Further report on the chemical constituents of the n-hexane extract of Leonotis nepetaefolia (L.) R. Br (Lamiaceae)

Abstract

Introduction: Leonotis nepetifolia (L.) R. Br, a species of the family Lamiaceae, has some interesting biological activities, such as antibacterial, antioxidant, and anti-inflammatory activities. This paper describes the structural elucidation of seven compounds isolated from Leonotis nepetifolia collected in Xuyen Moc district, Ba Ria–Vung Tau Province, in June 2018.

Methods: Phytochemical investigations of the n-hexane extract of Leonotis nepetifolia led to the isolation of seven pure compounds. Their chemical structures were elucidated by extensive MS, 1D and 2D-NMR spectroscopic analysis and comparison with previously published data.

Results: Seven compounds, namely, (E)-10-oxooctadeca-8-enoic acid (1), ergosterol peroxide (2), turmeronol A (3), methyl (E)-3-(3,4-dimethoxyphenyl)propenoate (4), methyl 4-hydroxybenzoate (5), methyl (E)-3-(4-hydroxy-3-methoxyphenyl)propenoate (6), and alpinumisoflavone (7), were identified.

Conclusions: Although these compounds are known in other species, this is the first time they have been reported in Leonotis nepetifolia.

INTRODUCTION

Leonotis nepetifolia (L.) R. Br (family Lamiaceae) is native to tropical Africa and southern India. In traditional medicine, L. nepetifolia is used to treat bronchial asthma, diarrhea, fever, rheumatism, and malaria as an analgesic in menstrual cramps 1 . Previous studies of this plant reported that extracts and compounds isolated from this species possessed interesting biological activities: antioxidant 2 , inhibition of MCF-7 (human breast cancer cell line) and Hep-2 (human larynx epithelioma cancer cell line) 3 , anti-inflammatory 4 , and antibacterial 5 activities. Previous phytochemical studies of L. nepetifolia showed the presence of laballenic acid, allenic acid, labdane diterpenoids, iridoids, and coumarins 6 , 7 , 8 , 9 .

In the search for chemical constituents of Leonotis nepetifolia , herein, we reported a continuation study on an n -hexane extract to isolate seven compounds: ( E )-10-oxooctadeca-8-enoic acid ( 1 ), ergosterol peroxide ( 2 ), turmeronol A ( 3 ), methyl ( E )-3-(3,4-dimethoxyphenyl)propenoate ( 4 ), methyl 4-hydroxybenzoate ( 5 ), methyl ( E )-3-(4-hydroxy-3-methoxyphenyl)propenoate ( 6 ), and alpinumisoflavone ( 7 ). Their chemical structures were elucidated by extensive MS, 1D and 2D-NMR spectroscopic analysis and comparison with previously published data.

MATERIALS AND Methods

General experimental procedures

HR–ESI–MS was recorded on an HR–ESI–MS MicrOTOF–Q mass spectrometer. The LC–MSD was recorded on an 1100 Series LC/MSD Trap SL mass spectrometer. The LC–MS/MS was recorded on a ^TM TSQ Quantum ^TM Access MAX triple quadrupole mass spectrometer. Optical rotations were measured on a Krüss (Germany) polarimeter with a tube length of 0.5 decimetres.

The ¹ H-NMR 500 (MHz), ¹³ C-NMR (125 MHz) and 2D-NMR spectra were recorded on a Bruker Avance 500 ^III spectrometer. Chemical shifts are expressed in ppm using a residual solvent signal as the internal reference (CDCl ₃ d _H 7.26, d _C 77.1).

Thin-layer chromatography (TLC) was carried out on precoated silica gel 60 F ₂₅₄ or silica gel 60 RP–18 F ₂₅₄ (Merck), and the isolated compounds were visualized by spraying with 5% vanillin/ethanol solution followed by heating. Column chromatography (CC) was performed by gravity using glass columns of appropriate sizes with silica gel (230–400 mesh RM7484-500G, Himedia) or Sephadex ^® LH-20 (Sigma–Aldrich).

Plant material

The Leonotis nepetifolia plant was collected in Xuyen Moc district, Ba Ria–Vung Tau province, in June 2018. The scientific name of the plant was authenticated by M.Sc. Hoang Viet, Department of Ecology and Evolutionary Biology, Faculty of Biology, University of Science, Vietnam National University Ho Chi Minh City.

