A New Polyoxygenated Cyclohexene From the Rhizomes of Globba pendula Roxb.

Abstract

A new polyoxygenated cyclohexene, globbanol A (1), 2 other cyclohexene derivatives, crotepoxide (2) and boesenboxide (3), along with a chalcone, 2ʹ- hydroxy-4,4ʹ,6ʹ-trimethoxy-chalcone (4) were isolated from the rhizomes of Globba pendula Roxb. (Zingiberaceae) of Vietnam. Their structures were elucidated on the basis of extensive analysis of high-resolution electrospray ionization mass spectrometry and NMR spectral data. Absolute configuration of new compound was determined by circular dichroism spectra. All these compounds are first reported from the genus Globba.

The genus Globba (100 species) is one of the largest genera in the primarily tropical Zingiberaceae and widely distributed from India and eastward through southern China, Indochina, and the Malaysian region. ^1,2 Previous phytochemical investigations on the genus Globba have revealed the presence of sesquiterpenes, diarylheptanoid, labdane diterpene, triterpene, benzofuran, phenolics, and steroids. ¹ Globba pendula Roxb. (called Ngải mọi in Vietnam) is a herb belonging to the genus Globba (Zingiberaceae). In Vietnamese traditional medicine, the rhizomes of G. pendula are used to treat rheumatism and osteoarthritis. ³ In Malaysia, people use the decoction of the rhizomes for postpartum women and deworming. In Indonesia, it has been used to treat flatulence. ^4,5 Very few studies on phytochemistry of G. pendula have been seen up to date. A report on chemical constituents presented the isolation of 10 compounds including labdane diterpene, benzofuran, phenolics, and steroids. ⁶ In our latest investigation, 25 compounds were identified from the essential oil of G. pendula by gas chromatography-mass spectrometry (GC-MS) with δ-selinene (22.09%) as the main component. The essential oil was found to possess moderate NO inhibitory effect with IC₅₀ of 41.68 ± 4.51 µg/mL and significant cytotoxic activity against MCF7 and Hep3B cell lines with IC₅₀ of 28.15 ± 1.08 and 35.24 ± 0.06 µg/mL, respectively. ⁷ In the present paper, we report the isolation and structural identification of a new polyoxygenated cyclohexene, globbanol A (1), together with 2 other cyclohexene derivatives, crotepoxide (2) and boesenboxide (3) and 2ʹ-hydroxy-4,4ʹ,6ʹ-trimethoxy-chalcone (4). The known compound structures were identified by 1D and 2D spectra (supplemental Figures S9-S22) in comparison with the literature data, ^{8 -10} and all these compounds (compounds 1-4) were isolated from the genus Globba for the first time.

