Chemical composition and glucose uptake effect on 3T3-L1 adipocytes of Ligustrum lucidum Ait.flowers

Lili Cui,Jinmei Wang,Mengke Wang,Baoguang Wang,Zhenhua Liu,Jinfeng Wei,Wenyi Kang

a National R&D Center for Edible Fungus Processing Technology,Henan University,Kaifeng,475004,China

b Kaifeng Key Laboratory of Functional Components in Health Food,Henan University,Kaifeng,475004,China

c Joint International Research Laboratory of Food&Medicine Resource Function,Henan Province,Henan University,Kaifeng,475004,China

ABSTRACT Ligustri lucidi Fructus is a traditional Chinese medicine and possesses various bioactivities,including hypoglycemic effect.Ligustrum lucidum Ait flowers are poorly investigated.Thus,we hypothesized that L.lucidum flowers also could have hypoglycemic effect.Chemical composition and glucose uptake effect of L.lucidum flowers on 3T3-L1 adipocytes were investigated.In this study,the components of L. lucidum flowers were investigated by various chromatographic and spectroscopic methods and the effects of L. lucidum flowers on the induction of glucose uptake were investigated by 3T3-L1 adipocytes.Seven compounds were isolated and identified from L. lucidum flowers,including ursolic acid (1),kaempferol-7-O-α-Lrhamnoside (2,KR),β-sitosterol (3),β-daucosterol (4),kaempferitrin (5,KF),10-hydroxy oleuropein (6,HO),and kaempferol-3-O-β-D-glucopyranoside-7-α-L-rhamnopyranoside (7,KGR).The results of glucose uptake showed that total extract (TE),KR,KF and HO from L. lucidum flowers significantly enhanced glucose uptake.This study demonstrated that compounds 2,5-7 were isolated for the first time from this plant and compounds 2 and 7 were isolated for the first time in genus Ligustrum,L. lucidum flowers,KR,KF and HO may possess potential hypoglycemic effect.

Keywords:Ligustrum lucidum flowers Chemical composition 3T3-L1 adipocytes

1.Introduction

Obesity and type 2 diabetes,two common metabolic diseases worldwide,have become global public health problems[1-3].Obesity is a complex,multifactorial chronic disease considered to be a risk factor associated with type 2 diabetes.It arises from an imbalance between energy intake and energy expenditures that lead to the pathological growth of adipocytes.Differentiation and hypertrophy of adipocytes are the fundamental process of obesity[4].Obesity is a condition under which adipocytes accumulate a large amount of fat.Meanwhile,the preadipocytes play a role by differentiation into mature adipocytes and increase of fat mass.Recent reports have proposed mechanisms to reduce the incidence of obesity,including inhibition of differentiation of 3T3-L1 preadipocytes and the increased lipolysis in 3T3-L1 mature adipocytes [5].Obesity-induced insulin resistance increases the likelihood of type 2 diabetes.Insulin plays a key regulatory role in stimulating the transport of blood glucose into peripheral tissues,such as skeletal,muscle and adipose.The insulin-mediated glucose uptake is impaired in insulin resistance,resulting in the decreased glucose uptake into muscle or adipose cells[6,7].

Ligustri lucidi Fructusis a traditional Chinese medicine commonly used to treat deficiency of liver-yin and kidney-yin,vertigo,tinnitus,soreness and weakness of waist and knees,premature graying hair,blurred vision,inner heat of diabetes,bone steaming and hectic fever in clinic.Ligustri lucidi Fructushas also been reported to possess various bioactivities,including antioxidant,anti-inflammatory,anti-cancer,hypoglycemic and immune regulation [8].However,the effects ofL.lucidumflowers remain practically unknown.Zhang et al.[9]reported that a new seco-iridoid fromLigustri lucidi Fructuscan inhibit triglyceride accumulation.Gao et al.[10]reported the antidiabetic potential of oleanolic acid inL.lucidum.So,we hypothesized thatL.lucidumflowers might have hypoglycemic effect and selected it to investigate.

In this study,chemical composition and glucose uptake effect ofL.lucidumflowers were investigated.Its chemical compositions were investigated by various chromatographic and spectroscopic methods.A total of seven compounds were isolated and identified fromL.lucidumflowers,including ursolic acid (1),kaempferol-7-O-α-L-rhamnoside (2,KR),β-sitosterol(3),β-daucosterol (4),kaempferitrin (5,KF),10-hydroxy oleuropein (6,HO),and kaempferol-3-O-β-D-glucopyranoside-7-α-Lrhamnopyranoside(7,KGR).Among which,compounds 2,5-7 were isolated for the first time from this plant and compounds 2,7 were isolated for the first time inLigustrumgenus.In addition,glucose uptake effect of these compounds and total extract (TE) fromL.lucidumflowers were evaluated with a 3T3-L1 adipocytes model.

