Juanjuan Zhang, Li Zhou, Lili Cui, Zhenhua Liu, Jinfeng Wei,e,?,Wenyi Kang,?

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

bKaifeng Key Laboratory of Functional Components in Health Food, Kaifeng, 475004, China

cJoint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng, 475004, China

dZhengzhou City Key Laboratory of Medicinal Resources Research, Huanghe Science and Technology College, Zhengzhou, 450063, China

eMinsheng College, Henan University, Kaifeng, Henan, 475004, China

ABSTRACT

Antioxidant and α-glucosidase inhibitiory active compounds of Cercis chinensis flowers were investigated with bio-assay guiding method. Ethyl acetate fraction (CLEa) and n-butanol fraction (CLBu) exhibited antioxidant and α-glucosidase inhibitiory activity in vitro, and the corresponding active fractions, EaFr.3,EaFr.5 and BuFr.1, exhibited higher antioxidant and α-glucosidase inhibitiory activity than those of other fractions. Eight compounds, ethyl gallate (1), stearic acid (2), docosanoic acid (3), 5α-stigmast-9(11)-en-3β-ol (4), kaempferol-3-O-α-rhamnopyranoside (5), vanillic acid (6), fisetin (7), and β-sitosterol (8),were isolated and identified from CLEa and CLBu. Ethyl gallate shown the highest antioxidant activity by scavenging DPPH radical and reducing ferric compared. Docosanoic acid and vanillic acid shown stronger α-glucosidase inhibitory activity than that of acarbose.

Keywords:

Antioxidative activity

Cercis chinensis bunge

Chemical component

α-glucosidase

1. Introduction

Cercis chinensis Bunge, is widely distributed in most regions of China with furs, woods, roots, and flowers used as Chinese Traditional Medicine (TCM) [1]. Its main chemical constituents were reported to be flavonoid, phenolic acid, toluylene, xylogen and polysaccharides [2–4]. Mu et al. [5] found three new dibenz [b, f] oxepins: 6-methoxy-7-methyl-8-hydroxydibenz [b,f] oxepin, 1,8-dimethoxy-6-hydroxy-7-methyldibenz [b, f] oxepin,and 1-hydroxy-6,8-dimethoxy-7-methyldibenz [b, f] oxepin from C. chinensis. Shi et al. [6] determined the contents of myricetrin,quercitrin and afzelin in C. chinensis leaves by High Performance Liquid Chromatograph (HPLC) analysis. Pharmacological investigation showed that C. chinensis had antibacterial, antioxidant,hypoglycemic and tyrosinase activity [7–9]. Some reports have found that the ethonal extract of the C. chinensis leaf showed more remarkable anti-in flammatory and analgesic effects than that of water extract, and the ethonal extract and water extract also showed remarkable anti-hypoxia and anti-fatigue effects [10]. The red pigment from C. chinensis flowers could regulate blood glucose in diabetic rats induced by alloxan and the levels of lipids in hyperlipidemia rat induced by high-fat cream [11].

α-Glucosidase inhibitors have been used as agents in the treatment for diabetes mellitus type 2 that work by preventing the digestion of carbohydrates such as starch and table sugar [12]. It has been known that a number of antidiabetic medicinal plants can be an important source of α-glucosidase inhibitors [13]. Free radicals is mostly considered to be associated with pathogenesis and be responsible for the initiation or/and development of many diseases such as atherosclerosis, in flammation, cancer, hypertension,ischemia-reperfusion, autoimmune diseases, agingand age-related diseases [14]. Antioxidants may have an important role in preventing or alleviating chronic diseases by reducing the oxidative damage to cellular components caused by free radicals [15]. A number of reports on the isolation and testing of plant-derived antioxidants in the maintenance and improvement of health and wellness have been described during the past decade [16,17]. At present, the studies of C. chinensis mainly focused on evaluating the biological activity of crude extracts and red pigment. On the bases of those, antioxidant activity and α-glucosidase inhibitiory activity of 70% ethanol extracts of C. chinensis flowers were investigated with activity guiding method. Eight compounds were identified from active fractions as ethyl gallate (1), stearic acid (2),docosanoic acid (3), 5α-stigmast-9(11)-en-3β-ol (4), kaempferol-3-O-α- rhamnopyranoside (5), vanillic acid (6), fisetin (7), and β-sitosterol (8). Compounds 2–6 and 8 were isolated from this genus for the first time.