Extraction and isolation

Air-dried parts of Leonotis nepetifolia (46 kg) were ground and extracted with methanol by the maceration method at room temperature. After filtering the solution, the solvent was evaporated under reduced pressure to obtain a crude methanolic extract (1.7 kg). This crude was used to prepare the n -hexane extract using the liquid–liquid partition method. The -hexane extract (460 g) was subjected to silica gel column chromatography (CC) eluted with n -hexane:ethyl acetate (stepwise, 99:1, 98:2, 95:5, 90:10, 50:50, 0:100, v/v) and ethyl acetate:methanol (stepwise, 99:1, 98:2, 95:5, 90:10, 50:50, 0:100, v/v) to afford 32 fractions (H1-H32). This paper reports the purification of three fractions, H2, H6, and H8. Fraction H2 (31.0 g) was divided into eight subfractions (H2.1-H2.8) by silica gel CC using the mobile phase n -hexane:ethyl acetate (9:1, 8:2, 5:5, 0:10, v/v). Subfraction H2.8 (3 g) was repeatedly separated by preparative TLC to afford 1 (18 mg). Fraction H6 (8.28 g) was first chromatographed on Sephadex LH-20 to give ten subfractions (H6.1-H6.10). Subfraction H6.10 (900 mg) was subjected to silica gel CC eluted by n -hexane:acetone (9:1) to afford 2 (5 mg). Fraction H8 (8.0 g) was subjected to silica gel CC and eluted by n -hexane:acetone (9:1, 8:2, 5:5, 0:10) to give five fractions: H8.1 (93 mg), H8.2 (193 mg), H8.3 (1 g), H8.4 (698 mg), and H8.5 (2.7 g). Subfraction H8.4 (698 mg) was rechromatographed by silica gel CC and eluted with n -hexane:chloroform (8:2) to afford two compounds 3 (7 mg) and 4 (13 mg). The same procedure was applied to fraction H8.5 (2.7 mg) eluted by n -hexane:acetone (stepwise, 99:1, 98:2, 95:5) to obtain 5 (10 mg), 6 (4 mg) and 7 (20 mg), respectively.

RESULTS

The chemical investigation of the -hexane extract of Leonotis nepetifolia (L) R. Br led to the isolation of seven compounds whose physical properties are presented in the following. The ¹ H and ¹³ C-NMR data of compounds 1 , 2 , and 7 are presented in Tables 1 and 3 , and compounds 3 ‒ 6 are reported in Tables 2 and 3 .

• ( E ) - 10 - Oxooctadeca - 8 - enoic acid ( 1 ): White wax. ESI-MS/MS (negative mode) m/z 295.1 [M-H] ^- (calcd. for C ₁₈ H ₃₂ O ₃ -H, 295.2).

• Ergosterol peroxide ( 2 ): White needles, mp 179–181 °C. LC–MSD (positive mode) m/z 429.0 [M+H] ⁺ (calcd. for C ₂₈ H ₄₄ O ₃ +H, 429.3).

• Turmeronol A ( 3 ): Colorless oil. HR-ESI-MS (negative mode) m/z 231.1386 [M-H] ^- (calcd. for C ₁₅ H ₂₀ O ₂ -H, 231.1385). +248 ( c 0.43, acetone).

• Methyl ( E )-3-(3,4-dimethoxyphenyl)propenoate ( 4 ): Colorless needles, mp. 69–70 °C. HR-ESI-MS (positive mode) m/z 223.0963 [M+H] ⁺ (calcd. for C ₁₂ H ₁₄ O ₄ +H, 223.0970).

• Methyl 4-hydroxybenzoate ( 5 ): White crystalline solid, mp. 112–115 °C. HR-ESI-MS (negative mode) m/z 151.0397 [M-H] ^- (calcd. for C ₈ H ₈ O ₃ -H, 151.0395).

• Methyl ( E )-3-(4-hydroxy-3-methoxyphenyl)propenoate ( 6 ): Colorless needles, mp. 64–65 °C. HR-ESI-MS (positive mode) m/z 207.0663 [M-H] ^- (calcd. for C ₁₁ H ₁₂ O ₄ -H = 207.0657).

• Alpinumisoflavone ( 7 ): Pale yellow needles, mp. 210–213 °C. HR-ESI-MS (negative mode) m/z 335.0914 [M-H] ^- (calcd. for C ₂₀ H ₁₆ O ₅ -H, 335.0919).

