Chinese Chemical Letters  2015, Vol.26 Issue (08):1000-1003   PDF    
Three minor new compounds from the aerial parts of Leonurus japonicus
Wei-Mao Zhonga,d, Zhao-Meng Cuib, Zhi-Ke Liua,d, Yan Yanga, Da-Rong Wuc, Shao-Hua Liuc, Hui Longa,d, Han-Dong Suna, Yong-Jun Dangb , Wei-Lie Xiaoa     
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China;
b Key Laboratory of Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China;
c Chengdu No. 1 Pharmaceutical Group Co., Ltd., Chengdu 610031, China;
d University of Chinese Academy of Sciences, Beijing 100049, China
Abstract: Phytochemical investigation of the aerial parts of Leonurus japonicus led to the isolation of one new labdane diterpenoid, leojaponin D (1) and two new ionone derivatives, leojaponones A and B (2 and 3), together with seven known diterpenoids (4-10). Their structures were elucidated by extensive 1D and 2D NMR spectroscopic data and by comparison with data reported in the literature. Selected isolates were evaluated their effects on Jurkat IL2 secretion.
Key words: Leonurus japonicus     Diterpenoid     Ionone derivative     Jurkat IL2 secretion    
1. Introduction

Leonurus japonicus (Labiatae),commonly called Chinese motherwort,is a herbaceous flowering plant native to several regions in Asia,including China,Korea,Japan and Cambodia. For thousands of years in China,the aerial part of Leonurus japonicus has been primarily used to treat menoxenia,dysmenorrhea, amenorrhea,lochia,oliguresis,ulcerations and other diseases in women,and thus is named ‘‘Yi Mu Cao’’ [1, 2]. Phytochemical investigation on this species has led to discover various natural compounds with different structural patterns,including alkaloids, flavonoids,glycosides,diterpenoids and triterpenoids,among which diterpenoids are the major constituents [3]. Our previous research on the chemical constituents of L. japonicus has led to the isolation of three diterpenoids [4],especially leojaponin A,which is the first example of clerodane-type diterpenoid obtained from L. japonicus. As part of our ongoing programto discover structurally interesting and potential bioactive chemical constituents,we reinvestigated this plant,and obtained three additional new compounds,including one labdane-type diterpenoid,leojaponin D (1),and two ionone derivatives,leojaponones A and B (2 and 3), together with seven known diterpenoids,leojaponins A-C (4-6) [4],leoheteronin D (7) [5],villenol (8) [6],leoheterin (9) [7] and 3α-acetoxy-7β-hydroxy-15-O-methylleopersin C (10) [8] (Fig. 1). Herein,we report the isolation and structure elucidation of the new compounds,as well as the effect on Jurkat IL2 secretion of selected isolates.

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Fig. 1.Structures of compounds 1-10.
2. Experimental 2.1. Plant material

The aerial parts of L. japonicuswere collected in Xichang county, Sichuan Province,China,and identified by Prof. Xi-Wen Li, Kunming Institute of Botany. A voucher specimen (KIB 20120601) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany,Chinese Academy of Sciences.

2.2. Extraction and isolation

The air dried aerial parts of L. japonicus (15.0 kg)were extracted with 95% EtOH (3 × 40 L) at room temperature,and the combined solvents were evaporated in vacuo to yield a residue (1.5 kg). The residuewas subjected to a silica gel column (10 kg,100-200 mesh) eluting with CHCl3-Me2CO (1:0,9:1,8:2,2:1,1:1,0:1) to afford fractions A-F. Fraction A (120 g) was decolorized using MCI gel (90% MeOH-H2O),and the concentrated elute was chromato- graphed via a silica gel CC (1.2 kg,200-300 mesh) eluting with petroleum ether-Me2CO gradient (100:1-0:1) to afford subfrac- tions A1-A6. Fraction A2 (11.0 g) was subjected to a RP-18 CC (500 g,MeOH-H2O gradient,40%-100%) to afford subfractions A2.1-A2.5. Compound 1 (1.1 mg) was isolated from fraction A2.2 followed by repeated column chromatography over silica gel (17 g, CHCl3-Me2CO gradient,100:1-1:1). Fraction A3 (23.0 g) was separated by medium-pressure column chromatography on RP- 18 (600 g,MeOH-H2O gradient,40%-100%) to give subfractions A3.1-A3.3. Fraction A3.1.2 (4.2 g) was separated by Sephadex LH- 20 CC (CHCl3-MeOH),and then by repeated column chromatogra- phy over silica gel (3 g,petroleum ether-Me2CO gradient,30:1- 1:1) to give subfractions A3.1.2.1-A3.1.2.8. Compound 2 (1.2 mg) and 3 (1.1 mg)were finally purified by semi-preparativeHPLC (60% MeCN-H2O) from fraction A3.1.2.4 (306.2 mg). Fraction B (70.0 g) was subjected to MCI gel (90% MeOH-H2O) and chromatographed on silica gel (petroleum ether-Me2CO,30:1-0:1) to afford subfractions B1-B7. Fraction B3 (11.3 g) was chromatographed via a RP-18 column (30%-100% gradient MeOH-H2O) to furnish B3.1-B3.7. Fraction B3.2 (0.06 g) was purified by semi-preparative HPLC (62% MeCN-H2O) to give compounds 4 (12.0 mg) and 7 (3.0 mg). Fraction B3.3 (0.25 g) was purified by Sephadex LH-20, and finally by semi-preparative HPLC (50% MeCN-H2O) to give compounds 5 (14.0 mg),8 (8.1 mg) and 9 (6.3 mg). Fraction B3.4 (0.08 g) was purified by LH-20 and semi-preparative HPLC (42% MeCN-H2O) to yield compounds 6 (6.0 mg) and 10 (11.4 mg).

