Yongmei Xio,Ming LiPu MoLingru YngLingo Qu

a School of Chemistry and Chemical Engineering,Henan University of Technology,Zhengzhou 450001,PR China

b School of Chemistry and Molecular Engineering,Zhengzhou University,Zhengzhou 450001,China

Keywords:

Troxerutin

Enzymatic synthesis

Fatty acid esters

Antioxidation

Oil-water distribution coefficient

ABSTRACT

Troxerutin fatty acid esters were prepared using troxerutin and fatty acid vinyl esters as substrates in pyridine through enzymatic route.The structures of as-prepared compounds were identified by FT-TR,NMR,and ESI-HRMS.Using alkaline protease(≥30 mg/mL)as enzyme,maximum yields reached 58%at 3:1(vinyl hexanoate to troxerutin)in pyridine(water content ≤1%).The yields gradually declined as chain length of acyl donors rose.The antioxidation abilities of the as-obtained compounds were confirmed by both DPPH free radical scavenging and potassium ferricyanide reduction methods.The antioxidation ability of troxerutin fatty acid esters was found lower than that of troxerutin.However,the logP values of troxerutin fatty acid esters varied from 0.15 to 1.94,suggesting that troxerutin fatty acid esters had better lipophilicity than troxerutin(logP=-2.12)when compared to their oil-water distribution coefficients.Overall,these findings look promising as reference for further development of future troxerutin.

1.Introduction

Flavonoids are phenolic compounds issued during secondary metabolism.They have many pharmacological functions,including antitumor,immunomodulatory,and antiseptic properties [1].Today,flavonoids attracted widespread attention in various fields,including medical treatment and diet[2,3].In addition,flavonoids are polyphenol compounds with high antioxidant capacity [4].As natural antioxidants,flavonoids are easily accepted by people than the synthetic antioxidant counterparts,such as butylated hydroxytoluene (BHT),butylated hydroxyanisole(BHA),and tert-butylhydroquinone(TBHQ)[5].However,most flavonoids have poor water solubility or lipid solubility due to their structural characteristics.These features limit the utilization rates of flavonoids physiological activities[6].

A number of studies[7-9]have reported that flavonoid fatty acid esters could improve lipophilicity of flavonoids through esterification and transesterification.For instance,Ishihara et al.[10]synthesized rutin myristate with myristic acid as an acyl donor using transesterification to improve ruin lipophilicity.Ardhaoui et al.[11]used different fatty acids as acyl donors to prepare flavonoid fatty acid esters by esterification.Ma et al.[12]found that the esters based on isoorientin and isovitexin could be synthesized by fatty acids in presence of enzyme,which played an important role in increasing their lipid solubilities but reduced their antioxidation properties.Similarly,Li et al.[13]prepared dihydromyricetin esters through enzyme catalysis to improve its lipid solubility.They noticed that introduction of esters at 3-OH position of dihydromyricetin induced lower effect on the antioxidation characteristics.Therefore,flavonoid fatty acid esters can not only enhance lipid solubility but may influence the antioxidation properties.

Some studies[14]have reported that usage of fatty acids or fatty acid esters as acyl donors might extend the application scope of flavonoids.Fatty acids are mainly composed of saturated,monounsaturated,and polyunsaturated fatty acids.These play important roles in regulating health of the human body in terms of anti-inflammatory function,enhancing immunity,promoting infant development,and boosting memory[15].Besides,saturated fatty acids can be used as energy sources to empower the human body[16].The combination of fatty acids or fatty acid esters with flavonoids to synthesize flavonoid fatty acid esters would not only improve lipid solubility of flavonoids but also maintain health benefits of fatty acids.In addition,some investigations[17-19]reported that increased ester solubility of acylated flavonoids enhances their ability to interact with and penetrate the cell membranes.Two main methods are currently utilized to prepare flavonoids fatty acid esters.The first consists of chemical route and the second of enzymatic synthesis[20,21].Compared to chemical synthesis,the enzymatic route is more widely used due to its mild reaction conditions,high efficiency,and elevated selectivity[22,23].Flavonoids are polyhydroxyl compounds and their antioxidant capacities are mainly determined by the location and quantity of available hydroxyl groups on flavonoid aglycones[24].Hence,the enzymatic approach to prepare flavonoid fatty acid esters could yield the required flavonoid esters[25,26]and retain some hydroxyl groups with antioxidant ability.

In this study,troxerutin fatty acid esters with troxerutin and fatty acid vinyl esters were prepared through transesterification using the enzymatic route.The antioxidant properties of troxerutin and its fatty acid esters were investigated using both DPPH free radical scavenging and potassium ferricyanide reduction methods.As oil-water distribution coefficient is an important parameter to evaluate the transmembrane transport of compounds[27],it was investigated for all prepared troxerutin fatty acid esters and troxerutin.The results indicated that troxerutin fatty acid esters had better oil-water distribution coefficients than troxerutin.

