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Natural Product Research
Formerly Natural Product Letters
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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
171.2), indicating the location of O-acetyl group at C-3. The presence of a hydroxyl
group at C-6 was confirmed by detection of the HMBC cross-peaks between H-5 (d
0.85), H-7 (d1.49) and C-6 (d68.7). In addition, this fact was supported by the sequen- tial COSY correlations from H-5 (d0.85), H-6 (d4.53) to H-7 (d1.49 andd1.69).
The configuration at C-3 and C-6 stereogenic centres was deduced on the basis of NOESY measurement (Figure S10) and coupling constants of protons attached on them. The large coupling constant of Hax-3 (3Jax-ax ¼ 11.5 Hz) suggested an a-axial orientation for this proton, and thus the O-acetyl group at C-3 was assigned in a
b-equatorial position. This fact was confirmed by the NOESY cross-peak between H-3 (d 4.44) and H-5 (d 0.85). The NOESY correlations between H-6 (d 4.53) and H-5 (d
0.85), H-9 (d 1.61) supported the assignment of the axial position of the hydroxyl group at C-6. This result was proved by small coupling constants of H-6 (d4.53, brs),
Figure 1. The chemical structures of the isolated compounds1–4.
i.e. proton Heq-6 was split by a proton Hax-5 and two protons Hax-7 and Heq-7. Moreover, the broad singlet of H-6 would be attributed to the temperature during NMR experiment, which affects the relative conformer populations. As a result, the coupling constant changes. This pattern has been also observed in compound 2
(Figure S11) and the similar structure of 3b,6b,19a-trihydroxy-urs-12-en-28-oic acid (Calderon et al.2009). On the basis of the above spectroscopic evidence, the chemical structure of compound1 was elucidated as a new oleanane-skeleton triterpenoid, 3b- acetoxy-6b-hydroxyolean-12-en-28-oic acid, named coffecanolic acid.
The isolated compounds (1–4) were evaluated in vitro for a-glucosidase inhibitory activity. The activities are expressed as IC50(lM), and were compared with acarbose, used as a positive control. Compounds 1–4 inhibited a-glucosidase with IC50 values ranging from 83 to 203lM. In all cases, the compounds showed higher activity when compared with an acarbose standard (IC50¼ 209.8 ± 0.3mM). As summarized in Table S1, compounds 1 (IC50 ¼ 83.0 ± 1.2mM), 4 (IC50 ¼ 146.9 ± 1.2mM) bearing an acetyl group (CH3CO-) at C-3 exhibited higher activities than compounds 2 (IC50 ¼ 193.1 ± 0.6mM), 3 (IC50 ¼ 202.7.0 ± 0.9mM) having a hydroxyl (-OH) group. The pres- ence of hydroxyl group at C-6 resulted in a better activity against a-glucosidase. The highest inhibitory activity of the new compound 1 can be attributed to the presence of both an acetyl and a hydroxyl group at C-3 and C-6, respectively. Compound2con- tains a hydroxyl group at C-6 while this group is absent in3, and they displayed lower
activities in comparison with1, 4. This indicated that the acetylation on -OH group at
C-3 and the presence of -OH group at C-6 in oleanane-skeleton triterpenoids made a positive contribution to the antidiabetic activity.
3. Experimental
3.1. General experimental procedures
FT-IR spectrum was recorded on a Jasco FT-IR 4700 spectrometer (Jasco, Japan). NMR spectra were measured on a Br€uker Avance III spectrometer (Br€uker BioSpin, Switzerland, operating at 500 MHz for 1H and 125 MHz for 13C in CDCl3 as solvent). The residual solvent signals at dH 7.26 and dC 77.16 were referenced as an internal standard. The abbreviations of s (singlet), brs (broad singlet), dd (doublet of doublet) and t (triplet) were used to represent the patterns of the protons. HR-ESI-MS spectrum was recorded on a system of ExionLC UHPLC and X500R QTOF mass spectrometer (AB SCIEX, USA). Differential scanning calorimetry (DSC, NETZSCH DSC 214 Polyma, Germany): Temperature: 30 to 400C; Heating rate: 10C/min; Nitrogen flow: 60 mL/ min) was used for examination of melting point of the isolated compound. Column chromatography (CC) was performed on Merck normal phase silica gel (40–63mm). Thin-layer chromatography (TLC) was carried out on precoated silica gel 60 F254 plates from Merck (Darmstadt, Germany), and visualization of plates was carried out using a 10% H2SO4solution and heating. Sephadex LH-20 (25–100lm) was applied for gel filtration chromatography (GE Healthcare Bio-Sciences AB, Sweden). a-Glucosidase (EC 3.2.1.20) fromSaccharomyces cerevisiae(750 UN) and p-nitrophenyla-D-glucopyra- noside were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Acarbose and dimethyl sulfoxide were purchased from Merck (Darmstadt, Germany).
