Cycloartanes from leaves of Combretum quadrangulare growing in Vietnam

Abstract Two new cycloartanes, combretic acid C (1) and combretanone I (3), were isolated from the leaves of Combretum quadrangulare Kurz, together with the previously-reported combretic acids A-B (2 and 5) and combretanone A (4). An extensive set of spectroscopic methods were used to elucidate the structures of these compounds. Cytotoxicity against the K562 cancer cell line was evaluated. Compound 1 showed strong activity, with an IC50 value of 9.7 µM. The other compounds showed moderate activity. Alpha-glucosidase inhibition was also evaluated. The isolated compounds showed moderate inhibition, with IC50 values in the range 102.2-194.7 µM. Graphical Abstract


Introduction
Combretum quadrangulare Kurz is widely used as a traditional medicine in Vietnam. This plant had a range of biological properties: hepatoprotective, antipyretic, analgesic, antidysenteric, and anthelmintic (Dao et al. 2021). Chemical analyses have confirmed the presence of numerous triterpenes (mostly cycloartanes) and flavonoids (Adnyana et al. 2000(Adnyana et al. , 2001Banskota et al. 1998Banskota et al. , 2000aBanskota et al. , 2000bDao et al. 2021;Ganzera et al. 1998;Nguyen et al. 2021aNguyen et al. , 2021bToume et al. 2011). Over 30 cycloartane-type triterpenoids have been isolated from leaves of C. quadrangulare and structurally elucidated (Banskota et al. 1998(Banskota et al. , 2000aGanzera et al. 1998;Nguyen et al. 2021aNguyen et al. , 2021bToume et al. 2011). In Vietnam, Nguyen and co-workers isolated two new cycloartanes, combretanones G and H (Nguyen et al. 2021a). These compounds showed potential cytotoxicity against the cancer cell lines K562, HepG2, and MCF-7 while combretanone H is a potent antiparasitic. Our previous bioactivity-guided isolation study reported the presence of twelve flavones. In the same research, three synthetic flavones showed potent alpha-glucosidase inhibition, in contrast with natural flavones, which are weak or inactive. An investigation of the most bioactive fraction of C. quadrangular identified the presence of a new cycloartane, norquandrangularic acid D (Nguyen et al. 2021b). Continuing our quest to extract bioactive compounds from the leaves of C. quadrangulare growing in Vietnam, a phytochemical study of the ethyl acetate extract was undertaken. This paper describes the isolation and structural elucidation of two new cycloartanes, combretic acid C (1) and combretanone I (3), and three known compounds, combretic acids A-B (2 and 5) and combretanone A (4) (Toume et al. 2011). The isolated compounds were evaluated for cytotoxicity against the K562 cancer cell line and for alpha-glucosidase inhibition.

