Compounds from the flowers and fruits of Abelmoschus esculentus (L.) Moench

2021-11-05SihuiMiRuiGuoChuangXieXiaoxiaoHuang

Asian Journal of Traditional Medicines 2021年5期

Sihui Mi,Rui Guo,Chuang Xie,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 Abelmoschus esculentus (L.) Moench was previously reported to have various phytochemicals.Chemical fractionation yielded from the ethanolic extract of the A.esculentus ten compounds,including four alkaloids (4-6,8) and six phenolic acids (1-3,7,9-10).The structures of the compounds were elucidated through extensive spectra analyses and comparison of the experimental data with reported data.It is worth mentioning that compounds 1,2 and 5-10 were isolated from Abelmoschus esculentus (L.) for the first time.

Keywords:Abelmoschus esculentus (L.) Moench;phenolic acids;alkaloids

1 Introduction

Abelmoschus esculentuss

(L.) Moench belongs to Malvaceae family and is an annual herb of the Abelmoschus genus.The plant is native to Africa and has been planted in many countries around the world,mainly in tropical,sub-tropical,and warm temperate regions.In recent years,okra has also been widely cultivated in North and South China [1].It has become a part of the diet all over the world [2].Okra fruit has been paid increasing attention because of its health benefits [1].The edible part is rich in protein,unsaturated fatty acids,vitamins,and flavonoids [3,4].Previous investigations have revealed that

A.esculentuss

contains a wide variety of phytochemicals,including flavonoids,sterols,triterpenes,phenolic acids,amino acids,nucleotides and lipids [5,6].Besides,it also shows biological activity such as antioxidation,hypoglycemic,antiinflammatory and lipid-lowering effects [7,8].In order to search for the bioactive compounds in

A.esculentuss

,we herein reported the isolation and identification of four alkaloids (

4-6

) and six phenolic acids (

1-3

9-10

).It is important that compounds

and

5-10

were isolated from

A.esculentus

for the first time.

2 Materials and methods

2.1 Experimental instruments

NMR experiments were performed on Bruker ARX-400 and AV-600 spectrometers in CDCl(

7.26 and

77.2),with TMS as the internal standard.Chromatographic silica gel (200-300 mesh,Qingdao Marine Chemical Factory,Qingdao,China) and reversed phase C(RP-C) silica gel (50

m,Japan)were employed for column chromatography.RPHPLC separations were conducted using a Shimadzu LC-20AR series pumping system equipped with an SPD-20A UV detector,a YMC Pack ODS-A column(250 mm × 10 mm,5

m,YMC Company,Kyoto,Japan),TLC silica gel GF254 (Qingdao Marine Chemical Co.,China),Sephadex LH-20 (25-100

m,Green Herbs Science and Technology Development Co.,Ltd.China),and MCI resin (CHP20P,75-150 m,Mitsubish Chemical Corporation).

2.2 Plant material

The plants of

A.esculentus

were collected in 2017 in Bozhou,Anhui and identified as

A.esculentuss

dried flowers and fruits by professor Jincai Lu from the school of traditional Chinese medicine of Shenyang Pharmaceutical University.

2.3 Extraction and isolation

The dried flowers and fruits of

A.esculentuss

(30 kg) were extracted with 70% EtOH three times(2 h for each time).The resulting solutions were filtered,combined,and concentrated under reduced pressure to obtain methanol residue (1200 g).The residue was suspended in water and partitioned successively with petroleum ether,ethyl acetate and

-butanol to give the corresponding extracts:petroleum ether (40 g),ethyl acetate (360 g)and

-butyl alcohol layer (220 g).The extract ethyl acetate (360 g) was separated by silica gel CC,using the gradient elution of CHCl-MeOH (50:1-1:1) to obtain three fractions A1-A3.Fraction A1 was further separated by MCI,using gradient elution of MeOH-HO (90:10-100:0) to obtain three fractions A1A-A1C.Fractions A2 and A3 were further separated by ODS,using gradient elution of MeOH-HO (30:70-100:0) to obtain five fractions A2A-A2B and A3A-A3C.The above subfractions were further separated by silica gel CC,preparative HPLC and semipreparative HPLC.Compounds

(3.9 mg)

and

(16.0 mg) were

obtained from fraction A2A.Compound

(50.0 mg) was obtained from fraction A3C.Subfraction A2B was separated to give compounds

