13C NMR δ ppm 7 (CD3OD) DIBOA-glc 7 (CD3OD) DIBOA-glc 1, 4 - - - - 2 5.72 (1H; s) 5.89 (1H; s) 93.7 98.1 3 - - 160.1 157.5 5 7.38 (1H; dd; J = 1.5; 8.0) 7.09-7.48 (m) 114.4 115.5 6 7.12 (1H; ddd; J = 1.5; 7.5; 8.0) 7.09-7.48 (m) 123.8 126.6 7 7.09 (1H; ddd; J = 1.5. 7.5; 8.0) 7.09-7.48 (m) 125.6 124.8 8 7.05 (1H; dd; J = 1.5; 7.5) 7.09-7.48 (m) 118.5 118.4 9 - - 142.5 142.3 10 - - 129.7 129.1 Compound 7 (2,4-dihydroxy-1,4-benzoxazin-3-one) Compound 8 (pyrimidine-2,4(1H,3H)-dione) 7 5 O N 4 2 3 OH OH O N H1 2 NH 34 O O Ton Nu Lien Huong, Le Thi Diem Trinh, Van Nguyen Anh Duy 282 3.3. Compound 8 Compound 8 was obtained as yellow powder. The 1H NMR (DMSO-d6) spectra shows two secondary amine protons at 10.98 ppm and 10.97 ppm, along with two olefinic protons at 5.44 ppm and 7.38 ppm with the coupling constants of 7.5 Hz and 8.0 Hz, respectively. The 13C NMR and DEPT spectra indicates two quaternary and two tertiary carbons. Because of the chemical shifts of these carbons, 8 was assumed to be a heterocyclic compound. By comparing the spectral data of 8 with those of published literature [11] (Table 2), compound 8 was identified as pyrimidine-2,4(1H,3H)-dione (Uracil). Table 2. NMR spectral data of compound 8 in comparison with those of Uracil (DMSO-d6). Position 1H NMR (ppm, Hz) 13C NMR (ppm) 8 Uracil 8 Uracil 1 10.98 (1H; s; 1-NH) 10.98 (1H; s; 1-NH) - - 2 - - 151.5 151.5 3 10.97 (1H; s; 3-NH) 10.97 (1H; s; 3-NH) - - 4 - - 164.3 164.3 5 5.44 (d; J = 7.5) 5.44 (d; J = 7.5) 100.2 100.2 6 7.38 (d; J = 8.0) 7.39 (d; J = 8.0) 142.2 142.2 4. CONCLUSION Eight compounds were successfully isolated from the ethyl acetate extract of Scoparia dulcis L. and identified as apigenin, luteolin, baicalein, oroxylin A, oroxylin A 7-O-β-D- glucoside, vitexin, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and uracil. In this study, the presence of five compound as baicalein, oroxylin A, oroxylin A 7-O-β-D-glucoside, DIBOA and uracil were first report after isolating. The results have contributed to the chemical constituents of Scoparia dulcis L. growing in An Giang province, Vietnam. REFERENCES 1. Đỗ Tất Lợi - Những cây thuốc và vị thuốc Việt Nam, Nhà xuất bản Y học, Hà Nội, 2004, tr. 871-872. 2. Wu W. H., Chen T. Y., Lu R. W., Chen S. T., Chang C. C. - Benzoxazinoids from Scoparia dulcis (sweet broomweed) with antiproliferative activity against the DU-145 human prostate cancer cell line, Phytochemistry 83 (2012) 110-115. 3. Ahsan M., Islam S. N., Gray A. I., Stimson W. H. - Cytotoxic Diterpenes from Scoparia dulcis, Journal of Natural Products 66 (2003) 958-961. 4. Wankhar W., Srinivasan S., Rajan R., Rathinasamy S. - Evaluating in vitro antioxidant activity and GC-MS analysis of Scoparia dulcis Linn (Scrophulariacae), Journal of Applied Pharmaceutical Science 5 (2015) 029-034. 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Med. 2 (4) (2007) 149-153. 10. Hartenstein H. and Sicker D. - (2R)-2-β-D-Glucopyranosyloxy-4-hydroxy-2H-1,4-benzo xazin-3(4H)-one from Secale cereale, Phytochemistry 35 (3) (1994) 827-828. 11. Ngô H. Đ., Phùng V. T. và Nguyễn N. H. - Khảo sát thành phần hóa học của trái khổ qua (Momordica charantial), Tạp chí khoa học Đại học Cần Thơ 19a (2011) 53-59. TÓM TẮT THÀNH PHẦN HOÁ HỌC CAO ETHYL ACETATE CỦA SCOPARIA DULCIS LINN. Tôn Nữ Liên Hương*, Lê Thị Diễm Trinh, Văn Nguyễn Anh Duy, Lê Thành Công, Nguyễn Tân Quí, Nguyễn Duy Thanh Trường Đại học Cần Thơ, Khu 2, đường 3/2, Quận Ninh Kiều, Thành phố Cần Thơ *Email: tnlhuong@ctu.edu.vn Nghiên cứu này tập trung vào việc phân lập và nhận danh tám hợp chất tự nhiên từ cao ethyl acetate của cây Cam Thảo Đất Scoparia dulcis Linn. thu hoạch ở tỉnh An Giang, Việt Nam. Với các phương pháp tách chiết hợp chất sthiên nhiên thường qui, sáu hợp chất flavonoid là: apigenin (1), luteolin (2), baicalein (3), oroxylin A (4), oroxylin A 7-O-β-D-glucoside (5) và vitexin (6) cùng với hai hợp chất có nitrogen là: 2,4-dihydroxy-1,4-benzoxazin-3-one (7) và uracil (8) đã được cô lập thành công. Cấu trúc hóa học của các hợp chất được giải đoán căn cứ vào phổ NMR, MS cùng với đối chiếu dữ liệu trong tài liệu đáng tin cậy. Nghiên cứu đã đóng góp vào việc khảo sát hóa thực vật của loài Scoparia dulcis L. sinh trưởng tại Việt Nam với sự hiện diện của các chất lần đầu cô lập được từ nguyên liệu như (3), (4), (5), (7) và (8). Từ khóa: Scoparia dulcis Linn., ethyl acetate extract, apigenin, luteolin, baicalein, oroxylin A, vitexin, oroxylin A 7-O-β-D-glucoside, DIBOA, uracil.