Cao chiết IC50 (µg/mL) Cao n-hexane 13.5±0.3 Cao EtOAc 417.5±0.4 Cao MeOH 226.9± 0.3 Acarbose 135.4± 0.2
Bảng 3.3. Giá trị IC50 khả năng ức chế a-glucosidase của các hợp chất cô lập
Hợp chất IC50 (µM) HN1 83.0±1.2 Y01 193.1±0.6 HN2 202.7± 0.9 KK01 146.9± 1.2 Acarbose 209.8± 0.3
31
KẾT LUẬN
Từ thân cây cà phê robusta, Coffea canephora (Rubiaceae) thu hái ở Bảo Lộc, tỉnh Lâm Đồng, sử dụng phương pháp trích ly ngâm dầm ở nhiệt độ phòng, cùng các phương pháp sắc ký cột silica gel pha thường và sephadex đã cô lập được 4 hợp chất tinh khiết. Sử dụng các phương pháp hóa lý hiện đại như HR–ESI–MS, FT-IR, 1D và 2D–NMR và so sánh với các tài liệu tham khảo, chúng tôi đã xác định được cấu trúc hóa học của 4 hợp chất triterpene khung oleanane là coffecanolic acid (HN1), sumaresinolic acid (Y01), oleanolic acid (HN2), và 3-O-acetyloleanolic acid (KK01). Những hợp chất này lần đầu tiên được biết có hiện diện trong cây cà phê robusta,
Coffea canephora, hợp chất coffeanolic acid được cô lập lần đầu tiên trong tự nhiên.
Cao n-hexane và cả bốn hợp chất đều thể hiện hoạt tính ức chế a-glucosidase. Hợp chất HN1 có hoạt tính cao nhất và gấp 2.5 lần so với chứng dương acarbose (IC50 = 209.8±0.3 µM) trong thử nghiệm này.
Việc nghiên cứu cơ lập và xác định cấu trúc hóa học nhóm hợp chất triterpene khung oleanane nhằm cung cấp thêm thơng tin về thành phần hóa học của cây cà phê robusta, Coffea canephora (Rubiaceae) nói riêng và chi Coffea nói chung. Hoạt tính ức chế a-glucosidase của các hợp chất triterpene cô lập được cho thấy đây là nguồn dược liệu tiềm năng cho các nghiên cứu về chống bệnh đái tháo đường.
R1 R2
OAc OH 3β-Acetoxy-6β-hydroxy-olean-12-en-28-oic acid (HN1) OH OH Sumaresinolic acid (Y01)
OH H Oleanoic acid (HN2)
32 Ngồi ra, kết quả nghiên cứu cịn trở thành tài liệu cho các bài giảng chuyên môn về phân lập hợp chất hữu cơ và các phương pháp phổ nghiệm được giảng dạy tại Khoa Cơng nghệ Hố học và Thực phẩm, Trường Đại học Sư phạm Kỹ thuật Tp. Hồ Chí Minh. Nghiên cứu này góp phần nâng cao năng lực nghiên cứu cho nhóm tác giả trong lĩnh vực hóa học các hợp chất thiên nhiên, từ đó phục vụ đắc lực cho việc giảng dạy các môn học chuyên ngành tại trường.
Kết quả nghiên cứu đã được cơng bố trên tạp chí chun ngành quốc tế. Thơng tin nghiên cứu đăng tải trên bài báo góp phần chia sẻ kiến thức về thành phần hóa học của cây cà phê robusta, Coffea canephora cho những nhà nghiên cứu cùng hướng
chuyên ngành. Bên cạnh đó kết quả thử nghiệm hoạt tính sinh học trên các loại cao chiết và chất tinh khiết sẽ góp phần định hướng sử dụng hiệu quả thân cây cà phê, một nguồn sinh khối xem như bỏ đi sau thời gian thu hoạch trái cà phê. Bên cạnh đó đây cũng là thơng tin giúp các nhà nghiên cứu về tổng hợp hữu cơ đưa ra ý tưởng tổng hợp hoặc bán tổng hợp các hợp chất có hoạt tính sinh học từ phịng thí nghiệm, xa hơn là định hướng phục vụ cơng nghiệp dược phẩm.
Bài báo đăng trong tạp chí chuyên ngành quốc tế sẽ làm tăng uy tín học thuật cho trường Đại học Sư phạm Kỹ thuật TP. HCM.