2.Materials and methods

2.1.Chemicals and biochemicals

Dimethyl sulfoxide(DMSO),dexamethasone(DEX),3-isobutyl-1-methy lxanthine (IBMX) and insulin (INS) were obtained from Sigma (St.Lois MO,USA).Dulbecco’s modification of eagle’s medium (DMEM),trypsin,and penicillin-streptomycin (PS) were purchased from the Solarbio (Beijing,China).Fetal bovine serum(FBS)was obtained from Zhejiang Tianhang Biotechnology Co.,Ltd.(Hangzhou,China).FFA detection kit was purchased from Nanjing Senbeijia Biotechnology Co.,Ltd.(Hangzhou,Zhejiang,China).Glucose Assay Kit was purchased from Shanghai Rongsheng Biological Pharmaceutical Co.,Ltd.(Shanghai,China).Rosiglitazone tablets were purchased from Hengrui Pharmaceutical(Princeton,NJ,USA).

1H NMR and13C NMR spectra were measured on a Bruker Ascend TM 400 spectrometer (Málaga,Spain).ACXTHLC-3000 preparative HPLC equipped with binary pumps,an UV/VIS detector,and a manual injection valve was used for the high-performance column chromatography (HPLC).Moderate-pressure liquid chromatography(MPLC)was performed using a Sepacore glass column c-690(230 mm×40 mm,ID in 36 mm)filled with silica gel(mesh 60,50 g).Silica gel GF 254 TLC and silica gel(40-80 mesh,200-300 mesh) were purchased from Yantai Huiyou Development Co.Ltd.(Shandong,China).Column chromatography was performed on Sephadex LH-20 (Pharmacia).D101 macroporous resin was purchased from Haiguang Chemical Ltd(Tianjin,China).Centrifugation was performded on Shanghaianting TGL-16 grcentrifuge(Beckman Coulter,Brea,CA,USA).

2.2.Plant

L.lucidumflowers were collected from Huaxi Distric Wetland Park located in the Guiyang City,Guizhou Province,China and were identified asL.lucidumflowers by Professor Qianjun Zhang of Guizhou University(Guiyang,Guizhou,China).Specimens were deposited in National R & D Center for Edible Fungus Processing Technology,Henan University.The deposition number is 201506.

2.3.Extraction and isolation of compounds 1-7

The method is similar to our previous researches[11].L.lucidumflowers (475 g,dry weight) were extracted with petroleum ether by heating reflux for two times.Then,L.lucidumflowers were filtered with gauze to yield residues,which were extracted with 70% ethanol at room temperature for three times,three days for each time.The extracts were evaporated under reduced pressure to obtain TE.Subsequently,TE was dissolved by ethanol and continuously partitioned with water,20% ethanol-water,40%ethanol-water,60%ethanol-water and 95%ethanol on D101 macroporous resin column chromatography(Bio-Rad,Hercules,CA,USA).The eluent was concentrated and yielded to the water fraction,20%ethanol fraction(7 g),40%ethanol fraction(60 g),60%ethanol fraction(24 g)and 95%ethanol fraction(16 g),respectively.

Forty percent of ethanol fraction(60.0 g)was subjected to flash column chromatography on silica gel (200-300 mesh) and gradient elution with mixtures of dichloromethane-methanol (100:0,50:1,25:1,20:1,15:1,5:1,2:1,V/V) in sequence.The eluent was combined into two fractions by thin layer chromatography (TLC)monitoring.The first fraction(10 g)was further separated on a silica gel column,using dichloromethane-methanol (10:1,7:1,5:1,3:1,2:1,1:1)as mobile phase and two sub-fractions were obtained(F1and F2).F1was successively separated on Sephadex LH-20 to yield compound 5 (54 mg,KF) and F2was successively separated on RP-HPLC column to yield compound 6(71 mg,HO).The second fraction was successively separated on Sephadex LH-20(methanol)and silica gel column to obtain compound 7(547 mg,KGR).

