Study on the Chemical Constituents of Clerodendrum japonicum, a Zhuang Medicine

Jiangcun WEI, Zujie QIN*, Wenwei CAI, Haisheng ZENG, Liping QIN, Xinying MO, Yuping TAN, Guilin YANG

1. Guangxi International Zhuang Medical Hospital, Nanning 530201, China; 2. Guangxi University of Chinese Medicine, Nanning 530200, China; 3. Affiliated Hospital of Youjiang Medical University For Nationalities, Baise 533000, China

Abstract [Objectives] To make a preliminary study on the chemical constituents of Clerodendrum japonicum, a Zhuang medicine, so as to provide theoretical support and scientific basis for exploring its medicinal value and comprehensive development and utilization. [Methods] The compounds were separated by macroporous adsorption resin, silica gel, Sephadex LH-20, ODS chromatographic column and preparative high performance liquid chromatography, and their structures were identified by NMR and MS data. [Results] 8 compounds were isolated and identified from Clerodendrum japonicum extract with 80% ethanol solution: (1) fumaric acid, (2) trans-p-hydroxycinnamic acid, (3) cis-p-hydroxycinnamic acid, (4) ethyl caffeate, (5) acteoside, (6) kaempferol, (7) apigenin-7-O-β-D-glucopyranoside, (8) caffeic acid. [Conclusions] 8 compounds were isolated and purified from the ethanol extract of Clerodendrum japonicum.

Key words Clerodendrum japonicum, Chemical constituents, Structure identification

1 Introduction

Clerodendrumjaponicumis a plant in the Family Verbenaceae, and its dry aboveground part is used as a Zhuang medicine, also known as Honglongchuan, Mutonglian, Zhenzhuwutong, Hebaohua, Chouxuhong[1], and "Gepengbei" (Guangxi Zhuang language)[2]. It is produced in Guangxi, Guangdong, Guizhou and Yunnan, and mainly contains flavonoids, phenolic acids, tannins and volatile oils[3-4]. The whole plant can be used as medicine with sweet taste and cool nature. It has the effects of calming the mind, stopping bleeding, dispelling wind and removing blood stasis, detoxifying and promoting the subsidence of swelling, clearing lung heat, inducing diuresis and so on. It can treat rheumatism and bone pain, lung heat and cough, difficult urination and so on[5-6].

C.japonicumcontains a variety of chemical constituents, including flavonoids, alkaloids, phenolic acids, tannins and volatile oils, but there are few studies on flavonoids. In order to further develop the medicinal resources of the Zhuang medicineC.japonicumand clarify the material basis of its efficacy, some compounds were extracted from the Zhuang medicineC.japonicumand separated by various chromatographic methods and techniques[7], and the structure of the obtained monomer compounds was analyzed. 8 compounds were isolated and purified from the ethanol extract ofC.japonicum: (1) fumaric acid, (2) trans-p-hydroxycinnamic acid, (3) cis-p-hydroxycinnamic acid, (4) ethyl caffeic acid, (5) anthocyanin, (6) kaempferol, (7) apigenin-7 color O-β-D-glucoside, (8) caffeic acid.

2 Materials and methods

2.1 MaterialsVARIAN 500 MR superconducting nuclear magnetic resonance spectrometer (TMS-internal standard, Bruker, Switzerland); mass spectrometer: Waters Autospec Premier P776; nuclear magnetic resonance instrument: Avance III 600MHz, Avance III-800MHz (TMS-internal standard, δ-ppm, J-Hz); rotating evaporator: Shanghai Yarong RE-3000A rotary evaporator; PRACTUM224-KN SOP electronic analytical balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.; TLC high performance silica gel G plate (Qingdao Ocean Chemical Factory); Sephadex LH-20 (Ge Healthcare Bio-Sciences AB, Sweden); D101 macroporous adsorption resin (net grade, Tianjin Haiguang Chemical Co., Ltd.); preparation column Cosmosil 5C18-MS-II (250 mm × 10 mm, 5 μm) (Nacalai Tesque, Japan); dichloromethane, methanol and glacial acetic acid were chromatographically or analytically pure.

The Zhuang medicine used in this experiment was collected from Daming Mountain, Wuming District, Nanning City, Guangxi in July 2019. It was identified asClerodendrumjaponicum(Thunb.) Sweet in Family Verbenaceae by Zeng Chao, director of the Pharmacology Department of the First Affiliated Hospital of Guangxi University of Chinese Medicine. The whole plant could be used as medicine.

2.2 Methods21 kg ofClerodendrumjaponicumwas soaked in 80% ethanol (1∶10) for 24 h, then refluxed and extracted for 1.5 h and then filtered. After repeated extraction for 3 times, the drug residue was refluxed and extracted twice with 60% ethanol for 1.5 h each time. The filtrate was combined and concentrated until there was no ethanol smell. 228.67 g of ethyl acetate extract and 130.32 g of petroleum ether fraction were obtained fromC.japonicumby extraction with petroleum ether and ethyl acetate in turn.