2. Materials and methods

2.1. Chemical reagents and analytical instruments

1,1-diphenyl-2-picrylhydrazyl [(DPPH, Tokyo Chemical Industry Co., Ltd. (TCI)], [2,2′-azino-bis(3-ethylbenzothi-azoline)-6-sulphonicacid] diamonium salt (ABTS, Fluka), 6-hydroloxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Aldrich),2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ, Acros organics), butylated hydroxytoluene (BHT, Acros organics), butyl hydroxy anisd(BHA, Acros organics), propyl Gallate (PG, Acros organics), α-Glucosidase (EC 3.2.1.20), 4-N-trophenyl-α-D-glucopyranoside(PNPG, 026k1516), acarbose (Lot 20,120,523) and DMSO from Sigma. Sephadex LH-20 (Pharmacia), electronic balance (Mettler-Toledo), Multiskan MK3 microplate reader (Thermo Electron), NMR recoeded on a Bruker Avance Am-400 spectromete.

2.2. Plant materials

C. chinensis flowers were collected in Henan University, in April 2012. The samples were identified by Prof. Changqin Li (Henan University). A voucher specimen was deposited in National R & D Center for Edible Fungus Processing Technology, Henan University.

2.3. Sample preparation

Air-dried C. chinensis flowers (965 g) were extracted with 70%(V/V) ethanol at room temperature for 2 times, and 2 days for each time. An extract was obtained after removing ethanol. The extract (335 g) was suspended in deionized water and then successively partitioned with petroleum ether, ethyl acetate, and finally with n-butanol to obtain petroleum ether extract (CLPe, 8.33 g),ethyl acetate extract (CLEa, 11.71 g) and n-butanol extract (CLBu,23.00 g).

CLEa (11.71 g) was applied to a silica gel H medium-pressure liquid chromatography and successively eluted with petroleum ether/ethyl acetate (from 100:1 to 7:3) and chloroform/acetone (from 10:1–8:2) to obtain 7 fractions: EaFr.1, EaFr.2, EaFr.3, EaFr.4, EaFr.5,EaFr.6 and EaFr.7.

CLBu (23.00 g) was chromatographed on D101 macroporous resin, and successively eluted with 20% methanol, 40% methanol,60% methanol and methanol to obtain 4 fractions: BuFr.1, BuFr.2,BuFr.3 and BuFr.4.

2.4. Inhibiting activity of α-glucosidase in vitro

The α-glucosidase inhibitory activity was determined in accordance with a 96-well plate mothod described by Guo et al. [18,19].Every sample was dissolved in DMSO and the OD value was measured at 405 nm. Every reaction was carried out with three replications. Acarbose was used as a positive control. The inhibitory rates (%) were calculated according to the following formula:

2.5. Antioxidant activity in vitro

2.5.1. Scavening activity against DPPH radical

DPPH method was carried out according to the literatures [20,21]. The fractions and compounds were prepared into 2.0 mg/mL and 1.0 mg/mL with methanol as preliminary screening concentration, respectively. Then every sample was stepwise diluted into 5 different concentrations. An aliquot composed of 10 μL samples and 175 μL 200 μmol/L of DPPH solution were added into 96-well plates. Mixtures were vigorously shaken and left 20 min in the dark at room temperature. The absorbance was then measured at 515 nm. All reactions were carried out with three replications. Inhibition of DPPH radical was calculated as follows:

Where Ablankwas the absorbance of DPPH itself and Asamplewas the absorbance of the samples of DPPH with BHT, PG and BHA as positive control.