2.Experiment

2.1.Materials and instruments

Troxerutin was purchased from Xi'an Hui Bo Biological Technology,alkaline protease from Wuxi Xuemei Enzyme Preparation Science And Technology Co.,Ltd.,and all fatty acids were from Sinopharm Chemical Reagent.Vinyl acetate and pyridine were obtained from Tianjin Damao Chemical Reagent Factory and all solvents were analytical grade purity.

HRMS was used to identify the molecular weights(ion source:ESI).Fourier transform infrared spectroscopy(FT-IR,IR-200 Fourier transform spectrometer)was employed to confirm the chemical bonding.The chemical structures were determined by NMR (Bruker Avance 400 MHZ) using DMSO-d6+D2O as solvent.An ultraviolet spectrophotometer(UV-Vis)was employed for determination of antioxidation properties and HPLC was utilized to evaluate the influence of reaction conditions and logP.

2.2.Enzymatic synthesis of troxerutin fatty acid esters

The synthesis of troxerutin fatty acid esters mainly consisted of two steps:i)preparation of fatty acid vinyl esters,and ii)synthesis of troxerutin fatty acid esters.Firstly,vinyl acetate and fatty acids(molar ratio 9:1)were mixed in oil bath at 70 °C to prepare fatty acid vinyl esters.Secondly,560 mg (0.75 mmol) troxerutin,fatty acid vinyl esters (3 mmol) and 600 mg alkaline protease in pyridine were added to 100 mL conical bottle.The mixture left to oscillate for 80 h at 50 °C.The two-step reaction processes were monitored by TLC.After completion of reaction,alkaline protease was filtered off and pyridine was evaporated.Next,the products are separated by column chromatography(ethyl acetate:methanol:water=40:3.6:0.5 to 15:3.6:0.5,V/V/V)to collect pure troxerutin fatty acid esters: hexanoyl-troxerutin (abbreviated as,A),heptanoyl-troxerutin (B),octanoyl-troxerutin(C),nonanoyl-troxerutin(D),decanoyl-troxerutin(E),lauroyl-troxerutin(F),myristoyl-troxerutin(G),palmitoyl-troxerutin(H),stearoyl-troxerutin(I),oleoyl-troxerutin(J),linoleyl-troxerutin(K),and alpha-linoleyl-troxerutin(L).All compounds were prepared following similar method(Fig.1).

Hexanoyl-troxerutin(A)

Yellow solid,yield:58%,Rf=0.33,1H NMR(400 MHz,DMSO-d6+D2O,δ ppm):7.85-7.84(m,2H,1H of H2′,1H of H6′),7.16-7.13(d,1H,J=8.8 Hz,H5′),6.74(s,1H,H8),6.38(s,1H,H6),5.38-5.37(d,1H,J=4.4 Hz,H1′),4.44-4.41(m,3H,2H of A acylated,1H of B acylated),4.39(m,1H,H1‴),4.28-4.26(m,1H,H of B acylated),4.17-4.06(m,4H,H of A),3.77-3.72 (m,4H,H of B),3.37-3.06 (10H,H of rhamnosyl),2.36-2.30(m,2H,CH2CO-troxerutin),1.55-1.24(m,6H,other CH2ofhexanic acid),0.98-0.96 (d,3H,J = 6.4 Hz,CH3of rhamnosyl),0.85-0.81 (t,3H,CH3of hexanic acid);13C NMR: 14.4,18.1,22.2,24.6,31.1,33.8,59.8,60.0,62.5,67.3,67.5,67.6,68.7,70.6,70.8,70.9,71.1,72.2,74.6,76.4,76.8,93.3,98.9,101.3,101.9,105.5,113.9,115.8,122.8,123.8,147.7,150.8,151.0,156.9,161.3,165.2,173.4,178.0; IR (KBr): 3370 cm-1(OH),1735 cm-1(O=C-O),ESI-HRMS(m/z):calcd for C39H53O20+(M+H)+841.3125,found 841.3124.