Figure 1 . Chemical structures of seven isolated compounds

DISCUSSION

Compound 1 was obtained as white wax. The ¹ H-NMR spectrum of 1 displayed signals of two olefinic protons at δ _H 6.82 (1H, dt , 16.0, 6.5 Hz, H-8) and 6.08 (1H, d , 15.5 Hz, H-9). The large coupling constants of 16.0 Hz of protons H-8 and H-9 suggested the E -configuration of this double bond. The proton spectrum also showed a terminal methyl group [ δ _H 0.87 (3H, t , 7.0 Hz, H-18)] and two methylene groups next to two carbonyl groups [ δ _H 2.51 (2H, t , 7.0 Hz, H-11) and 2.44 (2H, t , 7.0 Hz, H-2)]. The ¹³ C-NMR spectrum ( Table 3 ) showed two olefinic carbons [ δ _C 146.9 (C-8), 130.5 (C-9)], one terminal methyl carbon [ δ _C 14.2 (C-18)] and some methylene groups at δ _C 22–41. The spectrum did not reveal the two quaternary carbonyl carbons C-1 and C-10.

The ESI-MS/MS spectrum of 1 ( Figure 3 ) showed a pseudomolecular ion peak at m/z 295.1 [M-H] ^- . The MS/MS (negative mode) fragment patterns ( Figure 3 ) suggested that 1 possessed a double bond at C-8, and this bond was adjacent to a ketone (C-10). These positions were supported by the HMBC experiment ( Figure 2 ) with cross-peaks of protons H-8, H-9, H-11 to the carbon signal at δ _C 200.1 (conjugated ketone carbon, C-10) and of protons H-2 to the carbon signal at δ _C 169.7 (carboxyl carbon, C-1). The comparison of NMR and MS data of 1 with those of ( E )-10-oxooctadeca-8-enoic acid 10 , a synthetic compound obtained from the oxidation of oleic acid, showed good compatibility. Therefore, the chemical structure of 1 was suggested to be ( E )-10-oxooctadeca-8-enoic acid.

Compound 2 was isolated as white needles. The LC–MSD-MS spectrum showed a pseudomolecular ion peak at m/z 429.0 [M+H] ⁺ (calcd. for C ₂₈ H ₄₄ O ₃ +H, 429.3). The ¹ H-NMR spectrum of 2 showed signals of six methyl groups [ δ _H 1.00 (3H, d , 7.0 Hz, H-21), 0.91 (3H, d , 6.5 Hz, H-28), 0.83 (3H, d , 7.0 Hz, H-27), 0.82 (3H, d , 7.0 Hz, H-26), 0.82 (3H, s , H-18) and 0.88 (3H, s , H-19)], one oxygenated methine [ δ _H 3.97 (1H, m , H-3)], two double bonds [ δ _H 5.15 (1H, dd , 15.5, 7.0 Hz, H-22),

Table 1 The ¹ H-NMR (CDCl ₃ , 500 MHz) data of compound 1, 2, 7

5.22 (1H, dd , 15.0, 7.5 Hz, H-23), 6.24 (1H, d , 8.5 Hz, H-6) and 6.50 (1H, d , 8.5 Hz, H-7)]. Its corresponding ¹³ C-NMR spectrum showed 28 signals with four olefinic carbons [ δ _C 130.9 (C-7), 132.5 (C-23), 135.6 (C-6) and 135.4 (C-22)] and two oxygenated quaternary carbons [ δ _C 82.3 (C-5) and 79.6 (C-8)] of an ergosterol peroxide derivative. The good compatibility of its NMR and MS data with those of ergosterol peroxide in the literature 11 suggested that compound 2 was ergosterol peroxide.

