New Biphenyls from Garcinia multiflora

Abstract

Three new biphenyls were isolated from Garcinia multiflora. The structures of these biphenyls were elucidated by spectroscopic methods, and their rotavirus activity was evaluated.

Keywords:
Garcinia multiflora; biphenyls; anti-rotavirus activity

Introduction

Garcinia multiflora (clusiaceae) is a dioecious evergreen tree that grows to a height between 3-10 m in Southern China. This is a widely adaptable species growing in various habitats and at various elevations. The seeds yield up to 50% oil (seed pulp up to 55% oil), which is used for manufacturing soap or as a lubricant for machines.¹ The bark is used as an external medicine to reduce inflammation, whereas the timber is used for making furniture, boats, and woodcuts.¹ Previous phytochemical studies of Garcinia multiflora revealed the presence of xanthones,^2,3 benzophenone derivatives,^4-6 and biflavonoids,^2,7-9 and a wide range of biological activities such as anti-hepatocellular carcinoma activity,⁴ anti-inflammatory,⁵ anti-HIV,⁹ antioxidant,^3,10 and anti-tuberculosis activities have been reported for this species.⁸ Motivated by the search for new bioactive metabolites from local plants, we investigated the chemical constituents of the stems of Garcinia multiflora growing in Xishuangbanna Prefecture, leading to the characterization of three new biphenyls (1-3) (Figure 1), along with maclurin (4),³ 2,4,6,3’-tetrahydroxybenzophenone (5),³ 4-methoxybenzoic acid(6),¹⁰ luteolin(7),¹⁰ and apigenin (8).¹⁰ The anti-rotavirus activity of 1-3 has also been evaluated.

Figure 1
New biphenyls from Garcinia multiflora.

Experimental

General experimental procedures

UV (ultraviolet) spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning infrared (IR) with KBr pellets. 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on a DRX-500 NMR spectrometer with tetramethylsilane (TMS) as internal standard (IS). Chemical shifts (d) are expressed in ppm with reference to the solvent signals. Electrospray ionization mass spectra (ESIMS), electron ionization mass spectra (EIMS) and high resolution electron ionization mass spectra (HREIMS) were performed on a VG Autospec-3000 spectrometer. Semi-preparative high performance liquid chromatography (HPLC) was performed on a Shimadzu LC-8A preparative liquid chromatograph with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C₁₈ (20 mm × 25 cm) columns. Column chromatography (CC) was performed using silica gel (200-300 mesh, Qing-dao Marine Chemical, Inc.), Lichroprep RP-18 gel (40-63 μm, Merck), 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) and middle chromatogram isolated (MCI) gel (75-150 μm, Mitsubishi Chemical Corporation). Fractions were monitored by thin layer chromatography (TLC), and spots were visualized by heating silica gel plates sprayed with 5% H₂SO₄ in ethanol (EtOH).

Plant material

Garcinia multiflora was collected in Xishuangbanna Prefecture, Yunnan Province, People’s Republic of China, in September 2012. The identification of the plant material was verified by PhD Huang Jian-Ping. A voucher specimen (YNNU 2012-09-16) has been deposited in our laboratory.

Extraction and isolation

The air-dried and powdered stems and leaves of Garcinia multiflora (5.5 kg) were extracted four times with 80% aqueous ethanol (4 × 50 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (260 g) was extracted by ethyl acetate and decolorized by MCI gel. The portion of the extract soluble in 90% methanol (85 g) was chromatographed on a silica gel column eluting with a CHCl₃-acetone gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions (A-F). Fractionation of fraction B (9:1, 4.6 g) by silica gel CC, eluted with petroleum ether-acetone (9:1-1:2), yielded fractions B1-B7. Fraction B2 (8:2, 0.86 g) was subjected to silica gel CC using petroleum ether-acetone and semi-preparative HPLC (70% MeOH-H₂O) to give 4 (4.8 mg), 5 (6.2 mg), and 6 (3.9 mg). Fraction C (8:2, 1.23 g) was subjected to silica gel CC using petroleum ether-acetone and semi-preparative HPLC (50% MeOH-H₂O) to give 1 (5.7 mg), 2 (5.8 mg), and 3 (4.9 mg). Fractionation of fraction D (7:3, 2.5 g) by silica gel CC, eluted with petroleum ether-acetone (9:1-1:2), yielded fractions D1-D7. Fraction D3 (7:3, 0.85 g) was subjected to silica gel CC using petroleum ether-acetone and semi-preparative HPLC (45% MeOH-H₂O) to give 7 (4.2 mg), 8 (4.5 mg).