Heptanoyl-troxerutin(B)

Yellow solid,yield: 53%,Rf = 0.35,1H NMR (400 MHz,DMSO-d6+ D2O,δ ppm): 7.86-7.84 (m,2H,1H of H2′,1H of H6′),7.16-7.14(d,1H,J=8.4 Hz,H5′),6.75(s,1H,H8),6.38(s,1H,H6),5.39-5.38(d,1H,J=4.4 Hz,H1′),4.44-4.39(m,3H,2H of A acylated,1H of B acylated),4.32(m,1H,H1‴),4.28-4.27(m,1H,H of B acylated),4.14-4.06(m,4H,H of A),3.77-3.75 (m,4H,H of B),3.42-3.04 (10H,H of rhamnosyl),2.36-2.32 (m,2H,CH2CO-troxerutin),1.54-1.23 (m,8H,other CH2of heptanic acid),0.98-0.96 (d,3H,J = 6.0 Hz,CH3of rhamnosyl),0.84-0.83(t,3H,CH3of heptanic acid);13C NMR:14.3,18.1,22.2,24.6,28.9,31.1,33.8,59.8,60.4,62.5,67.3,67.5,67.6,68.7,70.6,70.8,70.9,71.3,72.4,74.6,76.4,76.8,93.3,98.9,101.3,101.9,105.5,112.9,114.8,122.6,123.9,147.5,150.7,151.1,156.7,161.5,164.1,173.4,179.1;IR(KBr):3367 cm-1(OH),1735 cm-1(O=C-O),ESI-HRMS(m/z):calcd for C40H55O20+(M+H)+855.3281,found 855.3279.

Octanoyl-troxerutin(C)

Yellow solid,yield: 48%,Rf = 0.36,1H NMR (400 MHz,DMSO-d6+ D2O,δ ppm): 7.76-7.70 (m,2H,1H of H2′,1H of H6′),7.15-7.12(d,1H,J=8.8 Hz,H5′),6.71(s,1H,H8),6.36(s,1H,H6),5.38-5.37(d,1H,J=3.2 Hz,H1′),4.46-4.42(m,3H,2H of A acylated,1H of B acylated),4.39(m,1H,H1‴),4.27(m,1H,H of B acylated),4.12-4.07(m,4H,H of A),3.78-3.75(m,4H,H of B),3.45-3.08(10H,H of rhamnosyl),2.35-2.32(m,2H,CH2CO-troxerutin),1.52-1.21(m,10H,other CH2of octanoic acid),0.98-0.97(d,3H,J=5.6 Hz,CH3of rhamnosyl),0.81-0.80(t,3H,CH3of octanoic acid);13C NMR:14.4,18.1,22.4,24.8,28.8,28.9,31.5,33.8,59.9,60.0,62.8,67.2,67.4,68.7,70.6,70.7,70.9,71.1,72.2,74.6,76.4,76.8,93.3,98.8,101.3,101.8,105.4,113.7,115.4,122.7,123.4,147.4,150.7,151.4,156.9,161.3,165.1,173.4,177.9; IR (KBr):3365 cm-1(OH),1730 cm-1(O=C-O); ESI-HRMS (m/z): calcd for C41H57O20+(M+H)+869.3438,found 869.3443.

Nonanoyl-troxerutin(D)

Yellow solid,yield: 44%,Rf = 0.38,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm):7.87-7.84(m,2H,1H of H2′,1H of H6′),7.16-7.14(d,1H,J=8.8 Hz,H5′),6.74(s,1H,H8),6.37(s,1H,H6),5.39-5.38(d,1H,J=3.6 Hz,H1′),4.44-4.40(m,3H,2H of A acylated,1H of B acylated),4.33(m,1H,H1‴),4.28-4.27(m,1H,H of B acylated),4.14-4.07(m,4H,H of A),3.76-3.72 (m,4H,H of B),3.35-3.07 (10H,H of rhamnosyl),2.36-2.32(m,2H,CH2CO-troxerutin),1.53-1.23(m,12H,other CH2of nonanoic acid),0.98-0.97(d,3H,J = 5.6 Hz,CH3of rhamnosyl),0.86-0.82(t,3H,CH3of nonanoic acid);13C NMR:14.4,18.1,24.9,28.9,29.1,29.3,31.7,32.5,33.9,60.1,62.8,67.3,67.6,68.7,70.6,70.8,70.9,71.2,72.4,74.5,76.3,76.8,93.3,98.6,101.2,101.8,105.6,113.8,115.4,122.7,123.6,147.5,150.6,151.4,156.9,161.2,165.2,173.5,177.9; IR (KBr): 3370 cm-1(OH),1735 cm-1(O=C-O); ESIHRMS(m/z):calcd for C42H59O20+(M+H)+883.3594,found 883.3591.

Decanoyl-troxerutin(E)

Fig.1.The enzymatic synthesis route of troxerutin fatty acid esters.