150 g of ethyl acetate extract ofC.japonicumwas dissolved and mixed with methanol and pretreated with appropriate amount of silica gel column, and the sample was injected after drying. The sample was separated by silica gel column chromatography and eluted with dichloromethane-methanol methanol (100∶0→70∶1→50∶1→30∶1→20∶1→15∶1→10∶1→7∶1→5∶1→2∶1→1∶1→methanol)[8]. It was eluted with dichloromethane-methanol (70∶1), dichloromethane-methanol (50∶1), dichloromethane-methanol (30∶1), dichloromethane-methanol (20∶1) and dichloromethane-methanol (10∶1). The elution parts were concentrated and freeze-dried. Dry extracts from each part were obtained, sealed and stored for later use.

12 compounds were isolated by silica gel column chromatography (CC), preparative thin layer chromatography (PTLC), dextran (Sephadex LH-20) column chromatography, polyamide column chromatography, preparative high performance liquid chromatography and recrystallization: compound 1 (15.97 mg), 2 (9.56 mg), 3 (11.28 mg), 4 (7.86 mg), 5 (24.15 mg), 6 (16.09 mg), 7 (10.21 mg) and 8 (7.83 mg). 8 compounds were identified by spectral techniques such as IR, EI-MS, HR-ESI-MS, 1D NMR (′H NMR, BC NMR, DEPT NOE) and 2D NMR (′H-′H COSY, HSQC, HMBC, NOESY), and compared with the standard compounds and related literature data.

3 Structure identification

Compound 1: White crystal, ESI-MS m/z 117[M+H]+.1H NMR data showed an alkenyl proton signalδH 6.76 (1H, s),13C NMR data showed a set of symmetrical carbonyl carbon signalsδC 168.3 (1-COOH, 2-COOH) and a set of trans alkenyl carbon signals δC 135.8 (C-1, C-2). The1H NMR and13C NMR data of the compound were basically consistent with those reported in reference[9], so the compound could be identified as fumaric acid.

Compound 2: Yellowish crystal, ESI-MS m/z 187[M+Na]+.1H NMR data showed that δH 7.45 (2H, d, J=8.6 Hz, H-3, H-5), 6.81 (2H, d, J=8.6 Hz, H-2, H-6) indicated the existence of a symmetrically substituted benzene ring in the molecule. The13C NMR data not only showed six benzene ring carbon signals, but also included a set of alkenyl carbon signals δC 127.5 (C-7), 115.9 (C-8), and a carbonyl carbon signal δC 171.3 (C-9). The1H NMR and13C NMR spectral data of compound 3 were basically consistent with trans-p-hydroxycinnamic acid reported in literature[10], so the compound could be identified as trans-p-hydroxycinnamic acid.

Compound 3: White acicular crystal, ESI-MS m/z 187[M+Na]+.1H NMR data showed that H 7.62 (2H, d, J=8.6 Hz, H-2, H-6), 6.81 (2H, d, J=8.6 Hz, H-3, H-5) indicated the existence of a symmetrically substituted benzene ring in the molecule. The13C NMR data not only showed six benzene ring carbon signals, but also included a set of alkenyl carbon signals δC 145.7 (C-7), 115.9 (C-8), and a carbonyl carbon signal δC 170.4 (C-9). The1H NMR and13C NMR spectral data of compound 3 were basically consistent with cis-p-hydroxycinnamic acid reported in literature[11], so the compound could be identified as cis-p-hydroxycinnamic acid.

Compound 4: Yellow crystalline powder, ferric trichloride-potassium ferricyanide reaction was positive, indicating that there were phenolic hydroxyl groups in the compound. HR-ESI-MS m/z：207.0657[M-H]-, the known molecular weight was 208, it was speculated that its molecular formula was C11H12O4.1H-NMR(600MHz, CD3OD) δ：7.03(d, J=1.8Hz,1H, H-2), 6.77(d, J=8.2 Hz,1H, H-5), 6.92(dd, J=8.2, 1.8 Hz, 1H, H-6), δ7.52(d, J=15.9 Hz, 1H, H-7), 6.23(d, J=15.9 Hz, 1H, H-8), 4.20(q, J=7.1 Hz, 2H, H-10), 1.29(t, J=7.1 Hz, 3H, H-11)；13C-NMR(150 MHz, CD3OD) δ：123.03(C-1), 115.23(C-2), 146.86(C-3), 149.66(C-4), 116.62(C-5), 127.86(C-6), 146.93(C-7), 115.39(C-8), 169.47(C-9), 61.55 (C-10), 14.76(C-11). The above data were basically consistent with those reported in reference[12-13], so the compound was identified as ethyl caffeate.