2.5.2. Scavening activity against ABTS radical

ABTS assay was carried as described in reference [22]. Before the analysis, ABTS radical cation was produced by reacting 7 mmol/L stock solution of ABTS with 2.45 mmol/L potassium persulfate and then the mixture was stood in the dark at room temperature for another 12 h. ABTS radical cation solution was diluted with methanol to produce a solution with an absorbance of 0.800 ± 0.03 at 734 nm. Then 10 μL samples were mixed with 200 μL ABTS radical cation solution. After 20 min response period at room temperature in darkness, the absorbance of the resulting solution and blank (with same chemicals, except for the sample) was measured at 734 nm. All reactions were carried out with three replications. ABTS radical inhibition was calculated in the following way:

Where Ablankwas the absorbance of ABTS itself and Asamplewas the absorbance of the samples on ABTS.

2.5.3. Scavening activity against FRAP radical

FRAP assay was determined according to the method in references [23,24]. Every sample was determined in a series of different concentrations with methanol. 10 μL samples were mixed with 200 μL TPTZ working liquid that was freshly prepared. After the mixture was incubated at 37°C for 30 min, the absorbance of the resulting solution was measured by microplate reader at 595 nm with trolox as a reference. Results were expressed in μmol Trolox equivalents (TEAC) (TE)/g sample. All reactions were carried out with three replications.

2.6. Statistical analysis

The results were expressed as arithmetic mean ± standard deviation (SD). Statistical analysis was performed using SPSS19.0 software, and comparison between any two groups was evaluated using one-way analysis of variance (One-Way ANOVA). The difference between groups with P < 0.05 was regarded as statistically significant.

2.7. Compounds isolation

EaFr.2 (1.32 g) was subjected to a silica gel H medium-pressure liquid chromatography and eluted with petroleum ether/ethyl acetate (from 20:1 to 8:2) to obtain from A to C fractions. Fraction A was separated on a silica gel H with petroleum ether/chloroform(50:1 to 8:2), and further chromatographed on Sephadex LH-20 to give compound 3 (5 mg). EaFr.3 (0.63 mg) was chromatographed on a silica gel H and eluted with petroleum ether/chloroform/ethyl acetate (7:2:0.5) to obtain D and E fractions. Fraction E was applied to a silica gel H with petroleum ether/ethyl acetate(from 40:1 to 7:3), and further chromatographed on Sephadex LH-20 (petroleum ether/chloroform/ methanol = 9:9:2) to yield compound 2 (11.2 mg). EaFr.4 (0.82 g) was separated on silica gel H with petroleum ether/chloroform (20:1) to obtain F and G fractions. Fraction F was separated on silica gel H with petroleum ether/acetone (20:1) and Sephadex LH-20 to give compound 8 (7.0 mg). Fraction G was chromatographed on Sephadex LH-20 (petroleum ether/chloroform/ methanol = 9:9:2) to yield compound 4 (12.0 mg). EaFr.5 (0.70 g) was purified by silica gel H with petroleum ether/chloroform/ethyl acetate (5:4:1), and further chromatographed on Sephadex LH-20 (chloroform/methanol = 1:1)to yield compound 1 (15.0 mg).

BuFr.1 (3.04 g) was separated on silica gel H with chloroform/acetone (50:1–8:2) to obtain H and I fractions. Fraction I was separated on silica gel H with chloroform/acetone/methanol(20:1:0.5) and chromatographed on Sephadex LH-20 (methanol)to give compound 5 (6.8 mg). BuFr.3 was chromatographed on Sephadex LH-20 (methanol), further chromatographed on ODS to obtain compound 6 (5.0 mg) and 7 (6.0 mg).

2.8. Identification of the compounds

After comparing the melting points and spectral data (1H-NMR,13C-NMR, and MS) of the literature values, compounds 1 to 8 were identified as ethyl gallate (1) [25], stearic acid (2) [26], docosanoic acid (3) [27], 5α-stigmast-9(11)-en-3β-ol (4) [28], kaempferol-3-O-α-rhamnopyranoside (5) [29], vanillic acid (6) [30], fisetin (7) [31],and β-sitosterol (8). And the chemical structures of compounds 1 to 8 are shown in Fig. 1.