Đề xuất hướng nghiên cứu tiếp theo:
Qua các kết quả nghiên cứu, chúng tơi nhận thấy rằng nhóm hợp chất triterpene là nhóm hợp chất hiện diện trong lồi cà phê Coffea canephora với nét đặc trưng của các cây thuộc lồi Coffea. Bên cạnh đó, nhóm hợp chất diterpene, cũng là nhóm hợp chất có nhiều hoạt tính sinh học tiềm năng và bước đầu phát hiện sự hiện diện của chúng trong qúa trình khảo sát. Do vậy, đề xuất hướng nghiên cứu tiếp theo cô lập và thử nghiệm hoạt tính sinh học nhóm hợp chất diterpene.
Kết quả nghiên cứu:
Kết quả nghiên cứu đã được công bố một bài báo trên Natural Product Research thuộc tạp chí chuyên ngành quốc tế trong hệ thống SCIE, Q2,
Minh Hao Hoang, Thi Anh Tuyet Nguyen, Nguyen Kim Tuyen Pham, Van Son Dang, Thi Nga Vo (2021), “A new oleanane-skeleton triterpene isolated from Coffea
33
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Phụ lục 1a: PHỔ HR-ESI-MS CỦA HN1
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Natural Product Research
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ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new oleanane-skeleton triterpene isolated from
Coffea canephora
Minh Hao Hoang, Thi Anh Tuyet Nguyen, Nguyen Kim Tuyen Pham, Van Son Dang & Thi Nga Vo
To cite this article: Minh Hao Hoang, Thi Anh Tuyet Nguyen, Nguyen Kim Tuyen Pham, Van Son
Dang & Thi Nga Vo (2021): A new oleanane-skeleton triterpene isolated from Coffea�canephora, Natural Product Research, DOI: 10.1080/14786419.2021.1921767
To link to this article: https://doi.org/10.1080/14786419.2021.1921767
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A new oleanane-skeleton triterpene isolated from Coffea canephora
Minh Hao Hoanga, Thi Anh Tuyet Nguyenb, Nguyen Kim Tuyen Phamc, Van Son Dangd and Thi Nga Voa
a
Department of Chemical Technology, Ho Chi Minh University of Technology and Education, Ho Chi Minh City, Vietnam;bDepartment of Chemistry, Ho Chi Minh University of Education, Ho Chi Minh City, Vietnam;cFaculty of Environmental Science, Sai Gon University, Ho Chi Minh City, Vietnam;
d
Institute of Tropical Biology, Vietnam Academy Science and Technology, Ho Chi Minh City, Vietnam
ABSTRACT
Extensive fractionation of n-hexane extract from the dried pow-
dered-trunks ofCoffea canephoraPierre ex A.Froehner (Rubiaceae) led to the isolation of a new oleanane-skeleton triterpene, coffe- canolic acid (1), along with three known analogues sumaresinolic acid (2), oleanolic acid (3), and 3-O-acetyloleanolic acid (4). The chemical structures were elucidated using FT-IR, 1D and 2D NMR and HR-ESI-MS data analysis. The isolated compounds were assayed for in vitroa-glucosidase inhibitory activity by determin- ing their half-maximal inhibitory concentration (IC50, mM). Compounds1–4 exhibited higher inhibitory activities when com- pared with acarbose, a positive control. Compound1 was found to be the most potent molecule against a-glucosidase, with the IC50 ¼ 83.0 ± 1.2mM, which improved by 2.5-fold over acarbose (IC50¼209.8 ± 0.3mM) in this assay.
ARTICLE HISTORY Received 15 February 2021 Accepted 19 April 2021 KEYWORDS Coffea canephora; triterpene; oleanane skeleton; NMR analysis; a-glucosidase inhibition 1. Introduction
Coffee is the most popular beverage worldwide, and clinical trials demonstrated that drinking coffee within usual levels can be likely to improve health benefits (Higdon and Frei 2006). Coffea canephora Pierre ex A.Froehner (known as Robusta coffee)
CONTACTThi Nga VO ngavt@hcmute.edu.vn
Supplemental data for this article can be accessed online athttps://doi.org/10.1080/14786419.2021.1921767.
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NATURAL PRODUCT RESEARCH
belongs to the genus Coffea, which comprises five species grown in Vietnam (Pham
2000). Robusta coffee is the primary coffee in Vietnam, and it contributes significantly to Vietnamese economy. The coffee beans and leaves are investigated from both the chemical and biological perspectives. The previous study reported the metabolites of caffeine, caffeic acid, trigonelline, alkaloids, chlorogenic acids, lipids, steroids and ter- penoids fromCoffea canephora (Oestreich-Janzen2019). Metabolites from coffee show a diversity of biological effects including the reduced risk of Alzheimer’s (Quintana et al. 2007) and Parkinson’s diseases (Hu et al. 2007), anti-inflammatory (Pergolizzi et al.2020), anti-oxidant activities (Natella et al. 2002; Svilaas et al.2004). Interestingly, the beneficial effect of coffee consumption on the reduced risk of type II diabetes (T2D) has been reported (Kim2015; Mellbye et al.2015; Herawati et al.2019).