2.3. The immune activity assay

Proliferation assay: Jurkat T cells (5000 cells/well) were seeded into 96 well plate. Compounds 4-10 were dissolved in DMSO and added into cells with final concentration of 20 μmol/L. The cell viability was measured with AlamarBlue (Invitrogen Inc.) after 72 h.

ELISA assay to detect the secretion of Interleukin 2 (IL-2): Jurkat T cells (105 cells/well) were seeded into 96 well plate and incubated with compounds as indicated in the presence of PMA (40 nmol/L) and Ionomycin (1 μmol/L) for 12 h. The amount of IL-2 inmediumwasmeasuredwith the kit fromBD biosciences. Briefly, the capture antibody in coating buffer (0.1 mol/L Sodium carbon- ate,pH 9.5) (1:500) was coated onto 96 well plate for overnight at 4 8C. After washing,medium from cultured cells was added into plate and incubated for 2 h at room temperature. Detector antibodies were used to incubate with captured IL-2 and developed with TMB substrate after stopping the reaction with stopping solution. Plates were read immediately at 450 nm with Envision from PE company.

3. Results and discussion

Compound 1 was obtained as a colorless oil,[α] 24:4 D - 92:2(c 0.11,MeOH); UV (MeOH) λmax (log ε) 203 (4.06) nm. Themolecular formula of 1 was assigned as C20H32O2 from its HR-EIMS (m/z 304.2402 [M] + ,calcd. 327.2295 [M+Na] + ) with five degrees of unsaturation. The IR spectrum revealed the presence of a hydroxyl group (3427 cm-1 ) and a carboxyl group (1724 cm-1 ). The 1 H NMR spectrum(Table 1) displayed signals of four tertiarymethyl groups and one olefinic proton signal (δH 5.17). The 13 C NMR and DEPT spectra (Table 1) exhibited 20 carbon resonances attributed to four methyls,seven methylenes,five methines (one carbonyl and two olefinics) and four quaternary carbons (two olefinics). On the base of the HSQC spectrum,all protonswere assigned unambiguously to their corresponding carbons. The 1 H-1 H COSY and HSQC spectra established the spin systems for themolecular structure fragments of C-1-C-2-C-3,C-5-C-6-C-7,C-9-C-11-C-12 and C-14-C-15,and the connectivity was confirmed by the HMBC experiment (Fig. 2). The HMBC correlations fromMe-18 to C-3,C-4 and C-5,fromH-19 to C-4,and fromH-5 to C-4 and C-18 indicated that the quaternary carbon C-4 was connected with C-18 and C-19 and C-3 was linked to C-5 through C-4. The HMBC correlations from H-1 and H-5 to C- 10 and C-20 indicated that the quaternary C-10 was connected with C-1 and C-5. The relationships fromH-20 to C-10 and C-9,from H-11 to C-10,and from H-11,H-12 and H-20 to C-9,suggestedthatC- 20 and C-12were linked to C-9 through C-10 and C-11 respectively. The above evidence implied that 1 was a labdane-type diterpenoid, which was highly similar to the known diterpenoid,villenol [6]. A careful comparison of their NMR data suggested that the main difference resulted from the hydroxymethyl (C-19,δC 64.7) in villenol oxidized to an aldehyde group (C-19,δC 206.3) in compound 1,which was further supported by theHMBC correlations ofH-3 and H-5with C-19. The ROESY correlation ofH-15 with Me-16 disclosed that the double bond between C-13 and C-14 was E geometry. The relative configurations of 1 were established by analysis of its ROESY data. Considering the structures of labdane-type diterpenoids previously isolated from the species L. japonicus,Me-20 was supposed to be β-oriented [5]. The correlations of Me-20 with H- 6β,H-11β and H-3β,of H-6α with H-5,of H-5 with H-9 andMe-18 revealed that H-5,H-9 and Me-18 were all α-oriented. Thus,compound 1 is elucidated as 15-hydroxylabda-7,13E-diene-4-al, named leojaponin D.