Fig. 1. Chemical structures of Compounds 1-8 isolated from C. chinensis flowers.

Compound 1, colorless crystal, m.p. 151～154°C, EI-MS m/s: 198[M]+.1H-NMR (400 MHz, CDCl3) δ: 7.74 (2H, s, H-2,6), 4.18 (2H, q,H-2′), 1.08 (3H, t, H-3′).13C-NMR (100 MHz, CDCl3) δ: 121.6 (C-1),110.3 (C-2, 6), 147.7 (C-3, 5), 136.0 (C-4), 167.2 (C-1′), 60.6 (C-2′),14.6 (C-3′).

Compound 2, white powder, m.p. 67～69°C, EI-MS m/s: 284 [M]+.1H-NMR (400 MHz, CDCl3) δ: 0.87 (3H, t, H-18), 1.20 (28H, m, H-4～17), 1.62 (2H, m, H-3), 2.34 (2H, t, H-2), 3.48 (1H, s, OH).13C-NMR(100 MHz, CDCl3) δ: 179.92 (C-1), 34.22 (C-2), 32.00 (C-3), 29.79-29.21 (C-4～15), 24.84 (C-16), 22.82 (C-17), 14.21 (C-18).

Compound 3, white powder, m.p. 80～81°C, EI-MS m/s: 340 [M]+.1H-NMR (400 MHz, CDCl3) δ: 0.87 (3H, t, H-22), 1.25 (36H, m, H-4～21), 1.62 (2H, m, H-3), 2.34 (2H, t, H-2).13C-NMR (100 MHz,CDCl3) δ: 180.00 (C-1), 34.20 (C-2), 32.08 (C-3), 29.83-27.31 (C-4～19), 24.85 (C-20), 22.83 (C-21), 14.23 (C-22).

Compound 4, white powder, m.p. 131～133°C, EI-MS m/s: 414[M]+.1H-NMR (400 MHz, CDCl3) δ: 0.67 (3H, s, H-18), 0.80 (3H,d, J = 8.4 Hz, H-29), 0.84 (6H, d, J = 7.6 Hz, H-26, H-27), 0.92 (3H, d,J = 6.4 Hz, H-21), 1.00 (3H, s, H-19), 3.52 (1H, m, H-3), 5.35 (1H, s,H-11).13C-NMR (100 MHz, CDCl3) δ: 37.44 (C-1), 31.85 (C-2), 71.98(C-3), 32.09 (C-4), 39.97 (C-5), 21.26 (C-6), 29.38 (C-7), 46.05 (C-8),140.97 (C-9), 36.69 (C-10), 121.89 (C-11), 42.49 (C-13), 56.96 (C-14), 24.47 (C-15), 28.41 (C-16), 56.26 (C-17), 12.15 (C-18), 19.56(C-19), 36.32 (C-20), 19.22 (C-21), 34.15 (C-22), 29.06 (C-23), 50.34(C-24), 26.32 (C-25), 19.97 (C-26), 18.95 (C-27), 23.27 (C-28), 12.07(C-29).

Compound 5, yellow powder, m.p. 172～174°C, EI-MS m/s: 432[M]+.1H-NMR (400 MHz, C5D5N) δ: 1.47 (3H, d, J = 4.8 Hz, H-6′′),3.00～5.00 (glycosyl-H), 5.13 (1H, s, H-1′′), 6.34 (1H, s, H-6), 7.20(1H, s, H-8), 7.27 (2H, d, J = 8.8 Hz, H-3′, H-5′), 8.10 (2H, d, J = 12 Hz,H-2′, H-6′).13C-NMR (100 MHz, C5D5N) δ: 157.51 (C-2), 134.81 (C-3), 178.83 (C-4), 162.79 (C-5), 99.57 (C-6), 165.66 (C-7), 94.37 (C-8), 157.71 (C-9), 105.29 (C-10), 121.62 (C-1′), 131.27 (C-2′, C-6′),116.16 (C-3′, C-5′), 103.62 (C-1′′), 72.34 (C-2′′), 71.83 (C-3′′), 73.06(C-4′′), 71.77 (C-5′′), 18.11 (C-6′′).