Yellow solid,yield: 40%,Rf = 0.40,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm):7.87-7.85(m,2H,1H of H2′,1H of H6′),7.15-7.13(d,1H,J=8.8 Hz,H5′),6.71(s,1H,H8),6.36(s,1H,H6),5.38-5.37(d,1H,J=4.0 Hz,H1′),4.41-4.39(m,3H,2H of A acylated,1H of B acylated),4.31(m,1H,H1‴),4.27(m,1H,H of B acylated),4.12-4.07(m,4H,H of A),3.79-3.74 (m,4H,H of B),3.48-3.06 (10H,H of rhamnosyl),2.35-2.30(m,2H,CH2CO-troxerutin),1.53-1.18(m,14H,other CH2of decanoic acid),0.98-0.97 (d,3H,J = 4.8 Hz,CH3of rhamnosyl),0.83-0.80(t,3H,CH3of decanoic acid);13C NMR:14.4,18.1,24.9,28.9,29.1,29.2,29.3,31.7,32.5,33.9,60.0,62.8,67.3,67.5 68.8,70.7,70.8,70.9,71.1,72.2,74.5,76.4,76.9,93.3,98.8,101.3,101.9,105.4,113.7,115.5,122.9,123.6,147.5,150.8,151.5,156.9,161.3,165.1,173.4,177.9;IR(KBr):3369 cm-1(OH),1735 cm-1(O=C-O);ESI-HRMS(m/z):calcd for C43H61O20+(M+H)+897.3751,found 897.3756.

Lauroyl-troxerutin(F)

Yellow solid,yield: 34%,Rf = 0.44,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm): 7.85-7.84(m,2H,1H of H2′,1H of H6′),7.16-7.13(d,1H,J=8.4 Hz,H5′),6.74(s,1H,H8),6.39-6.38(s,1H,H6),5.41-5.40(d,1H,J=4.0 Hz,H1′),4.43-4.39(m,3H,2H of A acylated,1H of B acylated),4.32(m,1H,H1‴),4.26(m,1H,H of B acylated),4.12-4.06(m,4H,H of A),3.76-3.71 (m,4H,H of B),3.38-3.06 (10H,H of rhamnosyl),2.35-2.32(m,2H,CH2CO-troxerutin),1.52-1.21(m,18H,other CH2of lauric acid),0.97-0.96(d,3H,J=5.2 Hz,CH3of rhamnosyl),0.84-0.83(m,3H,CH3of lauric acid);13C NMR:14.4,18.1,22.6,24.9,29.0,29.2,29.5,31.8,33.9,59.9,62.8,67.2,67.5,68.7,70.6,70.7,70.8,71.2,72.3,74.7,76.4,76.8,93.3,98.7,101.3,102.0,105.5,113.4,115.5,122.7,123.7,147.5,148.1,151.5,156.9,161.3,165.1,173.4,177.9;IR(KBr):3365 cm-1(OH),1735 cm-1(O=C-O); ESI-HRMS (m/z): calcd for C45H65O20+(M+H)+925.4064,found 925.4064.

Myristoyl-troxerutin(G)

Yellow solid,yield: 30%,Rf = 0.47,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm): 7.87-7.85(m,2H,1H of H2′,1H of H6′),7.14-7.12(d,1H,J=6.8 Hz,H5′),6.71(s,1H,H8),6.36(s,1H,H6),5.42-5.41(d,1H,J=5.6 Hz,H1′),4.44-4.39(m,3H,2H of A acylated,1H of B acylated),4.31-4.26(m,1H,H1‴),4.31-4.26(m,1H,H of B acylated),4.11-4.07(m,4H,H of A),3.76(m,4H,H of B),3.45-3.09(10H,H of rhamnosyl),2.35-2.32(m,2H,CH2CO-troxerutin),1.52-1.21(m,22H,other CH2of myristic acid),0.99-0.97 (d,3H,J = 4.8 Hz,CH3of rhamnosyl),0.91-0.83(m,3H,CH3of myristic acid);13C NMR:14.4,18.1,22.6,24.9,29.0,29.2,29.4,29.5,29.6,31.8,33.9,60.0,62.8,67.2,67.5,68.7,70.6,70.8,70.9,71.1,72.2,74.7,76.4,76.8,93.3,98.8,101.3,102.0,105.6,113.4,115.5,122.8,123.6,147.5,148.1,151.5,156.9,161.3,165.1,173.4,177.9; IR (KBr): 3369 cm-1(OH),1735 cm-1(O=C-O); ESIHRMS (m/z): calcd for (M + H)+: calcd for C47H69O20+(M + H)+953.4377,found 953.4371.