Results and Discussion

Globbanol A (1) was obtained as a white amorphous powder. Its molecular formula was determined to be C₂₂H₂₂O₇ by quasi-molecular ion peak at m/z 399.1456 [M+H]⁺ (calcd. for C₂₂H₂₃O₇, 399.1444) and m/z 421.1260 [M+Na]⁺ (calcd. for C₂₂H₂₂O₇Na, 421.1263) in the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (supplemental Figure S1). The ¹H NMR spectra of 1 (supplemental Figure S2) showed signals of 3 oxymethines at δ _H 4.20 (1H, t, J = 5.0 Hz), 5.68 (1H, br. t, J = 3.0 Hz), 3.96 (1H, d, J = 3.5 Hz), 2 olefinic methines at δ _H 5.90 (1H, dd, J = 10.5, 3.0 Hz), 6.11 (1H, ddd, J = 10.5, 3.5, 1.5 Hz), an oxymethylene at δ _H 4.70 (1H, d, J = 12.0 Hz) and 4.79 (1H, d, J = 12.0 Hz), a methoxy group at δ _H 3.51 (3H, s) together with those for 2 benzoyl groups. The ¹³C NMR spectrum (supplemental Figure S3) revealed 22 carbon signals including a methoxy group (δ _C 58.24), 5 oxygenated carbons (δ _C 66.85, 71.62, 74.17, 75.86, 77.96), 2 olefinic carbons (δ _C 126.84 and 128.08), and 2 benzoyl groups (Figure 1). These predictions are completely based on the data obtained from the HSQC (Heteronuclear Single Quantum Coherence) HMBC (Heteronuclear Multiple Bond Correlation), and COSY (Correlated Spectroscopy) spectra (supplemental Figures S4-S6). In the ¹H-¹H COSY spectra, a cyclohexene ring structure was established by the correlations of neighboring protons including H-2 (δ _H 4.20)/H-3 (δ _H 5.68)/H-4 (δ _H 5.90)/H-5 (δ _H 6.11)/ H-6 (δ _H 3.96). The location of the methoxy group was at C-6 based on the HMBC correlation between the methoxy group (δ _H 3.51) and the oxymethine carbon at δ _C 77.96 (C-6). Besides, the long-range correlations from the oxymethylene protons at δ _H 4.70, 4.79 (H₂-7) to the quaternary oxygenated carbon at δ _C 75.86 (C-1) and 2 neighboring oxymethine carbon at δ _C 71.62 (C-2), 77.96 (C-6) suggested the connectivity of C-7 to C-1. Furthermore, HMBC correlations of the H₂-7 oxymethylene protons and the aromatic protons at δ _H 7.94 (H-2′,6′) with the ester carbonyl carbon at δ _C 166.95 indicated the location of the benzoyl group to be at C-7. However, no HMBC correlations of the other ester carbonyl carbon at δ _C 167.25 with 3 oxymethine protons were observed, suggesting that the other benzoyl group was at C-1. From the above evidence, the planar structure of 1 was identified (Figure 1). The relative configuration of 1 (Figure 2) was assigned based on NOESY (Nuclear Overhauser Effect Spectroscopy) correlations (supplemental Figure S7). The strong NOESY cross-peaks between H-2 (δ _H 4.20) and H-3 (δ _H 5.68), H-2 (δ _H 4.20) and H-7 (δ _H 4.70, 4.79) suggested that those protons are located on the same side of the cyclohexene ring, whereas the absence of any NOE correlations from H-2 and H-3 to H-6 (δ _H 3.96) revealed H-6 to be oriented toward the opposite side of the cyclohexene ring. Furthermore, the coupling constant of H-2 with H-3 (³ J _^2,3 = 5.0 Hz) also indicated the cis relationship between H-2 and H-3. The medium NOE observed for MeO-6 (δ _H 3.51) and H-7 (δ _H 4.70, 4.79) provided further confirmation for the C-6 configuration. The circular dichroism (CD) spectrum of compound 1 exhibited a positive Cotton effect at 233 nm (∆ԑ = +11.93) and a negative Cotton effect at 208 nm (∆ԑ = −5.48), indicating that 2 benzoyl groups were oriented in a clockwise manner (supplemental Figure S8; Figure 3). Therefore, the absolute configuration of globbanol A (1) was established as 1R,2S,3S,6S.

Figure 1

Key HMBC and COSY correlations of 1.

Figure 2

The relative structure of 1.

Figure 3

The chirality of 2 benzoyl groups of 1.

Experimental

General

High-resolution electrospray ionization mass spectrometry was performed on an LTQ Orbitrap XLTM mass spectrometer. The ¹H NMR (500MHz) and ¹³C NMR (125MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer and tetramethylsilane was used as an internal standard. Circular dichroism spectra were acquired on a Chirascan spectrometer, λ in nm (∆ԑ in mdeg). Column chromatography (CC) was performed using a silica gel (0.040-0.063 mm) and YMC RP-18 resins (30-50 µm). Thin-layer chromatography used precoated silica gel 60 F254 and RP-18 F254S plates, and the compounds were visualized by spraying with the solution of 10% H₂SO₄ in ethanol and heating for 1 to 3 minutes.

Plant Materials

The fresh rhizomes of G. pendula Roxb. were collected in An Giang province, Vietnam in February 2019 and were identified by Dr Nguyen Van Du, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). The voucher specimens (NGM-02.2019) were deposited in the Pharmaceutical Chemistry Laboratory, Institute of Natural Product Chemistry, VAST.

