lsolation and identification of chemical constituents from Elephantopus scaber L

时间：2024-07-29

Wei Xu,Ming Bai,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 Elephantopus scaber L.,as a popular Chinese medicinal herb of the Compositae family,is widely distributed in America,Africa and Asia.In the present study,five compounds were isolated from the 70% EtOH extract of the whole herb from Elephantopus scaber L.,including two lignans and three flavones.Their chemical structures were identified based on NMR spectroscopic analysis and comparison with the data reported in literature.It is noteworthy that compounds 1-5 were isolated from Elephantopus scaber L.for the first time and their chemotaxonomic significance was discussed.

Keywords:Elephantopus scaber L.;separation and purification;chemical constituents;structural elucidation

1 Introduction

Elephantopus scaber

L.,belonging to

Elephantopus

plant of Compositae,is a common Chinese herbal medicine widely used in folk.Genus Elephantopus (Asteraceae) is distributed mainly in the Neotropical area and Europe,and partly in Africa and Asia [1,2].For a long time,it has become popular as a medicinal herb in many countries in Southeast Asia,Latin America and Africa [3,4].The whole plant of

E.scaber

(Compositae) is used in Chinese medicine as a diuretic,antifebrile,antiviral,and antibacterial agent as well as for treating hepatitis and bronchitis [5].The pharmacological properties of the leaf extracts have been evaluated,including antidiabetic,antitumor,antiasthmatic,hepatoprotective,anti-oxidative and analgesic activities [6-10].Phytochemical investigations have isolated sesquiterpenoids,sesquiterpene lactones,phenols,triterpenoids,alkaloid,sterol,flavonoids,anthraquinones,sugar derivatives,and organic acids compounds from

E.scaber

[11-13].In this study,five compounds were systematically extracted and isolated from

E.scaber

(Fig.1).It is noteworthy that compounds 1-5 were isolated from

E.scaber

for the first time.

2 Materials and methods

2.1 General procedures

The 1D NMR spectra were recorded on Bruker ARX-400 MHz spectrometers with tetramethylsilane(TMS) as an internal standard.Silica gel (100-200 mesh,200-300 mesh,Qingdao Marine Chemical Co.,China),Sephadex LH-20 (25-100

m,Green Herbs Science and Technology Development Co.,Ltd.China) and ODS gel (60-80

m,Merck,Germany) were used for column chromatography and silica gel GF254 (Qingdao Marine Chemical Co.,China) was used for TLC.Semipreparative RP-HPLC isolation was realized by an instrument(Shimadzu,Kyoto,Japan) equipped with LC-6AD pump and SPD-20A ultraviolet-visible light absorbance detector using YMC Ccolumn(250 mm × 10 mm,5

m).

2.2 Plant material

The whole herb of

Elephantopus scaber

was collected in September 2017 from Jingxi city,Guangxi Zhuang Autonomous Region,People’s Republic of China.The sample was authenticated by Professor Jincai Lu (School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University).A voucher specimen with the serial number 20170905 was deposited in the herbarium of the Department of Natural Products Chemistry,Shenyang Pharmaceutical University.

2.3 Extraction and isolation

Dried and grounded into powder,the whole herb of

Elephantopus scaber

(60 kg) was extracted four times with 70% EtOH,and the combined extract was concentrated under vacuum to afford a residue (1.8 kg).The residue was successively partitioned with petroleum ether (PE),EtOAc and

-BuOH to give a PE partition (100 g),an EtOAc partition (400 g) and an

-BuOH partition (480 g).The EtOAc partition was fractionated by vacuum flash chromatography on silica gel CC,and eluted with a gradient eluent of CHCl-MeOH from 100:1 to 3:1 to yield six fractions (A-F).Fraction C (63 g) was then chromatographed with HP-20 macroporous resin CC and eluted with aqueous ethanol (30%-90%) to obtain Fr.1 (16 g),2 (19.4 g),and 3 (5.2 g).Fr.2 was subjected to a Sephadex LH-20 column (120 cm × 3 cm) with 95% EtOH as the mobile phase to remove pigments,further chromatographed on an open-type ODS CC and eluted with EtOH-HO (20%-80%) to give three fractions (Fr.2-1,Fr.2-2,Fr.2-3).Fr.2-1,eluted with PE/EtOAc (10:1-1:1),was chromatographed on silica gel CC to afford five subfractions (Fr.2-1-Fr.2-1) on the basis of TLC analysis.Fr.2-1was separated by preparative reversed-phase HPLC using a mobile phase of CHOH-HO (55:45,

v/v

)to obtain six subfractions (Fr.2-1-Fr.2-1.).Fr.2-1was further subjected to semipreparative HPLC using CHCN-HO (40:60,

v/v

2.5 mL/min)as the mobile phase to give 4 (26.3 mg) and 5(40.7 mg).With a similar separation procedure,subfractions Fr.2-1,Fr.2-1were purified by RP-HPLC with the mobile phase CHCN-HO(35:65,

v/v

,2.5 mL/min) to produce compounds 1(48.2 mg) and 2 (64.9 mg).Compound 3 (18.5 mg)was separated from subfraction Fr.2-1by semipreparative HPLC and eluted with CHCNHO (30:70,

v/v

,2.5 mL/min).