Sixty percent of ethanol fraction (20.0 g) was subjected to silica gel column on middle-pressure liquid chromatography,and gradient elution was performed with mixtures of dichloromethane-acetone (50:1,40:1,30:1,20:1,10:1,1:1,V/V),dichloromethane-methanol(5:1,4:1,3:1,2:1,1:1,V/V)in sequence to obtain two fractions(F1and F2).F1(120 mg)was purified by silica gel column chromatography to yield compound 1(10 mg)while F2was isolated and purified by various types of chromatography to obtain compound 2(3.5 mg,KR).

Ninty-five percent of ethanol fraction (13.8 g) was partitioned on MPLC with petroleum ether-acetone(V/V,10:1-1:1)and dichloromethane-acetone (V/V,10:1-0:1) to obtain two fractions.Then the first fraction (1.4 g) was repeatedly purified by silica gel column chromatography to yield compound 3 (11 mg).The second fraction(2.3 g)was isolated and purified to yield compound 1(33 mg)and 4(196 mg),respectively.

2.4.Cell culture and differentiation

3T3-L1 preadipocytes were obtained from Cell Bank of Chinese Academy of Sciences(Shanghai,China)and cultured in DMEM containing 15%FBS and 1%PS at 37°C and a humidified 5%CO2incubator[5].3T3-L1 preadipocytes were induced to differentiate into mature adipocyte by the“hormone cocktail”method[12].Briefly,3T3-L1 preadipocytes were cultured in 96 well culture plates with complete medium (DMEM,15% FBS,1% PS).Upon 100% confluence,the media were replaced with adipogenic media(0.5 mmol/L IBMX,1 μmol/L DEX,and 10 μg/mL INS).The culture was incubated at 37°C,and 5% CO2for 3 days.Then,the media were replaced with growth media(10 μg/mL INS)for 2 days[2].Afterwards,the medium was changed into complete medium and renewed every 2 days.The mature adipocytes were harvested after the initiation of differentiation[13].

2.5.Cell viability assay

3T3-L1 preadipocytes were seeded in 96-well plates at a density of 1×105/well.After the cells adhered to the plates,preadipocytes were incubated with TE,KR,KF,HO,and KGR for 48 h,respectively [4].MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution)(5 mg/mL in PBS)was then added to the plate(10 μL/100 μL medium/well)and incubated for 4 h at 37°C.The resultant formazan product was dissolved in DMSO(200 μL/well)and measured at 570 nm by a microplate spectrophotometer[2](type:1510,the Fisher Scientific,Hampton,NH,USA).

Fig.1.The structures of compounds from L.lucidum flowers.

2.6.Glucose uptake

In 96-well plates,3T3-L1 preadipocytes were induced to mature adipocytes as described above.The mature adipocytes were cultured with test samples (TE,KR,KF,HO,and KGR) at 37°C in a humidified 5% CO2for 48 h.Thereafter,the supernatant medium was collected for measurement of glucose by glucose assay kit.

2.7.Statistical analysis

All the values are presented as mean±standard deviation(SD).The data was analyzed by one-way ANOVA using SPSS version 19.0.The differences between the experimental and control groups were assessed by independent sample tests.The difference between groups was considered statistical significance atP＜0.05.

3.Results

3.1.Structure identification

Seven compounds were isolated and identified fromL.lucidumflowers,including ursolic acid,KR,β-sitosterol,β-daucosterol,KF,HO,and KGR.The structures of these compounds were shown in Fig.1.

Compound 1 was isolated as a white powder and its molecular formula was C30H48O38,the structure was determined by1H-NMR and13C-NMR data according to literature [14,15],identified as ursolic acid.1H-NMR(C5D5N,400 MHz)δ:5.49(1H,s,H-12),3.47(1H,t,J=6.4 Hz,H-3),2.64(1H,d,J=11.2 Hz,H-18),1.25(3H,s,H-27),1.23(3H,s,H-25),1.06(3H,s,H-30),1.03(3H,s,H-29),0.95(3H,s,H-23),0.89 (3H,s,H-26).13C-NMR (C5D5N,100 MHz) δ:179.80(C-28),139.16(C-13),125.54(C-12),78.02(C-3),55.71(C-5),53.44(C-18),47.94 (C-9),47.94 (C-17),42.39 (C-14),39.86 (C-8),39.38(C-19),39.29 (C-4),39.27 (C-20),38.97 (C-1),37.34 (C-10),37.17(C-22),33.46(C-7),30.96(C-21),28.70(C-23),28.58(C-15),28.00(C-2),24.80(C-16),23.80(C-27),23.52(C-11),21.30(C-30),18.67(C-6),17.41(C-29),17.35(C-26),16.47(C-25),15.57(C-24).