Multibiphenyl A (1)

Pale yellow gum; [α]_Dc 0.07, MeOH); UV (MeOH) l_max / nm (log ε) 570 (2.16), 205 (4.71); IR (KBr) n / cm^-1 3422, 2939, 1721, 1611, 1589, 1498, 1443, 1357, 1266, 1172, 1102, 1045, 1023, 838; ¹H and ¹³C NMR data (400 and 100 MHz, CD₃OD), see Table 1; ESI-MS (positive mode) m/z 381 [M + Na]⁺; EI-HRMS (M⁺) calcd.: 358.1416; found: 358.1408 (C₂₀H₂₂O₆). –11.0 (

Table 1
¹H and ¹³C NMR data for compounds 1-3 (d in ppm, 1 and 2 in CD₃OD, 3 in CDC1₃, 100 and 400 MHz)

No.	1		2		3
	δC (m) / ppm	δH (m, J , Hz) / ppm	δC (m) / ppm	δH (m, J , Hz) / ppm	δC (m) / ppm	δH (m, J , Hz) / ppm
1	129.6 s		132.4 s		132.3 s
2	119.7 s		114.2 s		112.4 s
3	144.9 s		142.0 s		141.5 s
4	135.0 s		131.9 s		132.7 s
5	147.2 s		149.6 s		144.8 s
6	105.9 d	6.26 (s, 1H )	106.6 d	6.44 (s, 1H)	105.5 d	6.43 (s, 1H)
7	133.8 s		134.6 s		132.7 s
8	131.7 d	7.00 (dd, 1H, J 8.8 Hz, 2.4)	131.8 d	7.11 (dd, 1H, J 8.4 Hz, 1.9)	130.3 d	7.16 (d, 1H, J 8.6 Hz)
9	115.7 d	6.74 (dd, 1H, J 8.8 Hz, 2.4)	116.0 d	6.82 (dd, 1H, J 8.4 Hz, 1.9)	114.8 d	6.86 (d, 1H, J 8.6 Hz)
10	157.1 s		157.7 s		155.5 s
11	115.7 d	6.74 (dd, 1H, J 8.8 Hz, 2.4)	116.0 d	6.82 (dd, 1H, J 8.4 Hz, 1.9)	114.8 d	6.86 (d, 1H, J 8.6 Hz)
12	131.7 d	7.00 (dd, 1H, J 8.8 Hz, 2.4)	131.8 d	7.11 (dd, 1H, J 8.4 Hz, 1.9)	130.3 d	7.16 (d, 1H, J 8.6 Hz)
1'	26.8 t	3.21 (d, 2H, J 6.7 Hz, CH2)	124.7 d	6.41 (d, 1H, J 10.1 Hz)	21.1 t	2.59 (t, 2H, J 6.6 Hz, CH2)
2'	130.7 d	5.44 (t, 1H, J 6.7 Hz)	124.4 d	5.48 (d, 1H, J 10.1 Hz)	33.0 t	1.72 (t, 2H, J 6.6 Hz, CH2)
3'	134.6 s		77.6 s		74.7 s
4'	71.5 t	4.33 (s, 3H, CH3)	69.0 t	4.27 (d, 1H, J 11.5 Hz, CH2)	26.7 q	1.39 (s, 3H, CH3)
				4.14 (d, 1H, J 11.5 Hz, CH2)
5'	14.0 q	1.39 (s, 3H, CH3)	23.4 q	1.47 (s, 3H, CH3)	26.7 q	1.39 (s, 3H, CH3)
3-OMe	56.5 q	3.76 (s, 3H, OCH3)	56.5 q	3.85 (s, 3H, OCH3)	56.1 q	3.86 (s, 3H, OCH3)
5'- OAc	172.9 s		172.6 s
	20.8 q	1.99 (s, 3H, COCH3)	20.7 q	2.00 (s, 3H, COCH3)