Fig.1 Structures of compounds 1-5

(to be continued)

Continued fig.1

3 Results and discussion

TheH-NMR spectrum (Table 1) of compound 1 indicated the presence of four methoxy groups at

3.90 (12H,s),two oxygen-bearing methylene group signals [

3.83 (1H,m,H-9a),3.34 (1H,m,H-9b),4.13 (1H,d,

=9.6 Hz,H-9′a),3.85 (1H,m,H-9′b)],two pairs of equivalent aromatic protons[

6.60 (2H,s,H-2,6),6.59 (2H,s,H-2′,6′)],and two methine protons [

3.30 (1H,m,H-8′),2.90(1H,m,H-8)].TheH-NMR data also showed that compound 1 possessed typical hydrogen signals at 7,7′ position of bis-tetrahydroturan ring [

4.42 (1H,d,

=7.1 Hz,H-7),4.85 (1H,d,

=5.1 Hz,H-7′)].TheC-NMR data (Table 2) of compound 1 showed 22 carbon signals,including two phenyl carbons(

147.9×2,147.8×2,134.9,134.2,131.5,128.8,103.5×2,102.9×2),four oxygen-bearing carbons (

87.1,81.5,70.2,69.8),four methoxy carbons (

56.0×4) and two methine carbons (

53.9,49.3).Analysis of the above data showed the presence of symmetrical C-Cmoieties in the molecule.Besides,the signals at

87.1,81.5,70.2,69.8,53.9,49.3 constituted a typical unit of furofuran lignan.These results together showed that compound 1 might be a furofuran lignan.

Cis

configuration is charaterized by the C-8′,C-8 bond of naturally occurring furofuranic-type lignans.Therefore,the relative configuration of 1 was determined as 7′H,8′H-

trans

/7H,8H-

cis

according to Δ

-9=0.49 (H-9) and Δ

-9′=0.28 (H-9′) [14].After comparing the experimental spectroscopic data with that reported in literature [15],the structure of 1 was determined to be epi-syringaresinol.TheH-NMR spectrum of 2 revealed the presence of typical hydrogen signals at 7,7′position of bis-tetrahydroturan ring at

4.83 (2H,d,

=4.2 Hz,H-7′,7).Detailed analysis of theH andC-NMR spectra (Table 1,Table 2) showed that 2 and 1 had the same planar structure,but different configurations.The configuration of 2 was 7′H,8′H-

cis

/7H,8H-

cis

,which was confirmed by Δ

-9=0.36 (H-9) and Δ

-9′=0.36 (H-9′) [14].By comparing the NMR data with those reported in literature [16],the structure of 2 was determined to be dia-syringaresinol.According to theH andC-NMR data (Table 1,Table 2) of 3,the signals at

7.82 (2H,d,

=8.8 Hz,H-2′,6′),6.84 (2H,d,

=8.8 Hz,H-3′,5′)revealed the existence of a para-substituted benzene ring,the signals at

5.05 (1H,d,

=12.1 Hz) and 4.65 (1H,d,

=12.1 Hz) indicated that this was a 3-substituted flavanone type,and the deshielded shift of H-3 indicated that there was an oxygen-bearing atom at C-3,namely dihydroflavonol,which was confirmed by the presence of a conjugated carbonyl group (

181.0) and two methines of oxycarbons(

77.5,71.6).Two aromatic proton signals [

5.86(1H,d,

=2.2 Hz) and 5.91 (1H,d,

=2.2 Hz)]combined with two methine carbon signals (

99.8,94.6) proved that the A-ring was 5,7-disubstituted.All the above evidence was consistent with the physicochemical and spectroscopic data of those reported in literature [17].Therefore,compound 3 was identified as dihydrokaempferol.Analysis of the 1D NMR data (Table 1,Table 2) of 4 revealed that it possessed a pair of AXaromatic protons at

7.85 (2H,d,

=8.9 Hz,H-2′,H-6′),6.88 (2H,d,

=8.8 Hz,H-3′,H-5′),a methoxyl group signal at

3.89 (3H,s),twelve aromatic carbons (

161.5,95.2,161.7,107.6,157.6,120.9,128.2×2,116.1×2,161.1,102.3),two olefin carbons (