Palmitoyl-troxerutin(H)

Yellow solid,yield: 25%,Rf = 0.53,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm):7.86-7.85(m,2H,1H of H2′,1H of H6′),7.16-7.14(d,1H,J=8.4 Hz,H5′),6.74(s,1H,H8),6.38(s,1H,H6),5.37-5.36(d,1H,J=3.6 Hz,H1′),4.43-4.41(m,3H,2H of A acylated,1H of B acylated),4.31(m,1H,H1‴),4.26(m,1H,H of B acylated),4.12-4.07(m,4H,H of A),3.75 (m,4H,H of B),3.39-3.07 (10H,H of rhamnosyl),2.35-2.32(m,2H,CH2CO-troxerutin),1.52-1.20 (m,26H,other CH2of palmitic acid),0.98-0.97(d,3H,J=4.8 Hz,CH3of rhamnosyl),0.85-0.83(m,3H,CH3of palmitic acid);13C NMR: 14.4,18.1,22.6,24.9,29.0,29.3,29.6,31.8,33.9,60.0,62.8,67.2,67.5,68.7,70.6,70.8,70.9,71.1,72.3,74.6,76.4,76.9,93.3,98.8,101.3,102.0,105.6,113.4,115.5,122.8,123.6,147.5,148.1,151.5,122.8,123.7,147.5,148.1,151.4,156.9,161.3,165.1,173.4,177.9;IR(KBr):3369 cm-1(OH),1735 cm-1(O=C-O);ESI-HRMS(m/z):calcd for C49H73O20+(M+H)+981.4690,found 981.4686.

Stearoyl-troxerutin(I)

Yellow solid,yield,20%,Rf = 0.55,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm):7.86-7.84(m,2H,1H of H2′,1H of H6′),7.16-7.14(d,1H,J=8.8 Hz,H5′),6.74(s,1H,H8),6.38(s,1H,H6),5.36-5.35(d,1H,J=4.0 Hz,H1′),4.44-4.39(m,3H,2H of A acylated,1H of B acylated),4.31(m,1H,H1‴),4.26(m,1H,H of B acylated),4.12-4.06(m,4H,H of A),3.74-3.76 (m,4H,H of B),3.39-3.06 (10H,H of rhamnosyl),2.35-2.32(m,2H,CH2CO-troxerutin),1.52-1.22(m,30H,other CH2of stearic acid),0.98-0.97 (d,3H,J = 5.6 Hz,CH3of rhamnosyl),0.86-0.83(m,3H,CH3of stearic acid);13C NMR:14.4,18.1,29.0,29.2,29.4,29.6,31.8,33.9,60.0,62.8,67.3,67.5,68.7,70.6,70.8,70.9,71.1,72.2,74.6,76.5,76.9,98.8,101.3,102.0,105.5,113.4,115.5,122.8,123.5,147.5,148.1,151.4,156.9,161.3,165.1,173.4,177.9;IR(KBr):3369 cm-1(OH),1735 cm-1(O=C-O); ESI-HRMS (m/z): calcd for C51H77O20+(M+H)+1009.5003,found 1009.5004.

Oleoyl-troxerutin(J)

Yellow solid,yield 19%,Rf = 0.53,1H NMR (400 MHz,DMSO-d6+D2O,δ ppm):7.85-7.71(m,2H,1H of H2′,1H of H6′),7.15-7.13(d,J=8.8 Hz,1H,H of H5′),6.74-6.73(s,1H,H of H8),6.38-6.37(s,1H,H6),5.36-5.34(d,J=4.0 Hz,1H,H1″),4.42-4.39(m,5H,2H of A acylated,1H of Bacylated,2H of CHCH of oleic acid),4.32-4.26(m,2H,1H of H1‴,1H of Bacylated),4.13-4.15 (m,4H,H of A),3.77-3.74(m,4H,H of B),3.40-3.18(m,10H,H of rhamnoglucosyl),3.10-3.04(m,2H,CH2of oleic acid),2.70-2.73 (m,2H,CH2of oleic acid),2.35-2.29 (m,2H,H of CH2CO-troxeruin),2.02-1.95 (m,4H,CH2of oleic acid),1.52-1.23(m,22H,other CH2of oleic acid),0.97-0.96(d,J = 6.4 Hz,3H,CH3of rhamnosyl),0.86-0.83 (m,3H,CH3of oleic acid);13C NMR: 14.3,18.1,22.4,22.5,24.8,25.6,27.1,29.0,29.1,29.3,29.5,31.3,31.7,33.8,60.0,62.9,67.3,67.5,68.7,70.5,70.8,70.9,71.1,72.2,74.7,76.4,76.8,93.3,98.8,101.3,102.0,105.5,113.4,115.5,122.8,123.7,130.1,134.2,147.5,150.8,151.2,156.9,161.3,165.1,173.4,177.9; IR (KBr): 3369 cm-1(OH),1735 cm-1(O=C-O),1650 cm-1(C=C); ESI-HRMS (m/z): (M + H)+: calcd for C51H73O20+(M+H)+1007.4837,found 1007.4835.