Compound 2 was isolated as yellow needle (MeOH) and its molecular formula was C21H20O10,which was determined by1HNMR and13C-NMR data according to the literature[16],identified as KR.1H-NMR (DMSO-d6,400 MHz) δ:12.49(1H,s,5-OH),10.13(1H,s,4′-OH),9.52(3-OH),8.11(2H,d,J=8.8 Hz,H-2′,6′),6.95(2H,d,J=8.8 Hz,H-3′,5′),6.84 (1H,s,H-8),6.44 (1H,s,H-6),5.56 (1H,s,Rha,H-1′′),1.15(3H,d,J=6 Hz,Rha,H-6′′).13C-NMR(DMSO-d6,100 MHz)δ:176.02(C-4),161.39(C-7),160.34(C-5),159.32(C-4′),155.70(C-9),147.08(C-2),135.96(C-3),129.59(C-2′,6′),121.48(C-1′),115.41 (C-3′,5′),104.64 (C-10),98.79 (Rha,C-1′′),98.41 (C-6),94.32(C-8),71.58(Rha,C-4′′),70.24(Rha,C-3′′),70.01(Rha,C-2′′),69.80(Rha,C-5′′),and 17.85(Rha,C-6′′).

Compound 3 was isolated as a white needle and its molecular formula was C29H50O,which was determined by TLC,identified as β-sitosterol compared with standard β-sitosterol.

Compound 4 was isolated as a white granular powder and it has poor solubility,which was determined by TLC,identified as β-daucosterol compared with standard β-daucosterol.

Compound 5 was isolated as a yellow needle and its molecular formula was C27H30O14determined by1H-NMR and13C-NMR data according to the literature[17]identified as KF.1H-NMR(DMSO-d6,400 MHz)δ:7.79(2H,d,J=8.8 Hz,H-2′,6′),6.92(2H,d,J=8.8 Hz,H-3′,5′),6.79(1H,d,J=2 Hz,H-8),6.46(1H,d,J=2 Hz,H-6),5.55(1H,d,J=1.2 Hz,7-O-Rha,H-1),5.30(1H,d,J=1.2 Hz,3-O-Rha,H-1),1.13(3H,d,J=6 Hz,7-O-Rha,H-6),0.80(3H,d,J=5.6 Hz,3-O-Rha,H-6).13C-NMR(DMSO-d6,100 MHz)δ:177.97(C-4),161.73(C-7),160.96(C-5),160.26(C-4′),157.84(C-9),156.14(C-2),134.56(C-3),130.76(C-2′,6′),120.38(C-1′),115.47(C-3′,5′),105.82(C-10),101.90(7-O-Rha,C-1),99.51 (3-O-Rha,C-1),98.43 (C-6),94.63 (C-8),71.62(7-O-Rha,C-4),71.13 (3-O-Rha,C-4),70.73 (7-O-Rha,C-3),70.34(3-O-Rha,C-3),70.26 (7-O-Rha,C-2),70.11 (3-O-Rha,C-2),70.11(7-O-Rha,C-5),69.85 (3-O-Rha,C-5),17.97 (7-O-Rha,C-6),17.52(3-O-Rha,C-6).

Compound 6 was isolated as a white solid and its molecular formula is C25H32O14,determined by1H-NMR and13C-NMR data referring to the literature[18]and identified as HO.1H-NMR(DMSO-d6,400 MHz) δ:6.64 (1H,d,J=7.6 Hz),6.60 (1H,s),6.47(1H,d,J=8.0 Hz),which was a proton on benzene ring,7.53 (1H,s),6.00 (1H,t,J=6 Hz),which was two alkene protons,4.66 (1H,d,J=7.6 Hz),which was glucose terminal proton,3.64 (1H,s,C-11-OCH3).13C-NMR (DMSO-d6,100 MHz) δ:170.70 (C-7),166.15(C-11),153.42 (C-3),145.15 (C-3′),143.82 (C-4′),129.24 (C-1′),128.36(C-8),128.26(C-9),119.54(C-6′),116.22(C-2′),115.59(C-5′),107.61 (C-4),99.14 (Glc-1′′),92.66 (C-1),77.42 (Glc-3′′),76.63(Glc-5′′),73.32(Glc-2′′),69.95(Glc-4′′),65.16(C-β),61.13(Glc-6′′),57.28 (C-10),51.34 (C-11-OCH3),39.90 (C-6),33.71 (C-α),30.69(C-5).