Multibiphenyl B (2)

Pale yellow gum; [α]c 0.05, MeOH); UV (MeOH) l_max / nm (log ε) 570 (2.32), 205 (4.36); IR (KBr) n / cm^-1 3430, 2926, 2930, 1720, 1609, 1578, 1495, 1442, 1351, 1263, 1178, 1109, 1049, 1020, 830; ¹H and ¹³C NMR data (400 and 100 MHz, CD₃OD), see Table 1; ESIMS (positive mode) m/z 379.1163 [M + Na]⁺; EI-HRMS (M⁺) calcd.: 356.1260; found: 356.1274 (C₂₀H₂₀O₆). –7.2 (

Multibiphenyl C (3)

Pale orange gums; [α]c 0.02, MeOH); UV (MeOH) l_max / nm (log ε) 570 (1.76), 266 (3.70), 226 (3.80), 204 (3.98); IR (KBr) n / cm^-1 3423, 2973, 2931, 1612, 1494, 1446, 1418, 1369, 1346, 1319, 1252, 1220, 1169, 1150, 1113; ¹H NMR and ¹³C NMR data (400 and 100 MHz, CDCl₃), Table 3; ESI-MS (positive mode) m/z 323 [M + Na]⁺; EI-HRMS (M⁺) calcd.: 300.1362; found: 300.1360 (C₁₈H₂₀O₄). (

Rotavirus bioassay

The human rotavirus Wa group was used to infect the cell culture MA104 in vitro, the 50% cytotoxicity concentration (CC₅₀) and half maximal effective concentration (EC₅₀) were evaluated.¹¹ Ribavirin was used as positive control. MA-104 cells (1 × 10⁵ cells per well) were grown in 96-well plates for 48 h. The media were removed and replaced by new media containing serial dilutions of compounds under test. After incubation for 72 h, the media were discarded, and 5 μL of MTT solution was added to each well. Plates were then incubated at 37 ºC for 4 h. The solution was removed, and 100 μL of 0.04 mol L^-1 HCl-isopropanol were added to each well to dissolve formazan crystals. Using a microplate reader, the absorbance of each well was measured at 540 nm. After subtracting the background absorbance at 655 nm, the 50% CC₅₀ of each compound was estimated by regression analysis.

In the mixed treatment assay, each compound was mixed with a 0.01 multiplicity of infection (MOI) of the rotaviruses at various concentrations (1-160 μg mL^-1) and incubated at 4 ºC for 1 h. The mixtures were inoculated in triplicates onto near confluent MA-104 cell monolayers (1 × 10⁵ cells per well) for 1 h with occasional rocking. The solution was removed and the cells replaced with eagles minimum essential medium (EMEM) containing 1 μg mL^-1 trypsin. The cells were incubated for 72 h at 37 ºC under 5% CO₂ atmosphere until the cells in the control showed complete viral cytopathic effect (CPE) by light microscopy. EC₅₀ was estimated by regression analysis.

Results and Discussion

Compound 1 was obtained as a pale yellow gum. The molecular formula was determined to be C₂₀H₂₂O₆ from the molecular ion peak [M]⁺ at m/z 358.1408 in the EI-HRMS. The IR spectrum indicated that 1 possesses hydroxy (3422 cm^-1), phenyl (2939, 1498 cm^-1), and carbonyl (1721 cm^-1) functional groups. The ¹H and ¹³C NMR spectra (Table 1) revealed the signals for a 1,2,3,4,5-pentasubstituted benzene ring [d_H 6.26 (1H, s, H-6); δ_C 129.6 (C-1), 119.7 (C-2), 144.9 (C-3), 135.0 (C-4), 147.2 (C-5), 105.9 (C-6)], one p-substituted benzene ring [d_H 7.00 (2H, dd, J 8.8, 2.4 Hz, H-8, H-12), 6.74 (2H, dd, J 8.8, 2.4 Hz, H-9, H-11); δ_C 133.8 (C-7), 131.7 (C-8, C-12), 115.7 (C-9, C-11), 157.1 (C-10)], one acetoxyprenyl group [d_H 3.21 (2H, d, J 6.7 Hz, H-1'), 5.44 (1H, d, J 6.7 Hz, H-2'), 4.33 (2H, s, H-4'), 1.39 (3H, s, H-5'), and 1.99 (3H, s, H-OAc); δ_C 26.8 (C-1'), 130.7 (C-2'), 134.6 (C-3'), 71.5 (C-4'), 14.0 (C-5'), 172.9, 20.8 (OAc)], and one methoxy group [d_H 3.76 (3H, s, OMe-5); δ_C 56.5 (OMe)], which implied that compound1 was a biphenyl derivative. This conclusion was confirmed by the heteronuclear multiple bond correlation (HMBC) correlations of H-6 with C-7, and of H-8 and H-12 with C-1 (Figure 2). HMBC correlations of H-1' with C-1, C-2, and C-3, and of H-2' with C-1 suggested the acetoxyprenyl group at C-2. The methoxy group was located at C-5 from the HMBC correlations of δ_H 3.76 (OMe) with C-5. Considering the signal for quarternary C-3, C-4, C-10 and the molecular formula of 1, three hydroxy groups were located at C-3, C-4, C-10, respectively. Thus, the structure of 1 was determined as shown (Figure 1), and named multibiphenyl A.