Linoleyl-troxerutin(K)

Yellow solid,yield:18%,Rf=0.49,1H NMR(400 MHz,DMSO-d6+D2O,δ ppm):7.84-7.71(m,2H,1H of H2′,1H of H6′),7.15-7.13(d,J=8.8 Hz,1H,H of H5′),6.74-6.73(s,1H,H of H8),6.38(s,1H,H6),5.36-5.35 (d,J = 4.0 Hz,1H,H1″),5.32-5.29 (m,2H,H of CHCH of linolieic acid),4.42-4.39(m,5H,2H of A acylated,1H of B acylated,2H of CHCH of linolieic acid),4.32-4.26(m,2H,1H of H1‴,1H of B acylated),4.13-4.05(m,4H,H of A),3.76-3.71(m,4H,H of B),3.40-3.23(m,10H,H of rhamnoglucosyl),3.10-3.04 (m,2H,CH2of linolieic acid),2.35-2.31 (m,2H,H of CH2CO-troxeruin),2.02-1.95 (m,4H,CH2of linolieic acid),1.52-1.23(m,16H,other CH2of linoleic acid),0.97-0.96 (d,J = 6.0 Hz,3H,CH3of rhamnosyl),0.86-0.83 (m,3H,CH3of linoleic acid);13C NMR: 14.3,18.1,22.4,24.8,25.6,27.0,29.0,29.1,29.4,29.5,31.3,33.8,60.0,62.8,67.3,67.5,68.7,70.6,70.8,70.9,71.1,72.2,74.7,76.4,76.8,93.3,98.8,101.3,102.1,105.5,113.4,115.5,122.8,123.7,128.1,130.1,134.2,147.5,150.8,151.5,156.9,161.3,165.1,173.4,177.9;IR (KBr):3369 cm-1(OH),1735 cm-1(O=C-O),1645 cm-1(C=C); ESI-HRMS (m/z): calcd for C51H73O20+(M+H)+1005.4690,found 1005.4686.

Alpha-linoleyl-troxerutin(L)

Yellow solid,yield:17%,Rf=0.45,1H NMR(400 MHz,DMSO-d6+D2O,δ ppm):7.84-7.71(m,2H,1H of H2′,1H of H6′),7.15-7.13(d,J = 8.8 Hz,1H,H of H5′),7.15-7.13 (d,J = 8.0 Hz,1H,H of H5′),6.74-6.73(s,1H,H of H8),6.38(s,1H,H6),5.36-5.34(d,J=4.0 Hz,1H,H1″),5.33-5.29 (m,4H,H of CHCH of alpha-linolenic acid),4.44-4.39(m,5H,2H of CHCH of alpha-linolenic acid,1H of B acylated,2H of A acylated),4.31-4.26 (m,2H,1H of H1‴,1H of B acylated),4.13-4.11 (m,4H,H of A),3.77-3.74 (m,4H,H of B),3.38-3.24 (m,10H,H of rhamnoglucosyl),2.77-2.74 (m,4H,H of = CHCH2CH=CHCH2CH= of alpha-linolenic acid),2.35-2.31(m,2H,H of CH2COtroxeruin),2.04-1.99 (m,4H,CH2of = CHCH2of alpha-linolenic acid),1.52-1.23 (m,10H,other CH2of alpha-linolenic acid),0.97-0.96 (d,J = 6.0 Hz,3H,CH3of rhamnosyl),0.93-0.89 (m,3H,CH3of alpha-linolenic acid);13C NMR: 14.5,18.1,20.4,22.4,24.8,25.8,25.6,27.0,28.9,29.0,29.1,29.4,31.3,33.8,60.0,62.8,67.3,67.5,68.7,70.6,70.8,70.9,71.1,72.2,74.7,76.4,76.8,93.3,98.8,101.3,102.0,105.5,113.4,115.5,122.8,130.1,131.9,134.2,147.5,150.8,151.5,156.9,161.3,165.1,173.4,177.9; IR (KBr):3369 cm-1(OH),1735 cm-1(O=C-O),1645 cm-1(C=C); ESIHRMS (m/z): (M + H)+: calcd for C51H71O20+(M + H)+1003.4533,found 1003.4534.