Compound 6, white powder, m.p. 211～212°C, EI-MS m/s: 168[M]+.1H-NMR (400 MHz, DMSO) δ: 3.81 (3H, s, ?OCH3), 6.85 (1H,d, J = 8.4 Hz, H-5), 7.43 (1H, s, H-2), 7.45 (1H, d, J = 7.2 Hz, H-6),9.84 (1H, s, ?OH).13C-NMR (100 MHz, DMSO) δ: 167.11 (?COOH),121.57 (C-1), 112.73 (C-2), 147.17 (C-3), 151.01 (C-4), 114.98 (C-5),123.41 (C-6), 55.52 (3?OCH3).

Compound 7, yellow powder, m.p. 330～331°C, EI-MS m/s: 286[M]+.1H-NMR (400 MHz, C5D5N) δ: 8.44 (1H, dd, J = 8.4 Hz, H-6′),8.09 (1H, d, J = 8.8 Hz, H-6), 7.27 (1H, d, J = 8.8 Hz, H-2′), 6.73 (1H,s, H-5′), 6.34 (1H, s, H-8), 13.37 (1H, s, ?OH).13C-NMR (100 MHz,C5D5N) δ: 149.07 (C-2), 135.75 (C-3), 176.56 (C-4), 123.75 (C-5),116.19 (C-6), 162.79 (C-7), 100.01 (C-8), 155.41 (C-9), 116.60 (C-10), 122.78 (C-1′), 106.80 (C-2′), 134.70 (C-3′), 134.68 (C-4′), 105.82(C-5′), 111.60 (C-6′).

Compound 8, white powder, m.p. 136～137°C, EI-MS m/s: 414[M]+. Compare it with β-sitosterol reference substance under TLC with three different spread layout, and they had the same Rf values and coloration. The fusion point of mixture of them had no change.

3. Results and discussion

3.1. α-Glucosidase inhibitory activity in vitro

α-Glucosidase inhibitory activity of C. chinensis fractions was screened to determine the active constituent by α-glucosidase inhibitory model in vitro. The results demonstrated that the C. chinensis fractions had certain inhibitory activity, of which screening inhibition rates are shown in Table 1.

Table 1α-Glucosidase inhibitory activity of the different extracts of C. chinensis.

As shown in Fig. 2, α-glucosidase inhibitory rates of CLEa and CLBu were 100.17 ± 2.30 (P < 0.01) and 99.83 ± 1.61 (P < 0.001),respectively, and dramatically higher than that of acarbose 1(59.62% ± 0.68%). In Fig. 3, EaFr.2, EaFr.3, EaFr.4, EaFr.5, EaFr.6,BuFr.1, BuFr.2 and BuFr.3 all shown stronger α-glucosidase inhibitory activity than that of acarbose 2. In Fig. 4, compound 6 (98.87% ± 1.16%) (P < 0.001) shows the highest α-glucosidase inhibitory activity, and compound 3 (76.14% ± 1.03%) (P < 0.01)shows stronger α-glucosidase inhibitory activity than that of and acarbose 3 (62.17% ± 0.72%). Therefore, compound 3 was active compound in EaFr.2 and compound 6 was active compound in BuFr.3.

Fig. 2. Inhibitory effect of extracts form C. chinensis against α-glucosidase.

Fig. 3. Inhibitory effect of fractions form C. chinensis against α-glucosidase.

Fig. 4. Inhibitory effect of compounds form C. chinensis against α-glucosidase.

For studying the glycosidases action mechanisms, glycosidase inhibitors are the important tools, for some degenerative diseases,glycosidase are also prospective therapeutic agents, for example, diabetes, viral attachment and cancer [32,33]. Currently, the structure types α-glucosidase inhibitor from plants were diversity,for example, terpenes, alkaloids, quinines, flavonoids, phenylpropanoids, steroids and organic acids, alcohols and allyls [34]. It was for the first time to report α-glucosidase inhibitory activity of compound 3 and compound 6.