163.1,99.8),and one carbonyl carbon (

181.0).These data indicated that it had a flavone skeleton and no hydroxyl was detected at C-3.The signal at

6.63 (1H,s) also indicated that it was a 3-unsubstituted flavanone type.Therefore,the signal at

6.27 (1H,s) was attributed to the proton of A-ring.The signals of

5.44 (1H,dd,

=12.0,2.2 Hz,H-1′′) and

63.1 (C-1′′),34.0 (C-2′′),66.9 (C-3′′),77.5 (C-4′′),71.6 (C-5′′),20.7(C-6′′) showed the presence of a glycoside,in which the large coupling constant [

5.44 (1H,dd,

=12.0,2.2 Hz,H-1′′)]indicated the axial orientation of H-1″and the

-linkage of the sugar moiety with flavone.Besides,the chemical shift of C-1′′ (

63.1) was much lower than

-linked glycosidic bond (

90-110),which indicated the presence of C-linked glycoside [18].The anomeric proton signal at

5.44 ppm was coupled with the methylene protons at 2.37 and 1.64 ppm,with the coupling constants of 12.0 and 2.2 Hz respectively.H-5′′ was coupled with the methyl doublet,indicating that the sugar was a 2,6-dideoxy sugar.Moreover,the coupling pattern of H-3″ (d,

=3.0 Hz) suggested the equatorial orientation of H-3″,and the coupling constant (

=9.6 Hz)of H-4″ and H-5″ indicated that these two protons had

trans

-diaxial orientations.The sugar was thus identified as digitoxose.Based on these results and the comparison of experimental spectroscopic data with those reported in literature [19],the structure of 4 was determined to be aciculatin.TheH-NMR data (Table 1) of compound 5 showed the presence of a benzyl moiety in the molecule,which was confirmed by the proton signals at

8.06 (2H,m,H-2′,6′),6.92 (2H,m,H-3′,5′),6.90 (1H,m,H-4′),and by the carbon signals at

128.0 (C-2′,6′),128.6 (C-3′,5′),128.1 (C-4′),and 66.6 (C-7′).The presence of an AMX spin system in the molecule was confirmed by the characteristic proton signals at

6.90 (1H,d,

=9.0 Hz,H-3),7.28 (1H,dd,

=9.0,3.0 Hz,H-4) and 7.44 (1H,d,

=3.0 Hz,H-6).The signals of

4.65 (1H,d,

=7.6 Hz,H-1′′) and

60.6 (C-6′′),69.5 (C-4′′),73.2 (C-2′′),77.0 (C-3′′),76.4 (C-5′′) and 102.4 (C-1′′) showed the presence of a

-D-glucopyranosyl.TheH andC-NMR data was consistent with the physicochemical and spectroscopic data of those reported in literature [20],and compound 5 was identified as benzyl 5-

-D-glucopyranosyl-2,5-dihydroxybenzoate.

Table 1 1H-NMR data (400 MHz) of compounds 1-5

(to be continued)

Continued table 1

Table 2 13C-NMR data (100 MHz) of compounds 1-5

(to be continued)

Continued table 2

4 Chemotaxonomic significance

Elephantopus

is a genus composed of about 30 species worldwide,mainly distributed in South America.Only 2 species,

E.scaber

and

E.tomentosus

,were found in Southwest China [21].In the previous studies,12 compounds were identified from the PE-soluble extracts of

E.scaber

and

E.tomentosus

using GC-MS.DNA fingerprinting analysis has indicated that

E.scaber

is closely related to

E.tomentosus

.These results may explain why these two species are mainly distributed in South China.In this phytochemical study on

E.scaber

,five compounds were obtained,including two lignans (1,2) and three flavones (3-5).To the best of our knowledge,all of these compounds were obtained from

E.scaber

for the first time.It is known that genus

Elephantopus

is a rich source of sesquiterpene lactones,the most important chemotaxonomic markers for this genus.The identification of compounds (1-5) reveals several potential chemotaxonomic markers for

E.scaber

,and thus provides new information for the chemical characteristics of Compositae family.Compounds 1 and 2 have been reported from Annonaceae Juss and Lauraceae respectively,compounds 3 and 4 have been isolated from Hypericaceae and Chrysopogon aciculatis respectively,and compound 5 has been obtained from Compositae.In conclusion,the existence of these compounds in other species indicates that there is a close chemotaxonomic relationship between

E.scaber

and the species in other genera.

5 Conclusion

In summary,this study reported the chemical components of ethanol extracts from

Elephantopus scaber

L..Compounds 1-5 were obtained by systematic extraction and isolation,and the structures of compounds were confirmed by comparing their spectral data with those reported in the literature.This study provided guidance for further research on the chemical constituents of

Elephantopus scaber

L..