Triterpene Derivatives from Garcinia oligantha and Their Anti-Cancer Activity

Phytochemical investigations of leaves and twigs from Garcinia oligantha Merr. resulted in the isolation of five undescribed triterpene derivatives (1–5) and six known analogs (6–11). Their structures were determined based on extensive spectroscopic data and high-resolution mass spectra analyses. Compounds 1–11 were tested for their in vitro cytotoxicity against three human cancer cell lines (HeLa, HepG-2, and MCF-7). Compounds 1, 2, 8, and 11 exhibited broad and significant cytotoxicity against the tested cell lines with IC50 values ranging from 5.04 to 21.55 μM. Compounds 5 and 9 showed cytotoxicity against HeLa and MCF-7 with IC50 values ranging from 13.22 to 19.62 μM. The preliminary structure–activity relationship for the 11 isolated compounds is also discussed.

G. oligantha, a 1-3 m tall shrub, has been used as a traditional Chinese herbal medicine to treat fevers, toothaches, and scalds [40]. Previous phytochemical investigations of the plant led to the isolation of many xanthone derivatives with anti-cancer and/or antiinflammatory activities [4,10,41,42], but triterpenoids were rarely reported from it.

Isolation and Structure Elucidation of Triterpenoids
The dried leaves and twigs of G. oligantha were extracted with acetone (30 L × 72 h × 3) at room temperature. After removing the acetone under reduced pressure, the residue was extracted sequentially with petroleum ether (PE), dichloromethane (DCM), and methanol. The PE-soluble and DCM-soluble fractions were separated by silica gel, Sephadex LH-20 column chromatography, and semipreparative HPLC and purified by crystallization to yield 11 triterpene derivatives.
The molecular formula of compound 1 was determined as C 34 (Table 1) of 1 displayed signals corresponding to six tertiary methyls at δ H 1.13 (3H, s), 1.08 (3H, s), 0.92 (3H, s), 0.90 (3H, s), 0.74 (3H, s), and 0.72 (3H, s), and two acetyl groups at δ H 2.08 (3H, s) and 2.00 (3H, s), as well as one olefinic proton at δ H 5.26 (1H, t, J = 3.8 Hz). The 13 C NMR spectrum (Table 2) of 1 showed 34 carbon signals assigned to eight primary, ten secondary, six tertiary, and ten quaternary carbons, including two acetyl groups and the typical olefinic signals at δ C 144.0 and 122.2 for the double bond at C-12 (13) of an oleanane-type triterpene. The 1 H and 13 C NMR spectra of 1 were similar to those of arjunolic acid except for the presence of two acetyl groups instead of two hydroxy groups in arjunolic acid [51]. The HMBC spectrum showed cross-peaks from H-2 and H 3 -32 to C-31 and from H-3 and H 3 -34 to C-33, confirming two acetyl groups located at C-2 and C-3, respectively ( Figure 2). The stereochemistry of 1 was determined by analyzing the coupling constants and the NOESY experiment. The coupling constant (J = 10.7 Hz) between H-2 and H-3 indicated the orientation of two protons possessing dual-axial bonds. The NOESY spectrum showed the cross-peaks of H-2 with H 3 -24, H 3 -24 with H 3 -25, H 3 -25 with H 3 -26, and H-18 with H 3 -30, indicating the β orientation in these protons. The NOESY correlations of H-3 with H-5 and H 2 -23 and H-9 with H-5 and H 3 -27 suggested their α-orientation ( Figure 3). After the hydrolysis of 1, compound 1a was obtained, and its 1D NMR data and specific rotation were the same as those of arjunolic acid, which further confirmed the above conclusion [51,52]. Thus, the structure of 1 was determined, as shown in Figure 1, and named 2α,3β-diacetyl arjunolic acid.  tallization to yield 11 triterpene derivatives. The molecular formula of compound 1 was determined as C34H52O7 based on th HRESIMS data at m/z 571.3633 [M − H] -(calcd 571.3640). The 1 H NMR spectrum (Table 1 of 1 displayed signals corresponding to six tertiary methyls at δH 1.13 (3H, s), 1.08 (3H, s 0.92 (3H, s), 0.90 (3H, s), 0.74 (3H, s), and 0.72 (3H, s), and two acetyl groups at δH 2.08 (3H s) and 2.00 (3H, s), as well as one olefinic proton at δH 5.26 (1H, t, J = 3.8 Hz). The 13 C NMR spectrum ( Table 2) of 1 showed 34 carbon signals assigned to eight primary, ten second ary, six tertiary, and ten quaternary carbons, including two acetyl groups and the typica olefinic signals at δC 144.0 and 122.2 for the double bond at C-12 (13) of an oleanane-typ triterpene. The 1 H and 13 C NMR spectra of 1 were similar to those of arjunolic acid excep for the presence of two acetyl groups instead of two hydroxy groups in arjunolic acid [51 The HMBC spectrum showed cross-peaks from H-2 and H3-32 to C-31 and from H-3 an H3-34 to C-33, confirming two acetyl groups located at C-2 and C-3, respectively (Figur 2). The stereochemistry of 1 was determined by analyzing the coupling constants and th NOESY experiment. The coupling constant (J = 10.7 Hz) between H-2 and H-3 indicate the orientation of two protons possessing dual-axial bonds. The NOESY spectrum showe the cross-peaks of H-2 with H3-24, H3-24 with H3-25, H3-25 with H3-26, and H-18 with H3 30, indicating the β orientation in these protons. The NOESY correlations of H-3 with Hand H2-23 and H-9 with H-5 and H3-27 suggested their α-orientation ( Figure 3). After th hydrolysis of 1, compound 1a was obtained, and its 1D NMR data and specific rotatio were the same as those of arjunolic acid, which further confirmed the above conclusio [51,52]. Thus, the structure of 1 was determined, as shown in Figure 1, and named 2α,3β diacetyl arjunolic acid.

