lsolation and identification of chemical constituents of Pithecellobium clypearia (Jack.) Benth

2021-03-04WanyiShiYuxiWangXiaoxiaoHuang

Asian Journal of Traditional Medicines 2021年1期

Wanyi Shi,Yuxi Wang,Xiaoxiao Huang

Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research &Development,Liaoning Province,School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University,Shenyang 110016,China

Abstract The twigs and leaves of Pithecellobium clypearia (Jack.) Benth are used as common Chinese traditional herbal medicine to treat upper respiratory tract infection,acute gastroenteritis and bacterial dysentery.The study isolated nine compounds from the EtOAc extract,all of which were obtained from Pithecellobium Mart.for the first time.Based on 1H-NMR and 13C-NMR data,the structures of these compounds,including two ladiosiplodins and two dihydroisocoumarins,were elucidated.

Keywords:Pithecellobium clypearia (Jack.) Benth;ladiosiplodins;dihydroisocoumarins

1 Introduction

Pithecellobium clypearia

(Jack.) Benth,distributed in the south of China,belongs to Mimosoaceaeis family.The twigs and leaves of

clypearia

are the traditional herbal medicine used for the treatment of upper respiratory tract infections,acute gastroenteritis and bacterial dysentery.Previous pharmaceutical research on

Pithecellobium

genus has found that it contains flavonoids [1],triterpenoids,steroids [2],lignans [3],phenolic acid and other components.Two known Lasiodiplodins were isolated from the stems and leaves of

clypearia

.It has been reported in many studies that Lasiodiplodin is one of fungal metabolites [4].It was also found in plants [5].The compound showed cytotoxicity against some cancer cell lines [6,7].

Pithecellobium

genus has many biological activities,including antibacterial [8],antiviral [9],anti-inflammatory [10],antioxidant [11],and antitumor activities [12].Studies showed that constituents of

clypearia

,especially the galloyl substituted flavonoid,exhibited antiviral activity [13].Besides,the plant also exhibited prominent anti-inflammatory activity.Monotherapy preparations from the plant have been on the market for many years with good clinical efficacy.Free radical scavenging assays have shown that both the galloyl substituted flavonoid and biphenyl have antioxidant activity [11].

2 Materials and methods

2.1 General material and instrument

NMR spectra (Bruker ARX-400,USA,TMS as an internal standard in DMSO-

or CDCl);Mass spectra (Bruker IFS-55 spectrometer,Germany);High performance liquid chromatography(Shimadzu,SPD-20A,UV detector,LC-6 high pressure pump,Japan);Preparative column (YMC Pack ODS-A,250 mm × 10 mm,5

m,USA);AE Electronic analytical balance (Mettler-Toledo,Shanghai,China);Silica gel (100-200 mesh,200-300 mesh,Qingdao Marine Chemical Co.,China);Silica gel GF254 (Qingdao Marine Chemical Co.,China);Reversed phase Csilica gel (60-80

m,Merck,Germany);Sephadex LH-20 (25-100

m,Green Herbs Science and Technology Development Co.,Ltd.China);HP20 Macroporous resin(Mitsubishi Chemical Co.,Japan);Polyamide (200-300 mesh,Shandong Yu Wang Co.,China).

2.2 Plant material

The twigs and leaves of

P.clypearia

were collected in Guangxi Province,People’s Republic of China,in May 2015.The plant was genetically identified by Professor Jincai Lu at Shenyang Pharmaceutical University.A voucher specimen (DG-20150529) has been deposited in the herbarium of the Department of Natural Products Chemistry,Shenyang Pharmaceutical University.

2.3 Extraction and isolation

The twigs and leaves of

P.clypearia

(60 kg)were extracted with 70% ethanol and refluxed 3 times to obtain 2.4 kg crude extract,and then 1.8 kg crude extract was suspended in water (15.0 L) and partitioned four times successively with EtOAc and