Compound 5: Yellowish solid, ESI-MS m/z 623[M-H]-. In the1H NMR (500 MHz, MeOD) hydrogen spectrum, there were two 1, 3, 4-substituted benzene ring proton signals δ6.73 (1H, d, J= 2.0 Hz, H-2), 6.71 (1H, d, J= 8.0Hz, H-5), 6.60 (1H, dd, J= 8.0, 2.0 Hz, H-6) and δ7.09 (1H, d, J= 1.5 Hz, H-2′), δ6.81 (1H, d, J= 8.0 Hz, H-5′), δ6.99 (1H, dd, J= 8.0, 1.5 Hz, H-6′), a conjugated alkene bond signal δ7.63 (1H, d, J= 16.0Hz, H-10), 6.30 (1H, d, J= 16.0, H-9), a methylene group signal δ2.82 (2H, m, H-8) and an oxygen-bound methylene group signal δ3.95 (1H, m, H-7), 3.75 (1H, m, H-7). In addition, there were two hexose signals, identified as glucose and rhamnose by comparison. δ1.21 (3H, d, J= 6.0 Hz, H-6‴) appearing in the high field was the hydrogen signal on rhamnose carbon 6. δ4.95 (1H, t, J= 9.0 Hz, H-4″) was the hydrogen signal on glucose carbon 4, which showed obvious shift due to its connection with the ester bond of caffeic acid. In the13C NMR (125 MHz, MeOD) carbon spectrum, there was a conjugated carbonyl signal δ168.3 (C-9), a caffeoyl signal δ127.6 (C-1), 114.7 (C-2), 149.8(C-3), 146.8 (C-4), 116.5 (C-5), 123.2 (C-6), 168.3 (C-9), 115.2 (C-10), 148.0 (C-11), a phenylethyl signal connected to oxygen δ131.5 (C-1), 116.3 (C-2), 144.7 (C-3), 146.1 (C-4), 117.1 (C-5), 121.3 (C-6), 72.0 (C-7), 36.5 (C-8). In addition, the chemical shift of carbon 3 on glucose was δ81.6, indicating that rhamnose was connected to glucose by 1, 3 glycosidic bond. The above data were basically consistent with the acteoside reported in reference[14-15], so compound 5 was determined to be acteoside.

Compound 6: Yellow powder. High resolution Q-TOF-ESI-MS gave its quasi-molecular ion peak m/z 287.0556[M+H]+(calcd for C15H11O6，287.055 0), and its molecular formula was determined to be C15H10O6.1H NMR(DMSO-d6, 500 MHz)：12.49(1H, br. s, 5-OH), 8.07(2H, d, J=9.0 Hz, H-2′,6′), 6.95(2H, d, J=9.0 Hz, H-3′, 5′), 6.45(1H, d, J=2.0 Hz, H-8), 6.21(1H, d, J=2.0 Hz, H-6).13C NMR(DMSO-d6, 125 MHz)：146.7(C-2), 135.7(C-3), 175.7(C-4), 160.7(C-5), 98.2(C-6), 164.1(C-7), 93.5(C-8), 156.2(C-9), 102.9(C-10), 121.7(C-1′), 129.4(C-2′, 6′), 115.4(C-3′, 5′), 159.1(C-4′). The above data were compared with reference[16], and the structure of the compound was identified as kaempferol.

Compound 7: Yellow powder. ′H NMR (DMSO, 400 MHz): 7.96 (2H,d,J=8.8Hz, H-2′, 6′), 6.94 (2H, d,J=8.8 Hz, H-3′, 5), 6.84 (1H, s, H-8), 6.45 (1H,s, H-6), 6.88 (1H, s, H-3), 5.08 (1H, d,J= 7.6 Hz, H-1″)."C NMR (DMSO, 100MHz): δcl81.9(C, C-4), 164.2 (C, C-2), 162.9(C, C-7), 161.3 (C, C-5), 115.9 (2CH,C-3′, 5″), 161.0(C, C-4′), 156.9(C, C-9), 128.5 (2CH, C-2′, 6′), 121.0 (C, C-1), 105.3 (C, C-10), 103.1 (CH, C-3), 99.5 (CH, C-6), 60.6 (CH2, C-6″), 77.1 (CH, C-5″), 69.5(CH, C-4″), 76.4 (CH, C-3″), 73.1 (CH, C-2″), 99.9 (CH, C-1″). The above data were basically consistent with those reported in the literature[17], so compound 7 was identified as apigenin-7-O-β-D-glucopyranoside.

Compound 8: Yellowish amorphous powder. ESI-MS m/z: 179.0[M-H]-.1H-NMR (600 MHz, DMSO-d6) δ: 7.40 (1H, d, J=15.9 Hz, H-7), 7.01 (1H, d, J=2.1 Hz, H-2), 6.95 (1H, dd, J=8.1, 2.1 Hz, H-6), 6.75 (1H, d, J=8.1 Hz, H-5), 6.16 (1H, d, J=15.9 Hz, H-8)；13C-NMR (150 MHz, DMSO-d6)δ: 167.9 (C-9), 148.0 (C-4), 145.5 (C-7), 144.3 (C-3), 125.6 (C-1), 121.0 (C-6), 115.6 (C-5), 115.2 (C-8), 114.5 (C-2). The above data were basically consistent with those reported in the literature[18-19], so compound 8 was identified as caffeic acid.