Isolation and Structure Elucidation of Triterpenoids
The dried leaves and twigs of G. oligantha were extracted with acetone (30 L × 72 h × 3) at room temperature. After removing the acetone under reduced pressure, the residue was extracted sequentially with petroleum ether (PE), dichloromethane (DCM), and methanol. The PE-soluble and DCM-soluble fractions were separated by silica gel, Sephadex LH-20 column chromatography, and semipreparative HPLC and purified by crystallization to yield 11 triterpene derivatives.
The molecular formula of compound 1 was determined as C34H52O7 based on the HRESIMS data at m/z 571.3633 [M − H] -(calcd 571.3640). The 1 H NMR spectrum (Table 1) of 1 displayed signals corresponding to six tertiary methyls at δH 1.13 (3H, s), 1.08 (3H, s), 0.92 (3H, s), 0.90 (3H, s), 0.74 (3H, s), and 0.72 (3H, s), and two acetyl groups at δH 2.08 (3H, s) and 2.00 (3H, s), as well as one olefinic proton at δH 5.26 (1H, t, J = 3.8 Hz). The 13 C NMR spectrum ( Table 2) of 1 showed 34 carbon signals assigned to eight primary, ten secondary, six tertiary, and ten quaternary carbons, including two acetyl groups and the typical olefinic signals at δC 144.0 and 122.2 for the double bond at C-12 (13) of an oleanane-type triterpene. The 1 H and 13 C NMR spectra of 1 were similar to those of arjunolic acid except for the presence of two acetyl groups instead of two hydroxy groups in arjunolic acid [51]. The HMBC spectrum showed cross-peaks from H-2 and H3-32 to C-31 and from H-3 and H3-34 to C-33, confirming two acetyl groups located at C-2 and C-3, respectively (  Figure 3). After the hydrolysis of 1, compound 1a was obtained, and its 1D NMR data and specific rotation were the same as those of arjunolic acid, which further confirmed the above conclusion [51,52]. Thus, the structure of 1 was determined, as shown in Figure 1, and named 2α,3βdiacetyl arjunolic acid.       (Table 2) showed 34 carbon signals, including three carbonyls at δ C 183.87, 172.32, and 171.03, and two olefinic carbons at δ C 143.8 and 122.4. The 13 C and 1 H NMR data of 1 and 2 are very similar, with major differences being the chemical shifts of C-2, and H-2 shifted upfield from δ C 69.6, and δ H 5.23 to δ C 67.6, and δ H 3.86; C-23 and H 2 -23 were shifted downfield from δ C 64.6, and δ H 3.37, 2.89 to δ C 65.5, and δ H 3.82, 3.65, respectively, suggesting that the acetoxy at C-2 in 1 was instead located at C-23 in 2. This finding was further supported by the HMBC correlation from H 2 -23 to C-31. We determined the relative configuration of 2 according to the NOESY correlations ( Figure 3) and the coupling constant (10.1 Hz) between H-2 and H-3. Hence, the structure of 2 was confirmed, as shown in Figure 1, and named 3β,23-diacetyl arjunolic acid.
Compound 3 was isolated as a white amorphous powder. The pseudomolecular ion peak at m/z 531.3694 [M + H] + in its HRESIMS suggested C 32 H 50 O 6 as its molecular formula. The 1 H NMR (Table 1) (Table 2) showed 31 signals, including a carbonyl at δ C 173.0 and two olefinic carbons at δ C 145.7 and 123.1, respectively. Comparing the 1D NMR data of 3 to those of tomentoid B (10) [48] indicated that the two compounds were closely related. The obvious spectroscopic difference between them resulted from the presence of the acetoxy group at C-2 in 3, instead of C-23 in 10. The shielded chemical shifts of C-23 and H 2 -23 of 3 further supported the above assignment. Its similar NMR data and same molecular formula as tomentoid B (10) indicated that 3 also had a carboxyl group at C-17, of which the carbon signal was missing. Fortunately, the carboxyl carbon signal was observed at 181.1 in the 13 C NMR spectrum measured in CDCl 3 ( Figure S25). The large proton spin-coupling constant (J = 10.8 Hz) of H-3 with H-2 suggests that the protons at C-2 and C-3 are trans-axial. Furthermore, the relative stereochemistry of 3 was established by the NOESY cross-peaks shown in Figure 3. Therefore, the structure of 3 was named 2α-acetyl arjunolic acid (Figure 1).
Compound 4 was obtained as a white amorphous powder. Its molecular formula was deduced as C 32 Table 1). The 13 C NMR spectrum ( Table 2) showed 32 carbon signals, including six methyls, two carbonyls, and two olefinic carbons. The 1 H and 13 C NMR spectra of 4 were similar to those of the known compound hovenic acid [53], with the most noticeable difference observed for the hydroxy at C-23 in hovenic acid replaced by an acetoxy in 4. The HMBC correlations from H 2 -23 and H 3 -32 to C-31 confirmed the above deduction. The stereochemistry of 4 was determined by analyzing the coupling constants and NOESY data. The large spin-coupling constant (J H-2, H-3 = 9.7 Hz) indicated that the 2,3-dihydroxyl groups should have a 2α,3βorientation, further supported by the NOESY correlations of H-2 with H 3 -24, and H-3 with H 2 -23 ( Figure 3). We can confirm the above conclusion by comparing the compound's specific rotation and NOESY spectrum to hovenic acid [53,54]. Thus, we determined the structure of 4 as 23-acetyl hovenic acid (Figure 1).
Compound 5 was obtained as a white amorphous powder. Its molecular formula was determined as C 34 H 52 O 7 based on the HRESIMS. The 1 H NMR spectrum (Table 1)