2.3.Evaluation of antioxidant activities

2.3.1.DPPH free radical scavenging method

The determination of antioxidant capacity by DPPH free radical scavenging described in the literature was used with some modifications[28].Basically,0.1 mmol DPPH methanol solution (1.5 mL) was mixed with 1.5 mL methanol solution containing different concentrations of compounds.The mixtures were left to oscillate for 5 min then placed at room temperature in the dark for 2 h.The absorbance was measured at 517 nm using UV-Vis spectroscopy.DPPH free radical scavenging rate (%) was calculated using the equation:[1-(Ai-Aj)/Ac]×100%,where Acis the absorbance of DPPH solution,Aiis the absorbance of mixed solutions of DPPH and test compounds,and Ajis the absorbance of test compounds.The antiradical activities of the compounds were expressed through their IC50values,presenting the concentration required to scavenge 50% of DPPH radicals.

2.3.2.Potassium ferricyanide reduction process

The potassium ferricyanide reduction assays were carried out as described by Kumar et al.[29]and Borneo et al.[30]with some modifications.Briefly,0.05 mmol ethanol sample solution (2.5 mL) was mixed with 2.5 mL phosphate buffer(pH=6.6,0.2 mol/L) and 2.5 mL of 1%K3Fe(CN)6solution(mass concentration).After water bath oscillation for 30 min at 50 °C in the dark,2.5 mL of 10%CCl3COOH solution(mass concentration)was added to the mixture.Next,after centrifugation for 20 min,the upper layer containing 2.5 mL in the test tube was taken out and 2.5 mL water with 0.5 mL of 0.1%FeCl3(mass concentration)were added to the supernatant to form true mixture.The absorbance was measured at 700 nm.Note that higher absorbance would reflect elevated antioxidant capacity[31].

2.4.Determination of oil-water distribution coefficient(P)

This was carried out according to the procedure reported by Luo et al.[32]with some modifications.The oil-water partition coefficients of all compounds were measured by shaking flask UV-Vis spectrophotometry[18].Two-phase saturated systems containing water and 1-octanol were prepared.The test compounds were added to 1-octanol to yield saturated solutions of compounds.Next,2 mL of saturated solution containing the compounds was added into a test tube and completed with 2 mL water.The mixture reached two- phase equilibrium after shaking process of 15 min.The upper 1-octanol and lower water solutions were then taken out and diluted properly to determine their peak areas and calculate product concentrations using HPLC.The oil-water distribution coefficient(P)values of the compounds were calculated using the formula:logP=log(C0/C1),where C0and C1are the concentrations of measured substance in 1-octanol and water,respectively.

3.Results and discussion

3.1.Enzymatic synthesis

Troxerutin fatty acid esters were synthesized by transesterification of troxerutin and fatty acid vinyl esters mixed in pyridine and in presence of alkaline protease enzyme.The structures of all compounds were confirmed by HRMS,FT-TR,and NMR.For example,the reaction of troxerutin and vinyl hexanoate led to hexanoyl-troxerutin based product.Its characterization through ESI-HRMS showed strong ion peak at m/z=841.3124.The chemical formula of the product was then identified as C39H52O20(M=840.3125).Hence,the ion peak at m/z(841.3124)indicated the formation of product (A),which was further confirmed by NMR.Compared to troxerutin,the1H NMR characteristic peaks of hexanoyl-troxerutin (δ 2.36-2.32,1.54-1.23,and 0.84-0.83 ppm)and13C NMR characteristic peaks of hexanoyl-troxerutin (δ 173.4,33.8,31.1,24.6,22.2,and 14.4 ppm)were all present.The FT-IR data of hexanoyl-troxerutin showed carbonyl absorption at 1735 cm-1,confirming the structure of hexanoyltroxerutin.All compounds were analyzed using the same procedures.

3.2.Reaction conditions

The influences of reaction conditions,such as water content,structure of acyl donors,enzyme load,and molar ratio of substrate on the enzymatic transesterification of troxerutin and fatty acid vinyl esters were all examined.The process consisted of changing one condition at a time and maintaining the others.The reactions were performed in an oscillating equipment for 80 h and the obtained yields were determined by HPLC.

3.2.1.Influence of water content

Water content has an important influence on enzymatic synthesis in non-aqueous media[33].Ara újo et al.[4]reported the acylation of hesperidin with decanoic acid catalyzed by CALB.Their data suggested a decrease in yield from 39.7 to 14.0%as water content increased from 200 ppm to 500 ppm.Here,the effect of water content present in the reaction medium on activity of alkaline protease was evaluated.Pyridine was dried in a molecular sieve for 20 h.Fig.2 showed 58%yield was achieved at water content of 1%.However,the activity of alkaline protease and yield(21%)both declined as water content rose to 2%.Further increase in water content reduced the yield gradually.This could be explained by the fact that higher water contents may surround the enzyme by water,hence preventing the reactants from interacting with the enzyme[34].Kontogianni et al.[35]noticed similar behavior and obtained higher yield at initial water activity of 0.11 or less.