Compound 7 was isolated as a yellow needle (MeOH) and its molecular formula was C27H30O15.The structure was determined by1H-NMR and13C-NMR data according to literature[19],identified as KGR.1H-NMR(DMSO-d6,400 MHz)δ:8.08(2H,d,J=8.8 Hz,H-2′,6′),6.89(2H,d,J=8.8 Hz,H-3′,5′),6.83(1H,s,H-8),6.45(1H,d,J=2 Hz,H-6),5.55(1H,s,3-O-Glu,H-1),5.48(1H,d,J=7.6 Hz,7-ORha,H-1),1.12(3H,d,J=6 Hz,7-O-Rha,H-6).13C-NMR(DMSO-d6,100 MHz)δ:177.70(C-4),161.62(C-7),160.92(C-5),160.17(C-4′),156.80 (C-9),156.03 (C-2),133.51 (C-3),130.07 (C-2′,6′),120.82(C-1′),115.20(C-3′,5′),105.71(C-10),100.77(3-O-Glu,C-1),99.44(7-O-Rha,C-1),98.41(C-6),94.54(C-8),77.62(3-O-Glu,C-5),76.45(3-O-Glu,C-3),74.26 (3-O-Glu,C-2),71.64 (7-O-Rha,C-4),70.28(3-O-Glu,C-4),70.12 (7-O-Rha,C-3),69.95 (7-O-Rha,C-2),69.87(7-O-Rha,C-5),60.89(3-O-Glu,C-6),17.98(7-O-Rha,C-6).

3.2.Cell viability

To determine the appropriate dose to evaluate glucose uptake effect ofL.lucidumflowers,the cell viability of TE(300,100,33,11 and 4 μg/mL),KF (300,100,33,11 and 4 μmol/L),HO (300,100,33,11 and 4 μmol/L),and KGR (300,100,33,11 and 4 μmol/L)were investigated on 3T3-preadipocytes with MTT assay[20].The results were shown in Figs.2-4.In Figs.2-4,KR,KF,and KGR had cytotoxicity at the concentration of 300 μmol/L,compared with control group.Therefore,the concentration of 100,33,11,4 μg/mL or μmol/L were selected to test glucose uptake effect.

3.3.Glucose uptake assay

Glucose assay kit was used to assay glucose utilization in 3T3-L1 mature adipocytes [21,22].100,33,11 and 4 μg/mL of TE and 300,100,33,11 and 4 μmol/L of KR,KF,HO,and KGR were evaluated.The results showed that TE at the concentration of 100,33 and 11 μg/mL could significantly reduce the glucose content in the culture medium (P＜0.001 or 0.001 ＜P＜0.01),compared with the control group,the details were shown in Fig.5.

Fig.2.Effect of TE on cell viability on 3T3-L1 preadipocytes(±SD,n=6).

Fig.3.Effects of KR and KF on cell viability on 3T3-L1 preadipocytes(±SD,n=6).

Fig.4.Effects of HO and KGR on cell viability of 3T3-L1 preadipocytes(±SD,n=6).

In Fig.6,KR could significantly reduce the glucose content in the culture medium at the concentration of 33,11 and 4 μmol/L(P＜0.001),compared with the control group,thereinto,the ability of reducing the glucose content of KR at the concentration of 11 and 4 μmol/L were better than that of rosiglitazone(9.52 μmol/L),which was used as positive control.

In Fig.7,the concentration of 100,33 and 4 μmol/L for KF and the concentration of 100,33,11 and 4 μmol/L for HO could significantly reduce the glucose content in the culture medium(P＜0.001 or 0.001 ＜P＜0.01),compared with the control group.

In Fig.8,all concentrations of KGR had no effect on glucose uptake in 3T3-L1 mature adipocytes,compared with the control group.

Fig.5.Effects of TE on glucose uptake effect on 3T3-L1 adipocytes(±SD,n=6).

Fig.6.Effects of KR on glucose uptake of 3T3-L1 adipocytes(±SD,n=6).

Fig.7.Effects of KF and HO on glucose uptake of 3T3-L1 adipocytes(±SD,n=6).