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Fig. 2.Key HMBC (H→C),1H-1H COSY (-) and ROESY (HH) correlations of 1.

Table 1
NMR data of compounds 1-3 (CDCl3,TMS,δ in ppm,J in Hz).a

Compound 2 was isolated as a colorless oil,[α] 17:7 D+3.4(c 0.07, MeOH); UV (MeOH) λmax (log ε): 202 (3.69) nm. Its molecular formula was determined as C16H24O3 by its HR-EIMS (m/z 264.1728 [M] + ,calcd. 264.1725),implying five degrees of unsaturation. The IR spectrumrevealed the existence of a carboxyl group (1724 cm-1 ). Its 1 H NMR and 13 C NMR (Table 1) spectra displayed signals of six methyls,one methylene,four methines (including two oxymethines) and five quaternary carbons (includ- ing two olefinic carbons). In addition to two methyl singlets at δC 27.4 and 29.0 and one quaternary carbon at δC 108.4 (s),indicating an isopropylidene group,the other 13 carbons implied an ionone derivative,in which C-3 and C-4 were oxygenated. Careful comparison of NMR data of 2 with known compound 3α,4a- isopropyliden-b-ionol showed close structural similarities[9].

The main difference resulted from the hydroxymethine (C-9,δC 68.1) in 3α,4α-isopropyliden-b-ionol oxidized to a carboxyl (C-9, δC 198.2) in 2,whichwas confirmed by the HMBC correlations of H- 7,H-8 and H-10 with C-9 (Fig. 3). The relative configurations of 2 were established by analysis of its ROESY data. The stereochemis- try of H-3 and H-4 were both assigned b-orientated according to the correlations between these two protons and Me-11. Thus, compound 2 is determined as 3α,4α-isopropyliden-b-ionone,and given the trivial name as leojaponone A.

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Fig. 3.Selected HMBC (H→C) and 1H-1H COSY (-) correlations of 2 and 3.

Compound 3 was isolated as a colorless oil with [α] 14.0 D -19:2(c 0.11,MeOH) and UV (MeOH) λmax (log ε) 225 (3.79) nm,which gave the molecular formula C13H20O3 from its HREIMS (m/z 224.1409 [M] + ,calcd. 224.1412),requiring four degrees of unsaturation. The IR spectrum revealed a hydroxyl group (3433 cm-1 ) and a carboxyl group (1666 cm-1 ). The 1 H NMR and 13 C NMR data (Table 1) of 3 were highly similar to those reported for crotalionoside C [10],which was also one ionone derivative. A careful comparison of their 1D NMR data,together with HMBC and 1 H-1 H COSY analysis indicated that the difference was due to a hydroxymethine (C-9,δC 69.3) in crotalionoside C replaced by a carboxyl group (C-9,δC 197.7) in 3. This was confirmed by HMBC correlations fromH-7,H-8 and H-10 to C-9. In the ROESY spectrum,the correlations of Me-11 with H-2β and H- 4β indicated that theywere all β-oriented. Correlations ofH-3with Me-13 implied that theywere all α-oriented. Therefore,compound 3 is determined as megatigma-7-en-3,6-epoxy-5-hydroxy-9-one, and named as leojaponone B.

Considering the immune activity of diterpenoids previously isolated from the plants of the genus Leonurus [11],compounds 4- 10 were tested for their in vitro effect on the secretion of IL-2 and the proliferation in Jourkat T cells [12]. Except for compound 7,all compounds exhibited the inhibition of IL-2 secretion upon the activation of T cell by PMA and ionomycin at the dose of 20 μmol/L. However,compound 10 also interfered the proliferation of Jurkat T cells (Fig. 4). New compounds (1-3) were not tested their bioactivities currently for the mass limitation.

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Fig. 4. Effect of compounds on the secretion of IL-2 and proliferation of Jurkat T cells. (a) Jurkat T cellswere seeded into 96well plate,and treatedwith compounds as indicated in the presence of PMA and ionomycin wasmeasured by Elisa after the treatment of the compounds as indicated for 12 h. (b) Jurkat T cells were plated into 96 well plate and the cell viability was measured by alamarblue after the treatment of the compounds as indicated for 72 h.
4. Conclusion

In summary,three new minor compounds,including one labdane-type diterpenoid (1) and two ionone derivatives (2 and 3), together with seven known diterpenoids,were isolated from the aerial parts of L. japonicus. This investigation could shed new light on the further understanding of the chemical constituents of L. japonicus.

Acknowledgments

This project was supported financially by National Natural Science Foundation of China (Nos. 81422046 and 31300293),the National Science and Technology Support Program of China (No. 2013BAI11B02),the Natural Science Foundation of Yunnan Province (No. 2012FB178) and the project sponsored by SRF for ROCS,SEM to W.L. Xiao.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.05.004.

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