3.2. Plant material
The trunk of Coffea canephora was collected in Lam Dong province, Vietnam, in August 2018. Plant material was authenticated by Dr. Van Son Dang from Institute of Tropical Biology, Vietnam Academy Science and Technology, Vietnam. A voucher spe- cimen was on deposit as UTE–A001 in the herbarium of Ho Chi Minh City University of Technology and Education, Vietnam.
3.3. Extraction and isolation
The dried powdered trunk (30 kg) of Coffea canephora was macerated with MeOH at room temperature for 72 h. The methanolic solution was filtered, and the solvent was evaporated under reduced pressure to afford the total MeOH residue. The preparation of
n-hexane and EtOAc extracts was performed according to the routine method for extrac-
tion of plant materials, in which metabolites possessing different polarities were parti- tioned in solvents with different polarities (Goad and Akihisa 1997). The total MeOH residue (1.4 kg) was soaked in n-hexane and then in EtOAc. Then n-hexane and EtOAc
were evaporated in vacuo to yield n-hexane extract mass (H, 300 g) and EtOAc extract
mass (EA, 180 g), respectively. Then-hexane extract mass (H, 300 g) was firstly selected for
further investigation in our present work. It was fractionated by silica gel CC, using a gra- dient ofn-hexane/EtOAc (85:15 to 50:50). From this extract, seven main fractions (H1–H7) were collected on the basis of their TLC profiles. Fraction H4 (8.87 g), were separated by silica gel CC, using a gradient ofn-hexane/EtOAc (8:2 to 0:1) to afford seven subfractions
(H4.1–H4.7). Subfraction H4.3 (0.92 g) was subjected to CC using silica gel as a stationary phase and eluted successively with n-hexane/EtOAc (9:1) to yield 4 (20 mg). Subfraction H4.4 (0.71 g) was fractionated by silica gel CC using a gradient of CHCl3/acetone (100:0 to 97:3) to obtain three fractions (H4.4.1–H4.4.3). Subfraction H4.4.1 (0.043 g) was chromato- graphed on a silica gel CC using CHCl3/acetone (9:1) as eluent and finally purified by Sephadex LH-20 chromatography eluting successively with CHCl3/MeOH (1:4) as eluent to give1 (10 mg). Subfraction H4.4.3 (0.140 g) was purified by silica gel CC using as mobile phase 9:1 CHCl3/acetone, then via a Sephadex LH-20 column eluting with a mixture of CHCl3/MeOH (1:4) to afford3(81 mg).
Fraction H6 (5.47 g) was chromatographed over silica gel by column chromatography and eluted withn-hexane/EtOAc (7:3) to give seven fractions (H6.1–H6.7). Subfraction H6.5 (1.75 g) was applied on silica gel CC, using a gradient ofn-hexane/acetone (8:2 to 5:5) to
yield five fractions (H6.5.1–H6.5.5). Compound 2 (6 mg) was isolated from the subfraction H6.5.3 (0.870 g) by silica gel CC, eluting withn-hexane/EtOAc (9:1).