-BuOH (15.0 L) to give 1.1 kg EtOAc extract.The extract was subjected to the silica gel column chromatography and eluted with a CHCl-CHOH gradient system (50:1→1:1) to give four fractions A (32 g),B (157 g),C (418 g) and D (130 g).Fraction B was separated by polyamide column chromatography using EtOH-HO (30:70,60:40,90:10,

v/v

) to obtain two fractions B1 (70 g) and B2 (63 g).Fraction B1 was separated by HP20 macroporous resin column chromatography with EtOH-HO gradient system to give two subfractions,B1-1 (45 g) and B1-2 (19 g).Subfraction B1-1 was separated by ODS column chromatography and eluted with EtOH-HO gradient system to yield three subfractions,B1-1-1 (26 g),B1-1-2 (4.2 g),and B1-1-3 (5 g).Subfraction B1-1-3 was purified by Silica gel column chromatography,eluted with CHCl-MeOH mixtures with increasing polarities(50:1→1:1,

v/v

),and was further subjected by preparative HPLC with MeOH-HO to obtain compounds 3 (16.7 mg),4 (25.6 mg),5 (9.8 mg),6 (10.8 mg),8 (4.5 mg),and 9 (16.8 mg).Subfraction B1-2 was subjected by ODS column chromatography and eluted with EtOH-HO gradient system to obtain two subfractions,B1-2-1 (6.4 g) and B1-2-2 (5.8 g).Subfraction B1-2-2 was purified by Silica gel column chromatography,eluted with CHCl-MeOH mixtures with increasing polarities (50:1→1:1,

v/v

),and was further subjected by preparative HPLC with MeOH-HO to obtain compounds 1 (20.8 mg),2 (30.1 mg),and 7 (11.4 mg).(+)-Lasiodiplodin (1):yellowish oil,+4.7(c 0.46,MeOH);H-NMR (400 MHz,CDCl) andC-NMR (100 MHz,CDCl) (Table 1);HRESIMS(

m/z

):[M+H]293.1766 (calcd.for CHO,293.1759).

(+)-Ficusine C (2):yellowish powder,+3.0(c 1.0,MeOH);H-NMR (400 MHz,CDCl) andC-NMR (100 MHz,CDCl) (Table 1).

(-)-8-methoxymellein (3):yellowish oil,-51.7(c 0.1,MeOH);H-NMR (400 MHz,DMSO-

) andC-NMR (100 MHz,DMSO-d) (Table 2).

(-)-3,5-dimethy l-8-methoxy-3,4-dihydroisocoumarin (4):yellowish oil,-238 (c 1.0,CHCl);H-NMR (400 MHz,CDCl) andC-NMR(100 MHz,CDCl) (Table 2).

Threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (5):colorless powder,H-NMR(400 MHz,DMSO-

) andC-NMR (100 MHz,DMSO-d) (Table 3).Erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (6):colorless powder,H-NMR(400 MHz,DMSO-

) (Table 3).

3 Results and discussion

(-)-Dihydrophaseicacid (7):colorless powder,-14.8 (c 0.0675,MeOH);H-NMR (400 MHz,DMSO-

) andC-NMR (100 MHz,DMSO-d)(Table 4).(+)-Dehydrovomifoliol (8):colorless powder,+139.7 (c 0.42,MeOH);H-NMR (400 MHz,DMSO-

) andC-NMR (100 MHz,DMSO-d)(Table 4).(+)-epiloliolide (9):colorless powder,+42.3 (c 0.058,MeOH);H-NMR (400 MHz,DMSO-

) andC-NMR (100 MHz,DMSO-d) (Table 4).

Fig.1 Structures of compounds 1-9

Compound 1 was a yellowish oil with the molecular formula of CHOThe positive ion at

m/z

293.1766 [M+H](calcd for CHO,293.1759) in the HRESIMS data indicated 6 indices of hydrogen deficiency.In theH-NMR spectrum,signals at

6.20 (1H,d,

=1.9 Hz,H-14),6.26(1H,d,

=1.9 Hz,H-12) were assigned to a 1,2,3,5-tetrasubstituted benzene ring.Proton signal at

5.21 (1H,m,H-3) indicated the presence of oxygenated methine.Additionally,two signals at

2.48 (1H,m,H-10a),2.57 (1H,m,H-10b) were observed,suggesting the presence of a methylene group.Proton signals at

1.23-1.65 (10H,overlap),1.88 (2H,m,H-4) indicated the presence of six spmethylenes.Besides,a methyl signal at

1.30 (3H,d,

=6.4,3-CH) and a methoxy signal at

3.70(3H,s,15-OCH) were observed.TheC-NMR data of 1 revealed 17 resonances,including six aromatic carbon signals (

96.9,108.0,116.5,142.6,157.4,158.1),one ester carbonyl signal (

168.7),one oxygenated carbon signal (

71.9),one methoxy carbon signal (

55.6),and seven methylene signals.Based on these data,and by comparing the experimental spectrum data with the values reported in the literature [14],the structure of 1 was determined as (+)-lasiodiplodin.TheH-NMR data of compound 2 revealed the presence of two olefinic hydrogen signals at

6.01(1H,d,

=1.4 Hz,H-12),5.54 (1H,d,

=1.4 Hz,H-14),an oxygenated methine at

4.77 (1H,m,H-3) and a hydrogen at

4.16 (1H,brs,16-OH)in the downfield region.