Figure 2 . Keys HMBC of compounds 1 and 7

Figure 3 . The (negative mode) MS/MS fragment patterns of compound 1

Table 2 The ¹ H-NMR (CDCl ₃ , 500 MHz) data of compound 3 ‒ 6

Compound 3 was isolated as a colorless oil. Its molecular formula was determined as C ₁₅ H ₂₀ O ₂ through its pseudomolecular ion peak at m/z 231.1386 [M-H] ^- (calcd. for C ₁₅ H ₂₀ O ₂ -H, 231.1385). The ¹ H-NMR spectrum ( Table 2 ) showed four methyl groups [ δ _H 2.20 (3H, s , H-15), 2.11 (3H, s , H-13), 1.86 (3H, s , H-12), 1.23 (3H, d , 7.0 Hz, H-14)], one olefinic proton [ δ _H 6.02 (1H, s , H-10)], a set of 1,3,4-trisubstituted benzene ring signals [ δ _H 7.02 (1H, d , 7.5 Hz, H-5), 6.70 (1H, dd , 7.5, 1.5 Hz, H-6) and 6.66 (1H, d , 1.5 Hz, H-2)], a methine adjacent to a methylene [ δ _H 3.24 (1H, sextet , 7.0 Hz, H-7), 2.70 (1H, dd , 15.5, 6.0 Hz, H-8a), 2.59 (1H, dd , 15.5, 6.0 Hz, H-8b)]. The ¹³ C-NMR spectrum of 3 showed six aromatic carbons [ δ _C 155.4 (C-3), 146.2 (C-1), 131.1 (C-5), 121.5 (C-4), 119.1 (C-6), 113.7 (C-2)], two olefinic carbons at δ _C 154.0 (C-11), 124.3 (C-10), four methyl groups [ δ _C 27.8 (C-12), 22.1 (C-14), 20.9 (C-13), 15.5 (C-15)], and one carbonyl group at δ _C 200.1 (C-9). Due to the compatibility of the specific optical rotation {Compound 3: +248 ( c 0.43, acetone) and turmeronol A: +63 ( c 0.1, chloroform) 12 } and NMR data with corresponding data in the literature 13 , 3 was turmeronol A.

Compound 4 was obtained as colorless needles. Compound 4 possessed a 1,3,4-trisubstituted benzene ring with proton NMR signals at δ _H 7.10 (1H, dd , 8.0, 2.0 Hz, H-6), 7.04 (1H, d , 1.5 Hz, H-2), 6.86 (1H, d , 8.5 Hz, H-5). The ¹ H-NMR spectrum also showed a six-proton singlet at δ _H 3.90 (6H, s ) for the two methoxy groups and two olefinic protons at δ _H 7.63 (1H, d , 16.0 Hz, H-7), 6.30 (1H, d , 16.0 Hz, H-8) of an E -configuration double bond. The ¹³ C-NMR spectrum of 4 showed signals of one carboxyl carbon ( δ _C 167.8, C-9), two olefinic carbons ( δ _C 144.9, C-7 and 115.7, C-8), six aromatic carbons [ δ _C 151.4 (C-4), 149.5 (C-3), 127.6 (C-1), 122.7 (C-6), 111.3 (C-5), 110.0 (C-2)] and three methoxy groups [ δ _C 56.1 (3-OCH ₃ and 4-OCH ₃ ) and 51.7 (9-OCH ₃ )]. In addition, the HR-ESI-MS spectrum of compound 4 showed a pseudomolecular ion peak at m/z 223.0963 [M+H] ⁺ (calcd. for C ₁₂ H ₁₄ O ₄ +H, 223.0970).

Table 3 ^{1 3} C-NMR (CDCl ₃ , 125 MHz) data of compound 1 ‒ 7

The comparison of these HR-MS and NMR data of 4 with those of methyl ( E )-3-(3,4-dimethoxyphenyl)acrylate in the literature 14 showed good compatibility. Therefore, 4 was methyl ( E )-3-(3,4-dimethoxyphenyl)acrylate with the systematic name methyl ( E )-3-(3,4-dimethoxyphenyl)propenoate.

Compound 6 was obtained as colorless needles. The molecular formula was determined to be C ₁₁ H ₁₂ O ₃ through its pseudomolecular ion peak at m/z 193.0501 [M+H] ⁺ (calcd. for C ₁₁ H ₁₂ O ₃ +H, 193.0864). The similarity in the NMR data ( Tables 2 and 3 ) of 6 and 4 with just one difference of the replacement of the methoxy group of the benzene ring in 4 [ δ _H 3.90 (3H, s , 4-OCH ₃ )] by a hydroxy group in 6 [ δ _H 5.89 (1H, brs , 4-OH)] suggested that the latter was methyl ferulate 15 or its systematic name, methyl ( E )-3-(4-hydroxy-3-methoxyphenyl)propenoate.