Coffecanolic acid (1), 3b-acetoxy-6b-hydroxyolean-12-en-28-oic acid: white amorph- ous powder; soluble in CHCl3; mp > 280C decomp; HR-ESI-MS (negative-ion mode):
m/z[M-H]- 513.3561, calcd 513.3580; FT-IRmmax(film) cm1: 3508, 2940, 1725, 1251;1H NMR (500 MHz, CDCl3); d5.31 (1H, t-like, 3.0 Hz, H-12), 4.53 (1H, brs, H-6), 4.44 (1H, dd, 11.5, 4.5 Hz, H-3), 2.82 (1H, dd, 14.0, 4.0 Hz, H-18), 2.05 (3H, s, H-20), 1.69 (overlapping, H-7a), 1.61 (overlapping, H-9), 1.49 (overlapping, H-7b), 1.32 (3H, s, H-25), 1.25 (3H, s, H- 24), 1.09 (3H, s, H-27), 1.06 (3H, s, H-26), 0.95 (3H, s, H-23), 0.92 (3H, s, H-30), 0.90 (3H, s, H-29), and 0.85 (1H, brs, H-5);13C NMR (125 MHz, CDCl3),d183.8 (C-28), 171.2 (C-10), 142.9 (C-13), 122.9 (C-12), 81.0 (C-3), 68.7 (C-6), 55.9 (C-5), 48.0 (C-9), 46.7 (C-17), 46.0 (C-19), 42.4
(C-14), 41.1 (C-18), 40.6 (C-7), 40.4 (C-1), 38.8 (C-8), 38.6 (C-4), 36.6 (C-10), 34.0 (C-21), 33.2 (C-29), 32.4 (C-22), 30.8 (C-20), 28.0 (C-15), 27.8 (C-23), 26.1 (C-27), 23.9 (C-11), 23.7 (C-16), 23.4 (C-2), 23.2 (C-30), 21.5 (C-20), 18.5 (C-24), 18.4 (C-26), and 17.2 (C-25).
3.4.a-Glucosidase inhibitory assay
The inhibitory activity of a-glucosidase was carried out as described previously by Apostolidis (Apostolidis et al. 2007) with some modifications. Samples 1–4 were dis- solved in dimethyl sulfoxide at different concentrations for thea-glucosidase inhibitory assay. The assay was performed in a 96-well plate with a total reaction volume of 200mL per well. The reaction components included 60mL 0.1 M phosphate buffer solu- tion (100 mM, pH 6.8), 20mL a-glucosidase (0.3 IU/mL), 20mL sample solution, 100mL p-
nitrophenyl a-D-glucopyranoside 200mM. The mixture incubated for 30 min at 37C. The reaction was terminated by adding 50lL of 50 mM sodium hydroxide solution. The solution without a test sample replaced by the buffer solution was used as the negative control, and acarbose was used as the positive control. Each experiment was carried out in triplicate. The absorbance of the sample, was measured with a BIOTEK microplate reader at 405 nm. The a- glucosidase inhibitory percentage, I (%), of the test sample was calculated through theEquation (1):
I ị ẳ% AcontrolAsample
Acontrol x100% (1)
where, Acontroland Asampleare the absorbances of the control and sample, respectively. The IC50(the half-maximal inhibitory concentration) was determined by the calibration curve equation between percentages of inhibition and sample concentration using Tablecurve software. IC50values are the mean values of three experiments.
4. Conclusion
A new compound, coffecanolic acid (1) and three known compounds, sumaresinolic acid (2), oleanolic acid (3), and 3-O-acetyloleanolic acid (4), were isolated from the n-
hexane extract of the trunk of Coffea canephora Pierre ex A.Froehner. The isolated compounds all exhibited stronger inhibitory activities of a-glycosidase than acarbose. Structure-activity relationship analysis indicated that the presence of acetyl and hydroxyl groups at C-3 and C-6, respectively, is responsible for higher inhibitory activ- ities against a-glycosidase. Remarkably, the new compound 1 showed better activity with an IC50 of 83.0lM when compared with acarbose (IC50 ¼209.8lM). Herein, we assigned the new compound1 as one of the active principles fora-glucosidase inhibi- tory activity in coffee trunk (Coffea canephora).
We deeply appreciate all who provided us with the possibility to complete this research.
Disclosure statement
The authors declare no conflict of interest.
Acknowledgements
This work belongs to the project grant No T2020-13TD funded by Ho Chi Minh City University of Technology and Education, Vietnam.
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