3.70 (3H,s,5-OCH)was the signal of a methoxy,

1.32 (3H,d,

=6.4 Hz,H-17) was the methyl signal.2.35 (2H,m,H-10b),1.15-1.57 (10H,overlap),1.87 (1H,m,H-4a),and 1.66 (1H,m,H-4b) were the methylene signals.Analysis of theC-NMR data indicated the existence of one ester carbonyl (

169.1),a quinone ring (

75.1,101.1,125.9,155.7,172.5,186.7),a methoxy (

56.3) and a chain of nine carbons moiety.Compared with the data of Ficusine C in literature [15],compound 2 was identified as(+)-Ficusine C.

Table 1 1H-NMR (400 MHz,CDCl3) and 13C-NMR (100 MHz,CDCl3) data for compounds 1 and 2

TheH-NMR data of compound 3 indicated the existence of a 1,2,3-trisubstited benzene ring at

7.05 (1H,d,

=8.5 Hz,H-5),7.52 (1H,dd,

=8.5,7.5 Hz,H-6),6.89 (1H,d,

=7.5 Hz,H-7),and a -CH-CH-CHmoiety at

1.35 (3H,d,

=6.2 Hz,3-CH),4.51 (1H,dqd,

=11.1,6.3,2.9 Hz,H-3),2.94 (1H,dd,

=16.2,2.9 Hz,H-4a),2.82 (1H,dd,

=16.2,11.1 Hz,H-4b).Furthermore,the presence of a singlet at

3.82(3H,s,8-OCH) suggested a methoxy substituent.TheC-NMR data of compound 3 showed 11 carbon signals,including one ester carbonyl (

161.2),a phenyl carbons (

111.2,113.1,119.2,134.5,142.1,160.4) and four spcarbons (

73.6,55.8,35.1,20.3).Compared with the data reported in literature [16],compound 3 was identified as(-)-8-methoxymellein.Compound 4 shared the structure skeleton with compound 3.Comparison of theH-NMR data of 3 and 4 indicated that compound 4 is the methyl substituted compound 3 on the benzene ring,and the difference in theC-NMR data was a methyl signal at

20.8.Compared with the data reported in literature [17],compound 4 was identified as (-)-3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin.

Table 2 1H-NMR (400 MHz,DMSO-d6) and 13C-NMR (100 MHz,DMSO-d6) data for compound 3 and 1H-NMR(400 MHz,CDCl3) and 13C-NMR (100 MHz,CDCl3) data for compound 4

TheH-NMR data of compound 5 showed the signals of two ABX coupled systems at

6.52 (1H,d,

=1.8 Hz,H-2′),6.57 (1H,d,

=8.1 Hz,H-5′),6.45 (1H,dd,

=8.1,1.8 Hz,H-6′),6.48 (1H,d,

=1.9 Hz,H-2′′),6.50 (1H,d,

=8.1 Hz,H-5′′),6.37 (1H,dd,

=8.1,1.9 Hz,H-6′′).

4.28 (1H,d,

=8.1 Hz,H-1),2.91 (1H,m,H-2),

3.77 (1H,dd,

=10.4,5.3 Hz,H-3a),3.71 (1H,dd,

=10.4,6.0 Hz,H-3b) are hydrogen signals of a group of CH-CH-CH.Furthermore,three methoxy signals at

3.08 (3H,s),3.60 (6H,s) existed in the upfield region.The coupling constant between H-1 and H-2 was 8.1 Hz,indicating that the relative configuration was

threo

.TheC-NMR data of 5 showed 18 carbon signals,including three aliphatic carbon (

84.3,53.7,62.4),three methoxy (

55.9,55.5,55.4)and twelve aromatic carbons (

131.6,114.5,145.4,144.5,114.6,121.5,130.8,113.5,146.9,146.6,120.2,111.5).Compared with the data reported in literature [18],compound 5 was determined as

threo

-2,3-

bis

(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol.