(24.0 mg) and

(40.0 mg).Compounds

(38.0 mg),

(69.0 mg) and

(42.0 mg) were purified from fraction A1C.Compound

(9.1 mg) was obtained from fraction A3B.Compound

(3.5 mg) was obtained from fraction A1B.

trans

-coumaroyl tyramine (

):white powder(MeOH);H-NMR (400 MHz,DMSO-

7.38(2H,d,H-2/6),

6.79 (2H,d,H-3/5),

7.32 (1H,d,H-7),

6.40 (1H,H-8),

7.01 (2H,d,H-2′/6′),

6.67(2H,d,H-3′/5′),

2.64 (2H,t,H-7′),

3.33 (2H,dd,H-8′),

8.03 (1H,t,N-H),

9.53 (2H,brs,OH-4/4′).

trans

-Feruloyltyramine (

):pale yellow solid(MeOH);H-NMR (400 MHz,DMSO-

7.12(1H,s,H-2),

6.80 (1H,d,

=8.0 Hz,H-5),

6.98(1H,s,H-6),

7.34 (1H,d,

=15.7 Hz,H-7),

6.46(1H,d,

=15.7 Hz,H-8),

7.02 (2H,d,

=8.3 Hz,H-2′/6′),

6.70 (2H,d,

=8.3 Hz,H-3′/5′),

2.66(2H,t,

=7.9 Hz,H-7′),

3.35 (2H,dd,

=12.7,6.5 Hz,H-8′),

8.02 (1H,s,N-H),

9.30 (1H,brs,OH),

3.80 (3H,s,OCH);C-NMR (100 MHz,DMSO-

129.6 (C-1),

110.8 (C-2),

147.9(C-3),

148.4 (C-4),

115.7 (C-5),

121.6 (C-6),

139.0 (C-7),

119.0 (C-8),

165.5 (C-9),

126.4(C-1′),

129.5 (C-2′/6′),

115.2 (C-3′/5′),

155.7(C-4′),

34.5 (C-7′),

40.8 (C-8′),

55.6 (OCH).Perlolyrine (

):yellow acicular crystal(MeOH);H-NMR (400 MHz,DMSO-

8.37(1H,d,

=5.1 Hz,H-3),

8.06 (1H,d,

=5.1 Hz,H-4),

8.26 (1H,d,

=7.8 Hz,H-5),

7.28 (1H,t,

=7.8 Hz,H-6),

7.59 (1H,t,

=7.6 Hz,H-7),

7.78 (1H,d,

=7.6 Hz,H-8),

3.69 (2H,m,H-9),

7.21 (1H,d,

=3.2 Hz,H-3′),

6.58 (1H,d,

=3.2 Hz,H-4′),

4.67 (2H,s,CH),

5.48 (1H,t,OH),

11.26 (1H,brs,NH);C-NMR (100 MHz,DMSO-

133.2 (C-1),

138.2 (C-3),

113.6 (C-4),

129.4 (C-4a),

120.6 (C-4b),

121.6 (C-5),

119.7 (C-6),

128.3 (C-7),

112.5 (C-8),

140.9(C-9),

130.5 (C-9a),

129.9 (C-1′),

156.8 (C-2′),

109.6 (C-3′),

109.0 (C-4′),

152.2 (C-5′),

55.9(CH).

-indole-3-carboxaldehyde (

):light yellow powder (MeOH);H-NMR (400 MHz,CDCl):

8.96 (1H,brs,H-1),

7.86 (1H,d,

=2.9 Hz,H-2),

8.33 (1H,m,H-4),

7.31 (1H,d,

=5.5 Hz,H-5),

7.35 (1H,d,

=5.5 Hz,H-6),

7.45 (1H,m,H-7),

10.07 (1H,s,CHO).2-hydroxyphenylacetic acid methyl ester (

):white powder (MeOH);H-NMR (400 MHz,CDCl):

7.39 (1H,d,

=7.6 Hz,H-3),

7.29 (1H,t,

=7.6 Hz,H-4),

7.07 (1H,t,

=7.6 Hz,H-5),

6.88 (1H,t,

=7.6 Hz,H-6),

7.79 (1H,brs,OH),

3.70 (3H,s,CH).

-methoxy benzoic acid (

):white needle crystal (MeOH);H-NMR (400 MHz,CDCl):

7.95(2H,d,

=8.5 Hz,H-2/6),

6.88 (2H,d,

=8.5 Hz,H-3/5),

3.89 (3H,s,OCH).