Figure 2
Selected HMBC (H→C) and ¹H-¹H correlation spectroscopy (COSY) (–) correlations of 1.

Compound 2 was obtained as a pale yellow gum and had the molecular formula C₂₀H₂₀O₆, as inferred from the EI-HRMS showing the molecular-ion peak at m/z 356.1274, indicating eleven degrees of unsaturation. The IR spectrum of 2 showed absorption bands at 3430 cm^-1 for free OH group. The ¹H NMR spectrum of compound 2 showed characteristic signals for an acetoxychromene ring, i.e., olefinic H-atoms at δ_H 6.41 (1H, d, J 10.1 Hz, H-1') and δ_H 5.48 (1H, d, J 10.1 Hz, H-2'), a Me group at δ_H 1.47 (s, Me-5'), an oxygenated methylene at δ_H 4.27, 4.14 (2H, d, J11.5 Hz, H-4'), and an acetoxy group δ_H 2.00 (s, OAc). A set of signals at δ_C 124.7 (C-1'), 124.4 (C-2'), 77.6 (C-3'), 69.0 (C-4'), 23.4 (C-5'), and 172.6/20.7 (OAc) in the ¹³C NMR spectrum provided further support for the presence of an acetoxy chromene system. The remaining unsaturation degrees suggested the presence of a typical biphenyl unit which was supported by the remaining 12 aromatic C-atom signals δ_C 132.4 (C-1), 114.2 (C-2), 142.0 (C-3), 131.9 (C-4), 149.6 (C-5), 106.6 (C-6), 134.6 (C-7), 131.8 (C-8, C-12), 116.0 (C-9, C-11), 157.7 (C-10). Further support for the determination of the structure was provided by the signal of H-6 showing a three-bond connectivity with C-7 (d_C 134.6), and H-8 and H-12 showing three-bond connectivities with C-1 in the HMBC plot. The HMBC experiment allowed to position the substituents at the chromene benzene ring. The H-1' signal at δ_H 6.41 showed a two-bond connectivity with C-2 (d_C 114.2), and a three-bond connectivity with C-1 (d_C 132.4) and C-3 (d_C142.0), whereas H-2' (d_H 5.48) correlated to C-2 in HMBC spectrum. Thus, the chromeme ring proved to be fused to the benzene ring at C-2 and C-3. The methoxy group was located at C-5 according to the HMBC correlation of OMe (d_H 3.85) with C-5 (d_C 149.6). A single H-atom at δ_H 6.44 (s, H-6) belonging to a 1,2,3,4,5-pentasubstituted benzene moiety showed a two-bond connectivity with C-1 (d_C 132.4) and C-5, and a three-bond connectivity with C-2 (d_C 114.2), and C-4 (d_C 131.9), which suggested that one OH group was attached to C-4. The existence of two phenolic OH groups was confirmed by the signals of two oxygenated quarternary aromatic C-atoms in the ¹³C NMR spectrum at δ_C 131.9 (C-4) and 157.7 (C-10), and the molecular formula of compound 2. The presence of the second benzene ring was deduced from the ¹H NMR spectrum, which showed a set of ds of orto-coupled H-atoms at δ_H7.11 (dd, J8.4 Hz, 1.9, H-8, 12) and 6.82 (dd, J 8.4 Hz, 1.9, H-9,11), typical for a para-substituted aryl moiety. Therefore, the other OH group was assigned to C-10. The configuration of C-3' was not assigned. Accordingly, the structure of compound 2 was determined as shown (Figure 1), and named multibiphenyl B.