Extraction and Isolation

The dried rhizomes of G. pendula (1.5 kg) were powdered and extracted with methanol at 50°C (3 times, 2 hours per time) on a heated ultrasonic machine to give 75.0 g residue, which was then suspended in water and successively partitioned with n-hexane and ethyl acetate (EtOAc). The EtOAc fraction (32.0 g) was separated by silica gel CC using the gradient of n-hexane-EtOAc (100:0, 75:25, 50:50, 25:75, 0:100, v:v) to obtain 5 fractions (E1→E5). The fraction E1 (5.1 g) was chromatographed on a silica gel column and eluted with n-hexane-EtOAc (25:1) to give 3 subfractions (E1.1-E1.3). The subfraction E1.3 (0.90 g) was first separated by a silica gel column and eluted with n-hexane-acetone (20:1), then purified by using YMC RP-18 column with methanol-water (1:3) to yield compound 4 (8.2 mg). The fraction E2 (6.5 g) was separated on a silica gel column using n-hexane-acetone (15:1) to obtain 4 subfractions (E2.1-E2.4). The subfraction E2.1 (1.0 g) was passed through on a silica gel column using chloroform-acetone (25:1) to give 3 subfractions (E2.1.1-E2.1.3). The subfraction E2.1.2 was further subjected to a silica gel column using the solvent system of n-hexane-dichloromethane (12:1) as a mobile phase to produce compound 2 (10.0 mg) and compound 3 (6.5 mg). The fraction E3 (4.2 g) was applied to a silica gel column with a solvent mixture of n-hexane-EtOAc (15:1) to obtain 4 subfractions (E3.1-E3.4). Compound 1 (15.0 mg) was obtained from the subfraction E3.2 by using a silica gel column with dichloromethane-acetone (16:1) as eluent.

Globbanol A (1)

White amorphous powder.

R _f: 0.65 (CH₂Cl₂:EtOAc, 10:1).

CD (CDCl₃) ∆ԑ (λ nm): +11.93 (233), −5.48 (208).

¹H NMR (500 MHz, CDCl₃): 3.51 (3H, s, OCH₃) 3.96 (1H, d, J = 3.5 Hz, H-6), 4.20 (1H, t, J = 5.0 Hz, H-2), 4.70 (1H, d, J = 12.0 Hz, Ha-7), 4.79 (1H, d, J = 12.0 Hz, Hb-7), 5.68 (1H, br. t, J = 3.0 Hz, H-3), 5.90 (1H, dd, J = 10.5, 3.0 Hz, H-4), 6.11 (1H, ddd, J = 10.5, 3.5, 1.5 Hz, H-5), 7.38 (4H, td, J = 8.0, 2.0 Hz, H-3ʹ, H-5ʹ, H-3ʺ, H-5ʺ), 7.53 (2H, t, J = 7.5 Hz, H-4ʹ, H-4ʺ), 7.95 (2H, d, J = 7.5 Hz, H-2ʹ, H-6ʹ), 8.01 (2H, d, J = 7.0 Hz, H-2ʺ, H-6ʺ).

¹³C NMR (125 MHz, CDCl₃): 167.25 (C-7ʺ), 166.95 (C-7ʹ), 133.26 (C-4ʺ), 133.11 (C-4ʹ), 130.04 (C-1ʺ), 129.88 (C-2ʺ,6ʺ), 129.69 (C-2ʹ,6ʹ), 128.43 (C-1ʹ), 128.41 (C-3ʹ,5ʹ,3ʺ,5ʺ), 128.08 (C-5), 126.84 (C-4), 77.96 (C-6), 75.86 (C-1), 74.17 (C-3), 71.62 (C-2), 66.85 (C-7), 58.24 (OCH₃).

HR-ESI-MS: m/z [M+H]⁺ calcd for C₂₂H₂₃O₇: 399.1444; found: 399.1456, m/z [M+Na]⁺ calcd for C₂₂H₂₂O₇Na: 421.1263; found: 421.1260.

Conclusion

A new polyoxygenated cyclohexene namely globbanol A (1) together with 3 known compounds crotepoxide (2), boesenboxide (3), and 2ʹ-hydroxy-4,4ʹ,6ʹ-trimethoxy-chalcone (4) (Figure 4) was isolated from the rhizomes of G. pendula Roxb. (Zingiberaceae) of Vietnam. All these compounds are first reported from the genus Globba.

Figure 4

The structure of 3 known compounds.

Supplemental Material

Figure S1 - Supplemental material for A New Polyoxygenated Cyclohexene From the Rhizomes of Globba pendula Roxb.

Supplemental material, Figure S1, for A New Polyoxygenated Cyclohexene From the Rhizomes of Globba pendula Roxb. by Ngo Thi Phuong, Do Thi Thanh Huyen, Bui Thi Hai Hoa, Le Ngoc Hung and Le Minh Ha in Natural Product Communications