Compound 6 was found to possess the molecular structure of CHO,which was the homolog of structure 5.Comparison of theH-NMR data of 6 and 5 indicated that these two compounds have similar proton signals,except for the difference caused by 1-OH.The coupling constant between H-1 and H-2 was 6.3 Hz,indicating that the relative configuration of compound 6 was erythro.TheC-NMR data of compound 6 showed the absence of 1-OCHsignal.Compared with the spectral data of the known compound Erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol reported in literature [19],compound 6 was identified as erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol.

Table 3 1H-NMR (400 MHz,DMSO-d6) and 13C-NMR (100 MHz,DMSO-d6) data for compounds 5 and 6

TheH-NMR data of compound 7 showed the presence of a group of

trans

-double bonds at

7.87 (1H,d,

=15.8 Hz,H-5),6.39 (1H,d,

=15.8 Hz,H-4),and an olefinic hydrogen at

5.68 (1H,s,H-2) in the upfield region.Analysis ofH-NMR data of compound 7 revealed that it possesses three methylenes at

1.70 (1H,dd,

=13.4,6.8 Hz,H-8a),1.53 (1H,dd,

=13.4,2.0 Hz,H-8b),1.86(1H,dd,

=13.5,6.9 Hz,H-10a),1.61 (1H,dd,

=13.5,10.2 Hz,H-10b),3.62 (1H,d,

=7.2 Hz,H-13a),3.54 (1H,d,

=7.2 Hz,H-13b) and three methyls at

1.99 (3H,d,

=1.3 Hz,H-12),1.00 (3H,s,H-15),0.80 (3H,s,H-14).TheC-NMR data of compound 7 showed 15 carbon signals,including one carbonyl at

167.2,four spcarbons at

118.9,129.5,134.6,148.4,four oxygenated spcarbons at

63.8,75.3,81.5,85.7 and six spcarbons at

48.0,45.5,43.9,20.8,19.5,16.1.Compared with the spectroscopic data reported in literature [20],compound 7 was determined as (-)-dihydrophaseic acid.

Table 4 1H-NMR (400 MHz,DMSO-d6) and 13C-NMR (100 MHz,DMSO-d6) data for compounds 7-9

TheH-NMR data of compound 8 revealed the presence of a

trans

-double bond at

6.97 (1H,d,

=15.8 Hz,H-7),6.27 (1H,d,

=15.8 Hz,H-8),an olefinic hydrogen at

5.86 (1H,brs,H-2),a methylene at

2.64 (1H,d,

=17.0 Hz,H-6a),2.16 (1H,d,

=17.0 Hz,H-6b) and four methyls at

2.26 (3H,s,H-10),1.80 (3H,d,

=1.4 Hz,H-13),0.96 (3H,s,H-11),0.92 (3H,s,H-12).TheC-NMR data of compound 8 showed 13 carbon signals,including two carbonyls at

198.2,197.0,four olefinic carbons at

161.6,147.1,130.5,126.5,one oxygenated carbon at

78.1 and six aliphatic carbons at

49.2,41.1,27.2,24.2,23.1,18.6.Compared with the spectroscopic data reported in literature [21],the structure of 8 was determined as dehydrovomifoliol.According to optical value and literature [22],the absolute configuration was finally determined as (+)-dehydrovomifoliol.TheH-NMR data of compound 9 displayed the existence of one olefinic hydrogen at

5.78 (1H,s,H-3),one oxygenated methine at

4.08 (1H,m),two methylenes at

1.86 (1H,dt,

=14.2,2.5 Hz,H-7),1.41 (1H,dd,

=14.2,3.6 Hz,H-7),

2.29 (1H,dd,

=13.4,2.5 Hz,H-5),1.62 (1H,dd,

=13.4,3.6 Hz,H-5) and three methyls at

1.66 (3H,s,H-10),1.37 (3H,s,H-8),1.19 (3H,s,H-9).TheC-NMR spectroscopic data of compound 9 revealed the presence of 11 carbon resonances,including one ester carbonyl,two olefinic carbons,two oxygenated carbons and six aliphatic carbons.Compared with the spectral data of the known compound epiloliolide reported in literature [23],compound 9 was identified as (+)-epiloliolide.

4 Conclusion

Two known compounds,Lasiosiplodins Lasiosiplodin (1),and Ficusine C (2),and seven other compounds,namely 8-methoxymellein (3),3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin(4),Threo-1,2-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (5),Erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (6),Dihydrophaseicacid (7),Dehydrovomifoliol (8),and epiloliolide (9),were isolated from the twigs and leaves of

P.clypearia

.All compounds were obtained for the first time from

Pithecellobium

Mart.

登录APP查看全文

Asian Journal of Traditional Medicines

2021年1期