3 Results and discussion

Structural elucidation of all isolated compounds from

A.esculentuss

(Fig.1) was performed by comparing their spectral data with literature values.Among these compounds,compounds

and

5-10

were reported as the first time isolated from the plant.

Fig.1 Chemical structures of compounds 1-10

Compound

was isolated as a pale yellow acicular crystal.TheH-NMR spectrum showed the signals of three ABX systems protons at

7.63 (dd,

=8.5,2.0 Hz,H-6′),6.81 (d,

=8.5 Hz,H-5′) and 7.51 (d,

=2.0 Hz,H-2′) on B-ring,two aromatic proton signals at

6.18 (s,H-6)and 6.39 (s,H-8) on A-ring protons,together with four phenolic hydroxyl protons at

12.61 (s),10.88 (s),9.76 (s) and 9.17 (s).From the above data,it can be easily deduced that there is a flavone skeleton in the molecule.In addition,an anomeric proton signal was found at

5.31 (d,

=7.8 Hz,H-1′′) and four proton signals at

3.38-4.07.An anomeric carbon signal of

-D-glucose was observed at

101.8 (C-1′′).TheH-NMR also showed one methyl proton signal at

1.73 (s).TheC-NMR spectrum showed 23 carbon signals.Six of the carbon signals were attributed to a glucose unit,while the remaining carbon signals included two benzene rings,two carbonyl carbon signals and one methyl carbon signal.The structure was confirmed based on analysis ofH andC-NMR data and the comparison of the experimental spectroscopic data with those in the literature [9].Therefore,the structure of compound

was identified as quercetin-3-

-(6’’-

-acetyl)-

-D-galactranoside.TheH andC-NMR spectrums of compound

displayed similar signals to compound

.According to the comparison of NMR data,they had the same structure skeleton.

The sugar moiety of compound

showed one hydroxyl proton signal at

5.12-4.43(m),the absence of signal of acetyl and the presence of a rhamnose.Based on the above results and the comparison with the data in the literature [10],compound

was identified as hyperin.TheH-NMR spectrum of compound

showed four AA′BB′ system protons at

7.99 (d,

=8.8 Hz,H-2′,H-6′),

6.85 (d,

=8.8 Hz,H-2′,H-6′),

7.36(d,

=8.5 Hz,H-2′′,6′′),and 6.79 (d,

=8.5 Hz,H-3′′,5′′),and two olefinic protons at

7.35 (1H,d,

=13.9 Hz,H-7′′′) and 6.10 (1H,d,

=13.9 Hz,H-8′′′).In addition,an anomeric proton at

5.45(d,

=7.3 Hz,H-1′′) and four signals at

3.17-3.37 (m) indicated the presence of

-glucose.The key to elucidating the structure of this compound is the two proton signals on glucose 6′′ at

4.28 (d,

=10.5 Hz,H-6′′) and 4.03 (m,H-6′′).TheH-NMR also showed two typical hydroxyl protons of flavonoid at

12.57 (s) and 10.35 (brs).TheC-NMR spectrum showed 30 carbon resonances consisting of two carbonyl carbons and three benzene rings.Based on the above data and the comparison with the data in the literature [11],compound

was identified as kaempferol-3-

-[6’’-

trans

-coumaroyl)]-

-D-glucopyranoside.TheH-NMR spectrum of compound

showed two olefinic protons at

7.32 (d,

=15.7 Hz,H-7)and 6.40 (d,

=15.7 Hz,H-8),four AA’BB’ system protons at

7.38 (d,

=8.5 Hz,H-2,6),6.79 (d,

=8.5 Hz,H-3,5) and

7.01 (d,

=8.3 Hz,H-2’,6’),6.69 (d,

=8.3 Hz,H-3’,5’),one proton signal at

8.03 (t,

=5.3 Hz,N-H) for NH and an active hydrogen proton signal at

9.53 (brs,OH).At the high field,two proton signals appeared at

2.64(t,

=7.3 Hz,H-7’) and 3.33 (t,

=7.3 Hz,H-8’).Based on the above data and the comparison with the data in the literature [12],compound

was identified as

trans

-coumaroyl tyramine.Compound

was obtained as a yellowish solid.Analysis of its NMR data showed that it was highly similar to compound

except for one more methoxyl group.TheH-NMR data of

revealed three ABX system protons at

6.98 (s,H-6),6.80 (d,

=8.0 Hz,H-5) and 7.12 (s,H-2) in contrast with four AA’BB’ system protons of compound