Compound 3 was obtained as a pale orange gum, and its molecular formula was established to be C₁₈H₂₀O₄ as deduced by the EI-HRMS molecular ion peak (M⁺) at m/z 300.1360, indicating nine degree of unsaturation. The IR spectrum revealed absorption bands of hydroxyl (3423 cm^-1) and methyl (1446 cm^-1) groups. The connectivity of the protons and C-atoms was established by the heteronuclear single-quantum correlation (HSQC) spectrum. There were eighteen resolved peaks observed in the ¹³C NMR spectrum (Table 1), including signals for twelve aromatic carbons, one methoxy group, two methylenes, and two methyl carbons. Its ¹H NMR spectrum indicated the presence of twenty protons including five aromatic protons at δ_H6.43 (1H, s, C-6), 7.16 (2H, d, J 8.6 Hz, C-8), 6.86 (2H, d, J 8.6 Hz, C-9), and two methyl protons at δ_H 1.39 (6H, s, C-3' and C-4') and 3.86 (3H, s, OMe). Analysis of the NMR data, indicated that the spectra of 3 were similar to those of 2 except for the absence of an acetoxy group and one double bond and the appearance of two methylenes, thus suggesting that 3 and 2 possess the same substitution pattern of the benzene rings. The ¹³C NMR spectrum of 3, when compared to that of 2, displayed an upfield shifted signal for C-4' (d_C 26.7), the absence of signals for OAc, and upfieldshifted signals for C-1' (d_C 21.1) and C-2' (d_C 33.0). These data indicated the presence of two methyl groups placed at C-4, and the absence of double bond between 1' and 2'. The structure of 3 was then established as shown and named multibiphenyl C.

The new biphenyls 1-3 were tested for their ability to prevent the cytopathic effects of rotavirus in MA104 cells, and their effects were measured in parallel with the determination of antiviral activity using ribavirin as positive control, showing potent anti-rotavirus activity with therapeutic index (TI) above 10 (Table 2).

Table 2
Anti-rotavirus activity of compounds 1-3^a

No.	CC50b / (μg mL-1)	EC50c / (μg mL-1)	TId
1	125.72 ± 6.41	11.56 ± 1.13	10.93 ± 1.27
2	134.65 ± 8.34	10.94 ± 1.65	12.35 ± 1.75
3	159.83 ± 7.46	12.73 ± 1.75	12.58 ± 1.68
Ribavirin	274.27 ± 11.07	13.61 ± 1.04	20.14 ± 1.16

• ^a
  All results are expressed as mean ± SD (n = 3);
• ^b
  CC₅₀: mean (50%) value of cytotoxic concentration;
• ^c
  EC₅₀: mean (50%) value of effective concentration;
• ^d
  TI: therapeutic index, CC₅₀/EC₅₀.

Conclusions

Three new biphenyls (1-3) were isolated from Garcinia multiflora, whose structures were elucidated by spectroscopic methods. The three compounds presented a promising anti-rotavirus activity.

Supplementary Information

Supplementary data (¹H, ¹³C, HSQC, HMBC, ¹H-¹H correlation spectroscopy (COSY) NMR and mass spectrometry (MS) spectra of 1-3) are available free of charge at http://jbcs.sbq.org.br as PDF file.

https://minio.scielo.br/documentstore/1678-4790/WqQVd4jbCq8N9YSJCrdbyzg/2c1c8dd1f6b5a1d3aa38017fe0bc03233b2118dc.pdf

Acknowledgements

This research was supported by the National Natural Science Foundation of China (21002085 and 21362044), and start-up funds of Yunnan University of Nationalities.

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Publication Dates

• Publication in this collection
  Jan 2016

History

• Received
  13 May 2015
• Accepted
  15 Sept 2015