Basically, different parts of a plant may have a chemical convergence. As a result, the similarity in bioactivities of metabolites is applicable. Furthermore, coffee tree is one of the perennial plants and its long-lasting growth impacts the biosynthesis trans- formation that results in the accumulation of interesting bioactive secondary metabo- lites in the trunk. Therefore, we focused on the investigation of chemical and biological perspectives of coffee trunk (Coffea canephora).
a-Glucosidase is an enzyme that breaks down carbohydrates to release glucose, which is absorbed in the small intestine. The inhibition of a-glucosidase delays the process of carbohydrate digestion and thus retards the liberation of glucose, which leads to chronic diabetes. As a result, this enzyme has been considered as an effective target for diabetic therapy. In recent years, many efforts have been made to discover effective a-glucosidase inhibitors from natural plants to develop lead compounds against diabetes (Kumar et al. 2011). In that respect, to identify substances in coffee having activities against diabetes, the work was intended to isolate and elucidate the structure of substances from coffee trunk (Coffea canephora). Subsequently, we tested the impact of pure compounds on a-glucosidase inhibition compared to an anti-dia- betic drug, acarbose.
2. Results and discussion
The dried and powdered trunk of Coffea canephora Pierre ex A.Froehner was macer- ated with methanol (MeOH) at room temperature (3days). The n-hexane and ethyl
acetate (EtOAc) extracts were prepared from the total methanolic extract. Purification of the n-hexane extract was performed using silica gel and Sephadex LH-20 column
chromatography to afford a new compound (1), along with three known compounds (2–4) (Figure 1), identified by comparison of their NMR data with published informa-
tion. Three known compounds were determined as sumaresinolic acid (2) (Calderon et al. 2009), oleanolic acid (3) (Mahato and Kundu 1994), and 3-O-acetyloleanolic acid (4) (Elujoba et al.1990).
Compound 1, obtained as a white amorphous powder, gave a pseudo-molecular
ion peak at m/z 513.3561 [M-H]- in the negative HR-ESI-MS, in agreement with a molecular formula of C32H50O5. The FT-IR spectrum of 1showed absorption bands cor- responding to hydroxyl (3508 cm1) and ester carbonyl (1725, 1251 cm1) groups. The
1
H NMR spectrum exhibited a doublet-doublet at d 4.44 (J¼11.5 and 4.5 Hz, H-3),
assigned to the oxygenated methine proton. A hydroxyl bearing methine proton appeared as a broad singlet atd4.53 (H-6). The most downfield triplet-like signal at d
5.31 (J¼3.0 Hz, H-12) was proposed to the olefinic proton. A remarkable doublet- doublet atd2.82 (J¼14.0 and 4.0 Hz, H-18) was assigned to the typical tertiary proton at C-18. The acetylation of the hydroxyl group on C-3 was deduced from a 3H singlet at d2.05 (H-20), while seven 3H singlets atd 1.32, 1.25, 1.09, 1.06, 0.95, 0.92 and 0.90 were ascribed to the C-25, C-24, C-27, C-26, C-23, C-30 and C-29 methyl protons, respectively. Briefly, an olefinic proton signal and seven methyl singlets in 1H NMR spectrum of1were characteristic of aD12oleanene skeleton.
The13C NMR spectrum showed resonances for 32 carbon atoms. The downfield res- onances at d 183.8 and 171.2 were assigned to the carboxyl and ester carbonyl car- bons. The C-13 and C-12 olefinic carbons were detected at d 142.9 and 122.9, respectively. The spectrum showed two oxygen-bearing carbons at d 81.0 and 68.7, suggesting the resonances of C-3 and C-6, respectively. The overall 13C NMR data were in good agreement with theD12oleanene skeleton of compound1 (Mahato and Kundu1994).
The COSY and HSQC experiments (Figures S5 and S6) were conducted to assign the
1
H NMR and13C NMR signals for the structure of compound1. Additionally, the HMBC
spectrum revealed correlations between various proton and carbon atoms (Figure S9). The H-20 (d 2.05) and H-3 (d 4.44) protons showed HMBC correlations with C-10 (d