Results and discussion
Compound 1, a colorless gum, had the molecular formula C 32 H 50 O 7 through its sodiated ion peak [M þ Na] 29.4, 21.9, 19.1, 18.6, 16.9, and 8.9), and nine methylenes (Table S1). The presence of the acetyl group was confirmed by HMBC correlation of the methyl at d H 1.98 with the carbonyl carbon at d C 170.8. These spectroscopic features indicated that 1 was a cycloartane-type triterpene with an additional acetyl group (Isaev et al. 1985;Khuong-Huu et al. 1975;Nguyen et al. 2021aNguyen et al. , 2021b. The planar structure was determined from a detailed analysis of COSY and HMBC experiments. HMBC correlations were found of the methyl at d H 1.13 (H 3 -29) with carbons at d C 180.2 (C-28), d C 75.0 (C-3), and 54.1 (C-4), and of the oxymethine at d H 4.11 (H-3) with carbons at d C 8.9 (C-29) and C-4. These findings located the hydroxy group at C-3 and the carboxylic acid group at C-4. The proton at d H 1.96 (H-8) gave HMBC cross peaks with carbons at d C 72.4 (C-7), 48.4 (C-14), and 19.1 (C-30), locating the acetyl group at C-7. This was supported by COSY correlations through H-5/H-6/H-7/H-8. These chemical features closely resembled those of combretic acids A (2) and B (5) (Toume et al. 2011). Careful comparison of NMR data from 1 with combretic acid B (2) (Toume et al. 2011) in the same deuterated solvent, chloroform-d, confirmed the consistency. Differences between 1 and 2 were found in the side chain, especially at CH-23, CH-24, C-25, CH 3 -26, and CH 3 -27. The olefinic protons H-23 and H-24 were shifted to lower fields (d H 5.51 and 5.38 in 1 vs. d H 5.40 and 5.15 in 2) while the methyl H 3 -27 was up-field shifted (d H 1.25 in 1 vs. 1.31 in 2). The 13 C NMR spectrum also showed significant differences in the side chain [d C 39.5 (C-22)/128.7 (C-23)/136.9 (C-24)/75.2 (C-25)/25.9 (C-26)/26.3 (C-27) in 1 vs 39.2 (C-22)/125.6 (C-23)/139.6 (C-24)/70.9 (C-25)/ 29.4 (C-26)/30.1 (C-27)]. This indicated the replacement of the hydroperoxy group -OOH at C-25 in 1 by the hydroxy group -OH in 2, a finding that was further supported by HRESI mass data. HRESI mass data indicated that 1 have one oxygen more than 2. The downfield chemical shift of C-25 was suggestive of the hydroperoxy group, which has been reported in triterpenes having the same side chain, e.g., euphorol L isolated from Euphorbia tirucalli (Duong et al. 2019) or quadrangularic acid F from C. quadrangulare (Banskota et al. 2000a). These phenomena could be found in related cycloartanes: methyl quadrangularate A (Banskota et al. 2000a), quadrangularic acid F (Banskota et al. 2000b), and musambin B (Lacroix et al. 2009). In 2000, Banskota and co-workers isolated methyl quadrangularates A and B from the methanolic extract of the Vietnamese leaves of Combretum quadrangulare. The only difference between two compounds is the substituent at C-25 ( Figure S21). These authors concluded that the 13 C difference of C-25 could determine the hydroperoxylated status of C-25 (d 80.9 in methyl quadrangularate A vs. 69.5 in methyl quadrangularate B) (Banskota et al. 2000a). In the same year, these authors used this conclusion to determine the peroxide group in quadrangularic acid F (Banskota et al. 2000b). In 2009, Lacroix and coworkers also determined the occurrence of the hydroperoxy group in musambin B ( Figure S21) by comparison of 13 C chemical shift of C-25 and HRESI mass data with those of the related compounds. The IR absorption band at 846 cm À1 of 1 also supported the presence the peroxide status of 1 (Vacque et al. 1997).
The .0, 18.6, and 17.6). These NMR data were reminiscent of those of combretanone A (4), indicating that they share the same planar structure (Table S2). The difference between them is the configuration of C-23 and C-24. This explained the obvious differences between 3 and 4 in the chemical shifts of CH-23, CH-24, and C-25 (Table S2). The J value of H-23 and H-24 of 3 was ca. 0 Hz, indicating the syn configuration of C-23 and C-24. A literature review confirmed that 23,24,25-trihydroxy-bearing cycloartanes with a 23,24-syn configuration would have the null J value, i.e., 23,24,25-trihydroxycycloartan-3-one (Joycharat et al. 2008), alisols A and P (Nakajima et al. 1994;Zhao et al. 2008), cumingianols A/B (Kurimoto et al. 2011), and cumingianosides G-J and L-M (Fujioka et al. 1997). The NMR data on the side chain of 3 were identical with those of combretanone B, indicating that they shared the same side chain (Toume et al. 2011). The absolute configuration of C-24 of 3 was proposed as 24 R, being similar to those of previously-reported 24-hydroxycycloartanes from the same bio-source: combretanones G and H (Nguyen et al. 2021a) or combretic acid B (5). Consequently, the chemical structure of 3 was elucidated as combretanone I.
Literature review of Combretum quadrangulare growing in Vietnam (Adnyana et al. 2000(Adnyana et al. , 2001Banskota et al. 1998Banskota et al. , 2000aBanskota et al. , 2000bDao et al. 2021;Nguyen et al. 2021aNguyen et al. , 2021bToume et al. 2011) indicated the presence of over 30 cycloartanes. All of them possess the C-10R configuration. From a biosynthetic perspective, compounds 1 and 3 should share the same 10 R configuration as the co-isolate combretic acid A (2) and combretanone A (4). Thus, the absolute configurations of C-3 and C-7 of 1 were proposed as 3S, 7 R. Likewise, 3 were proposed to have the (3S,7R, 23 R, 24R) configuration.
Compounds 1-5 were evaluated for a-glucosidase inhibition and cytotoxicity against K562. All tested compounds exhibited moderate a-glucosidase inhibition, with IC 50 values in the range 102. 2-194.7 lM, compared with the 360 lM of the acarbose positive control (Table S3). Compound 1 showed strong cytotoxicity toward the K562 cell line (IC 50 9.7 mM). The other compounds had lower cytotoxicity, with IC 50 values ranging from 20.2 to 51.3 lM (Table S4). The greater bioactivity of 1 indicated the important role played by the 24-hydroperoxy group in cytotoxicity.

General experimental procedures
The NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR) using residual solvent signals as internal references: chloroform-d at d H 7.26, d C 77.16. The HR-ESI-MS was recorded using an HR-ESI-MS MicrOTOF-Q mass spectrometer on an LC-Agilent 1100 LC-MSD Trap spectrometer. The IR spectrum was acquired using a Shimadzu FTIR-8200 infrared spectrophotometer. Thin layer chromatography (TLC) was carried out on precoated silica gel 60 F 254 or silica gel 60 RP-18 F 254S (Merck). Spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Gravity-column chromatography was performed on silica gel 60 (0.040-0.063 mm, Himedia).

Plant material
Leaves of Combretum quadrangulare were collected from Duc Hoa, Long An Province in March-April 2020 and identified as Combretum quandrangulare Kurz by Dr. Tran Cong Luan, Tay Do University, Vietnam. A voucher specimen (No UE-002) was deposited in the herbarium of the Department of Organic Chemistry, Faculty of Chemistry, Ho Chi Minh University of Education, Ho Chi Minh City, Vietnam.

Alpha-glucosidase inhibition assay
The a-glucosidase inhibition of 1-5 was determined using a method adapted from Dao et al. (2021). All samples were analyzed in triplicate at five different concentrations around the IC 50 values, and the mean values were retained.

Cytotoxicity assay
The cytotoxicity of 1-5 was evaluated against the K562 cancer cell line, as in our previous reports (Phan et al. 2021;Nguyen et al. 2021a).

Conclusions
Two new cycloartanes, combretic acid C (1) and combretanone I (3), were isolated from the leaves of Combretum quadrangulare Kurz and structurally elucidated. Three known compounds, combretic acids A-B (2 and 5) and combretanone A (4), were also isolated. The products were evaluated for alpha-glucosidase inhibition and cytotoxicity toward the K562 cell line. Compound 1 showed strong cytotoxicity with an IC 50 value of 9.7 mM. All compounds exhibited moderate alpha-glucosidase inhibition, with IC 50 values in the range 102.2-194.7 mM.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research is funded by Vietnam Ministry of Education and Training grant #B2021-SPS-03-HH). The study was supported by Van Lang University.