Evaluation of Biological Activity of Compounds 1-11
We tested the anti-tumor activities of compounds 1-11 against three cancer cell lines (HeLa, HepG-2, and MCF-7). Compounds 1-3, 10, and 11 are oleanane-type triterpenoids. Compounds 1, 2, and 11 with two acetoxys had stronger inhibitory activity than compounds 3 and 10 with one acetoxy, indicating that the more acetylated the hydroxyl groups, the stronger the activity.
Compounds 4-9 belong to lupane-type triterpenoids. Among them, compound 8 was the strongest inhibitor with IC 50 values of 5.04-9.76 µM, whereas compounds 6 and 7 exhibited no cytotoxicity to the three cell lines, indicating that 23-hydroxyl or 23-acetoxy group can increase inhibitory activity. Compound 4 exhibited moderate activity against HeLa and HepG-2 cell lines, while 5 and 9 had moderate activity against HeLa and MCF-7 cell lines (Table 3).

Discussion
In previous phytochemical investigations on the genus Garcinia, pentacyclic triterpenoids were rarely reported [55]. In this study, we isolated 11 pentacyclic triterpenoids, including five new natural products (1)(2)(3)(4)(5), from the leaves and twigs of G. oligantha. They belong to oleanane-and lupane-type triterpenes, most of which possess 23-acetoxyl or acetoxy groups. Compounds 1-11 were tested for their cytotoxic activity against HeLa, HepG-2, and MCF-7 cell lines. Compound 8 showed the highest anti-cancer activity against these three human cancer cell lines, with IC 50 values ranging from 5.04 to 9.76 µM. Among the five oleanane-type triterpenoids, the derivatives with two acetoxy groups showed more potent activity than those with one acetoxy group. For lupane-type triterpenes, the ones possessing 23-hydroxyl or 23-acetoxy groups were more active. The above results may support future investigations into the anti-tumor drug design of triterpenoids.

Extraction and Isolation
The air-dried leaves and twigs of G. oligantha (3.07 kg) were extracted with acetone (3 × 30 L) at room temperature to produce 387 g of dried resin, which was then extracted by PE, DCM, and methanol, successively.
The DCM-soluble part (128.0 g) was subjected to silica gel CC with a PE-EtOAc gradient system to produce six fractions (D1-D6   (1) Compound 1 (1.5 mg) was dissolved in methanol, and 1.5 µL of NaOH-H 2 O saturated solution was added and stirred at room temperature for 4 h. Then, the reaction solution was adjusted to pH 5-6 with 10% HCl and filtered to obtain a white solid 1a.

Cell Cultures
We determined the anti-cancer effects of compounds 1-11 using a CCK8 assay, as reported earlier [56]. The cells were seeded in a 96-well plate as 8 × 10 4 cells/mL (100 µL/well). In total, 100 µL/well of the serial dilutions of the tested compounds (50, 25, 12.5, 6.25, 3.125 µM) and adriamycin (2.5, 1.25, 0.625, 0.3125, 0.15625 µM) were added to the plate after the overnight incubation of the cells at 37 • C and 5% CO 2 . The cells were incubated for 48 h. Subsequently, 10 µL of CCK8 was added to each well, the plate was incubated for 1 h, and the absorbance of the wells was measured at 450 nm using a Biotech plate reader. Each experiment was repeated three times, and the standard deviation was calculated (±). The concentration that caused a 50% inhibition of cell growth (IC 50 ) was calculated for each compound.