3.2. Antioxidant activity in vitro

3.2.1. DPPH free radical scavenging assays

As shown in Fig. 5, CLEa and CLBu have ability to scavenge the DPPH radical (64.59% ± 0.45% and 84.26% ± 0.37%, respectively),which were stronger than that of BHA (56.84% ± 0.69%) as a positive control. In Fig. 6, all fractions exhibits better DPPH scavenging activity. Among the fractions, EaFr.3 showen higher antioxidant activity with inhibition ratio of 89.64% ± 0.68%, which was higher than that of BHT, BHA, and PG (58.35% ± 0.35%, 70.07% ± 0.64%, and 88.36 % ± 0.71%, respectively) as positive controls. In Fig. 7, compound 1 isolated from EaFr.5 exhibits strong activity against DPPH radical with the inhibition ratio 88.12% ± 1.03%, and was stronger than that of BHT and BHA (60.12% ± 0.75% and 79.03% ± 0.53%) but lower of PG (90.66% ± 1.14%).

Fig. 5. The DPPH and ABTS scavenging activity of extracts from C. chinensis.

Fig. 6. The DPPH and ABTS scavenging activity of fractions from C. chinensis.

Fig. 7. The DPPH and ABTS scavenging activity of compounds from C. chinensis.

3.2.2. ABTS free radical scavenging assays

In ABTS tests, as shown in Fig. 5, CLBu scavenged ABTS radicals with the inhibition ratio being 95.88% ± 1.12% as compared with 94.43 ± 0.75% for CLEa. Both of them were stronger than that of PG (56.84% ± 0.58%). In Fig. 6, EaFr.1 and EaFr.2 show the weaker ABTS radical scavenging ability. Moreover, no significant difference was observed among other fractions and positive controls. In Fig. 7,compound 1 isolated from EaFr.5 exhibits strong activity against ABTS radical with the inhibition ratio 94.96% ± 1.21%, and very close to that of positive controls.

3.2.3. Ferric reducing activity

FRAP values for investigated extracts are shown in Fig. 8.CLEa and CLBu (TEAC were (1172.24 ± 41.64) μmol/g and(2525.09 ± 29.92) μmol/g, respectively) had the higher ferric reducing activity than that of BHT (TEAC was(1047.11 ± 137.02) μmol/g), and weaker than that of PG (TEAC was (14,782.22 ± 257.02) μmol/g). As shown in Fig. 9, the ferric reducing activity of fractions and positive controls were in the order: PG > BuFr.1 > BuFr.2 > BuFr.4 > EaFr.5 > BHA > EaFr.3> EaFr.7 > BHA > EaFr.6 > BuFr.3 > EaFr.4. In Fig. 10, the ferric reducing activity of compound 1 (TEAC was (9592.60 ± 270.90)μmol/g) isolated from EaFr.5 was higher than that of BHT and BHA (TEAC were (1301.03 ± 64.32) μmol/g and (2744.80 ± 80.14)μmol/g, respectively), and weaker than that of PG (TEAC was(10,541.82 ± 79.50) μmol/g).

Fig. 8. The FRAP assay of extracts from C. chinensis.

Fig. 9. The FRAP assay of fractions from C. chinensis.

Fig. 10. The FRAP assay of compounds from C. chinensis.

4. Conclusion

CLEa, CLBu and fractions of C. chinensis flowers showed αglucosidase inhibitory and antioxidant activity. Based on the bio-assay guiding method was used and 8 compounds were isolated and identified from active fractions. Among them, docosanoic acid and vanillic acid had relatively stronger inhibitory effects on αglucosidase and were considered the active components of EaFr.2 and BuFr.3, respectively. Docosanoic acid and vanillic acid might be the potential inhibitors of on α-glucosidase for treatment of type 2 diabetes. Ethyl gallate had a significant antioxidant activity, and were the active components of EaFr.5. The above studies could provide theoretical basis for its application in α-glucosidase inhibitors and antioxidant activity.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by Key Project in Science and Technology Agency of Henan Province (192102110112, 192102110214 and 182102410083); Innovation Training Program for College Students(201910475107 and MSCXSY2019036).