Fig.8.Effect of KGR on glucose uptake on 3T3-L1 adipocytes(±SD,n=6).

4.Discussion

In this study,the chemical constituents ofL.lucidumflowers were investigated,including ursolic acid,KR,β-sitosterol,β-daucosterol,KF,HO,KGR.Wang et al.[23]investigated the chemical constituents ofL.lucidumflowers in Xi‘a(chǎn)n,five compounds were isolated and identified,including mannitol,rutin,quercetin,luteolin-7-O-β-d-glucoside and apigenin-7-Oβ-d-glucoside.Long et al.[24]investigated the chemical constituents ofL.lucidumflowers in Kunming,nine compounds were isolated and identified,including β-sitosterol,daucosterol,asergosta-7,22-dien-3β,5α,6β-triol,naringenin,luteolin,apigenin,quercetin,apigenin-7-O-β-D-glucopyranoside,luteolin-7-O-β-D-glucopyranoside,kaempferol-3-O-β-D-glucopyranoside,quercetin-3-O-β-D-glucopyranoside and rutin.In our study,ursolic acid,KR,KF,HO and KGR were first isolated fromL.lucidum.KR and KGR were isolated fromLigustrumgenus for the first time.The reasons for the difference in chemical composition may be due to the differences of geographical environment,biological environment,and extraction/separation methods[23-25].

Some of chemical compositions reported by Wang et al.[23]and Long et al.[24]possess hypoglycemic effect.Wei et al.[26]studied inhibitory effect of quercetin on pathological injury of pancreatic islet in db/db mice,and their results showed that quercetin could reduce the blood glucose and inhibit the pathological damage of islet on db/db diabetic mice.Shen et al.[27]investigated hypoglycemic composition ofSmilacis chinaerhizoma,the results showed that naringenin had inhibitory effect of α-glucose glycosidase and aldose reductasein vitro.Meanwhile,naringenin could significantly reduce the glucose content in alloxan-induced diabetic mice.Lee et al.[28]investigated anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model,and the results showed that rutin significantly decreased the serum glucose level,down-regulated the expression levels of proteintyrosine phosphatase 1B,negatively regulate insulin pathway both transcriptionally and translationally in myocyte C2C12 in a dosedependent manner.

In this study,the effect of glucose uptake of TE,KR,KF,Ho and KGR fromL.lucidumflowers were investigated on 3T3-L1 adipocytes.The results showed that TE,KR,KF and KGR could significantly reduce the glucose content in the culture medium,demonstrated thatL.lucidumflowers,KR,KF and HO possessed potential hypoglycemic effect.

Daniel et al.[29]studied the hypoglycemic effect of KF,KGR and KR by streptozotocin-induced diabetic mice.The results showed that KF had better hypoglycemic activityin vivo,while KGR and KR had weaker hypoglycemic effectin vivo.In addition,the effect of KF on glucose metabolism on mouse C2C12 cell were also studied in this paper.It was proved that KF could promote the utilization of glucose.C2C12 cell and 3T3-L1 preadipocytes cell are all good models for studying glucose uptakein vitro[30].In our researches,all dosage of KGR did not promote the absorption of 3T3-L1 adipocytes,while KF at the dosage of 100,33 and 4 μmol/L could promote the glucose uptake in 3T3-L1 adipocytes,which was consistent with the results reported in above literatures.

Our researches proved the potential hypoglycemic effect ofL.lucidumflowers.KF,HO and KR may be the active components ofL.lucidumflowers to promote the glucose uptake in 3T3-L1 adipocytes.

5.Conclusion

Seven compounds were isolated and identified fromL.lucidumflowers,including ursolic acid (1),KR (2),β-sitosterol (3),βdaucosterol (4),KF (5),HO (6),and KGR (7).Compounds 2,5-7 were isolated for the first time from this plant and compounds 2 and 7 were isolated for the first time inLigustrumgenus.L.lucidumflowers,KR,KF and HO possessed potential hypoglycemic effect.

Authors’contributions

WYK,JMW and LLC participated in the conception and design of the study.MMX performed bioactivity experiment and date analysis.LLC carried out the extraction and isolation process.JFW,MKW,BGW and ZHL directed the experiment.JMW and LLC wrote the first draft of the manuscript.WYK,JFW and ZHL revised the draft critically for important intellectual content.All the authors read and approved the final manuscript to be published.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by National Natural Science Foundation of China (31900292); Key Project in Science and Technology Agency of Henan Province(182102410083).