.TheC-NMR data showed 18 carbon signals,including one carbon of the amide group,two olefinic carbons and 12 carbons of aromatic rings.Based on the above results and the comparison with data in the literature [12],compound

was identified as

trans

-Feruloyltyramine.TheH-NMR spectrum of compound

showed an aryl hydroxyl methyl proton at

4.67 (s,H-6),four aromatic protons at

8.26 (d,

=7.8 Hz,H-5),

7.28 (t,

=7.8 Hz,H-6),

7.59 (d,

=7.6 Hz,H-7) and

7.78 (d,

=7.6 Hz,H-8),two olefinic protons at 8.37 (d,

=5.1 Hz,H-3),and

8.06 (d,

=5.1 Hz,H-4),and one signal for NH at

11.26(brs,N-H).The signals at

7.21 (d,

=3.2 Hz,H-3′) and 6.58 (d,

=3.2 Hz,H-4′) belonged to H-3′and H-4′ of the furan ring.TheC-NMR spectrum showed 16 carbon signals assigned to 5 quaternary carbons,8 methines,and 3 methylenes.Among the 16 carbon signals,there were 11 aromatic or olefinic carbons (

140.9,138.2,133.2,130.5,129.4,128.3,121.6,120.6,119.7,113.6,112.5),two oxygenated methines (

152.2,156.8) and an oxygenated methylene (

55.9).Based on the detailed comparison of the NMR data of compound

with those in the literature [13],compound

was identified as perlolyrine.TheH-NMR spectrum of compound

showed the existence of three aromatic proton signals at

6.91 (s,H-2),6.76 (s,H-5) and 6.69 (d,

=4.4 Hz,H-4′),two methylene protons at

2.54 (t,

=3.5 Hz,H-7′) and 1.68 (m,H-8′),an oxygenated methylene at 3.42 (t,

=6.4 Hz,H-9′),and two methoxy protons at

3.77 (s) and 3.74 (s).There were also typical proton signals at

5.41 (d,

=10.6 Hz,H-7),3.61 (dd,

=10.6,6.8 Hz,H-8)and 3.69 (m,H-9),accounting for the CH(O)-CH-CH(O) unit.All the assignments suggested compound

might be a dihydrobenzofuran-type neolignan.Based on the above results and the comparison with the data in the literature [14],compound

was identified as (−)-7

dihydrodehydro-coniferyl alcohol.TheH-NMR spectrum of compound

showed one aldehyde proton at

10.07 (s),a signal for NH at

8.96 (brs,N-H),and four aromatic protons at

8.33 (m,H-4),7.31 (m,H-5),7.35 (m,H-6),and 7.45 (m,H-7).Based on the above results and the comparison with the data in the literature [15],compound

was identified as 1

-indole-3-carboxaldehyde.TheH-NMR spectrum of compound

indicated the presence of one phenolic hydroxyl proton signal at

7.79 (brs),four aromatic protons at

7.39 (d,

=7.6 Hz,H-3),

7.29 (t,

=7.6 Hz,H-4),7.07 (t,

=7.6 Hz,H-5) and 6.88 (d,

=7.6 Hz,H-6),one methoxy proton at

3.70 (s,OCH),and one methylene proton signal at

2.95 (s,H-7).Based on these data and the comparison with the data in the literature [16],compound

was determined to be 2-hydroxyphenylacetic acid methyl ester.A detailed comparison of the NMR data of compound

with the data in the literature revealed that compound

was

-methoxy benzoic acid [17].TheH-NMR spectrum of compound

showed a set of AA’BB’ system protons at

7.95 (d,

=8.5 Hz,H-2,6) and 6.88 (d,

=8.5 Hz,H-3,5),and a methoxyl group at

3.89 (s,6’-OCH).Therefore,the structure of compound

was determined.

4 Conclusion

Ten compounds

(1-10)

were isolated and elucidated from the

Abelmoschus esculentuss

.It is noteworthy that compounds

and

5-10

were reported for the first time in

this plant and even in its family.Our findings might provide a valuable information source for further study on the biological activity of this plant.The present study also indicated that

A.esculentuss

might have broad prospects in the study of natural products.

Asian Journal of Traditional Medicines

2021年5期