Compound 5 was isolated as a white crystalline solid. Its MS spectrum showed a deprotonated molecular ion peak at m/z 151.0397 [M-H] ^- (calcd. for C ₈ H ₈ O ₃ -H, 151.0395). The ¹ H-NMR spectrum ( Table 2 ) showed four aromatic proton signals [ δ _H 7.95 (2H, d , 8.5 Hz, H-2, H-6) and 6.88 (2H, d , 9.0 Hz, H-3, H-5)] of a 1,4-disubstituted benzene ring, a phenolic hydroxyl [ δ _H 6.20 (1H, brs, 4-OH)] and one methoxy group [ δ _H 3.90 (3H, s, COOC H ₃ )]. The ¹³ C-NMR spectrum ( Table 3 ) showed six aromatic signals for one carboxyl carbon [ δ _C 167.4 (C-7)] and one methoxy group [ δ _C 52.1 ( C OOCH ₃ )]. Thus, 5 was assigned as methyl 4-hydroxybenzoate 16 .

Compound 7 was obtained as pale yellow needles. Its ¹ H-NMR spectrum showed proton signals at δ _H 13.08 (1H, brs, 5-OH), 7.81 (1H, s , H-2), 7.34 (2H, d , 8.5 Hz, H-2', H-6'), 6.83 (2H, d , 8.5 Hz, H-3', H-5'), 6.73 (1H, d , 10.5 Hz, H-4''), 6.34 (1H, s , H-8), 5.62 (1H, d , 10.0 Hz, H-3'') and 1.48 (6H, s , two methyl groups, 2''-CH ₃ ). The proton NMR data of 7 were almost identical to those of 4'- O -methylalpinumisoflavone, a compound previously isolated from a less polar H5 fraction, with one difference of lacking the methoxy signal at δ _H 3.84 in the former 17 . The comparison of the ¹³ C-NMR data of these two compounds as well as that of alpumisoflavone 18 showed good compatibility. This was further supported by the HR-ESI-MS spectrum with a deprotonated molecular ion peak at m/z 335.0914 [M-H] ^- (calcd. for C ₂₀ H ₁₆ O ₅ -H, 335.0919) and by all appropriate cross-peaks in the HMBC spectrum ( Figure 2 ). Therefore, the chemical structure of 7 was proposed as alpumisoflavone.

CONCLUSION

Further chemical studies on the n -hexane extract of Leonotis nepetifolia (L.) R. Br, collected at Xuyen Moc district, Ba Ria–Vung Tau province, using some column chromatographic separations, seven compounds were isolated. Their structures were elucidated as (E)- 10-oxooctadeca-8-enoic acid ( 1 ), ergosterol peroxide ( 2 ), turmeronol A ( 3 ), methyl ( E )-3-(3,4-dimethoxyphenyl)propenoate ( 4 ), methyl 4-hydroxybenzoate ( 5 ), methyl ( E )-3-(4-hydroxy-3-methoxyphenyl)propenoate ( 6 ), and alpinumisoflavone ( 7 ) . Although these compounds are known in other species, this is the first time they have been reported in L. nepetifolia .

ABREVIATIONS

LC–MSD: Liquid chromatograph/mass selective detector

LC–MS/MS: Liquid chromatography with tandem mass spectrometry

HR-ESI-MS: High resolution electrospray ionization-mass spectrometry

¹ H NMR: Proton Nuclear Magnetic Resonance

¹³ C NMR: Carbon-13 Nuclear Magnetic Resonance

HMBC: Heteronuclear Multiple Bond Correlation

s : singlet

d : doublet

dd : doublet of doublets

t : triplet

m : multiplet

COMPETING INTEREST

The authors declare no competing financial interest.

AUTHORS’ CONTRIBUTION

Nguyen T.K.H., Phan T.T., Le N.H.T. interpreted NMR and MS data and searched the bibliography. Nguyen K.P.P., Ngo T.T.D. contributed to conducting experiments and acquiring MS and NMR data and gave the final correction for the manuscript.

Corresponding author: Nguyen Thi Kim Huong, University of Science, National University-Ho Chi Minh City, 227 Nguyen Van Cu Street, ward 4 , district 5 , Ho Chi Minh city . Email: kimhuong241@gmail.com

ACKNOWLEDGMENTS

This research was funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number B 2019-18-05.