3.2.2.Effect of structures of acyl donors

Some studies[6,11]have been reported that the structures of acyl donors play important roles in enzymatic synthesis of flavonoids.Here,nine fatty acid vinyl esters with different chain lengths and three unsaturated fatty acid vinyl esters with the same chain length were employed as acyl donors to investigate the influence of acyl donors structure on yields.Fig.3 revealed that troxerutin and vinyl hexanoate synthesized by alkaline protease reached the highest yield of 58%.The reaction yield decreased from 58%to 21%as chain length of acyl donors rose from C6 to C18.Katsoura et al.[36]noticed similar trend during enzymatic synthesis of naringin and rutin with fatty acids using vinyl esters as acyl donors.They concluded that reaction yield declined as chain length increased.

Fig.3 also revealed that reaction yield did not undergo significant change in presence of unsaturated acyl donors with same chain length(C18).Salem et al.[6]reported enzymatic synthesis of isoquercitrin using stearic(C18)and oleic acid(C18:1)ethyl esters by CALB,and noticed no change in reaction yield.

3.2.3.Effect of enzyme load

Enzymes play important roles in modification of natural products thanks to their selectivities and elevated activities[37].Alkaline protease showed high activity for transesterification of troxerutin[38],hence was employed as enzyme.Enzyme load plays also an important role in enzymatic synthesis reactions.Here,the effects of different enzyme loads(10,20,30,40,and 50 mg/mL) on enzymatic synthesis of troxerutin and vinyl hexanoate were investigated.The results indicated that reaction yield increased with enzyme load of alkaline protease(Fig.4).At enzyme load of 30 mg/mL,the reaction yield reached 58%.However,further increase in enzyme load induced no significant change in reaction yield.

Fig.2.Effect of water content on the reaction.Conditions:0.01 mmol troxerutin,0.1 mmol vinyl hexanoate,1.5 mL pyridine with different proportions of water content(0,1%,2%,5%and 10%),80 mg alkaline protease,50 °C,and 80 h.The yields are determined by HPLC.

Fig.3.Effect of acyl donor chain lengths and unsaturated levels.Condition:0.01 mmol troxerutin,0.1 mmol fatty acid vinyl esters with different structures,1.5 mL pyridine,80 mg alkaline protease,50 °C,and 80 h.The yields are determined by HPLC.

3.2.4.Effect of molar ratio

The molar ratio of the substrates plays an important role in obtained reaction yield [39].The effect of molar ratio of substrates was studied through reaction of troxerutin and vinyl hexanoate in pyridine.The reaction yield increased from 14% to 58% as molar ratio of troxerutin and acyl donor enhanced from 1:1 to 1:3 (Fig.5).Further increase in molar ratio did not improve the reaction yield.It was indicated that the reaction reached equilibrium as acyl donor volume rose.

3.3.Antioxidant properties of troxerutin fatty acid esters

Fig.4.Effect of enzyme load.Condition:0.01 mmol troxerutin,0.1 mmol vinyl hexanoate,different enzyme loads of alkaline protease (10,20,30,40,and 50 mg/mL),1.5 mL pyridine,50 °C,and 80 h.The yields are determined by HPLC.

Fig.5.Effect of molar ratio of substrate.Condition:0.01 mmol troxerutin,different concentrations of vinyl hexanoate (0.01,0.02,0.03,0.04,0.06,0.08,and 0.10 mmol),80 mg alkaline protease,1.5 mL pyridine,50 °C,and 80 h.The yields are determined by HPLC.

The antioxidant properties of all compounds are listed in Table 1.Troxerutin showed better antioxidant abilities than all of its esters using both DPPH and potassium ferricyanide reduction methods.This was consistent with trends of acylated flavonoids reported elsewhere [24].Using DPPH assay,troxerutin recorded an IC50value of 3.073 mmol,and stearoyltroxerutin with IC50=13.219 mmol revealed the lowest free radical scavenging ability.Additionally,the antioxidant abilities of troxerutin fatty acid esters decreased as carbon chain of troxerutin fatty acid esters rose.This could be related to increased volume and steric hindrance of the esters by acylation,which made it more difficult to reach the radical active sites[34].

Lue et al.[24]reported the antioxidant ability of rutin and its esters,and noticed consisting trend.For troxerutin fatty acid esters with the same length chain,DPPH free radical scavenging ability of troxerutin unsaturated fatty acid esters was higher than that of stearoyl-troxerutin (C18,IC50= 13.219 mmol).The IC50values of alpha-linoleyl-troxerutin(C18:3),oleoyl-troxerutin(C18:1)and linoleyl-troxerutin(C18:2)were recorded as 3.175,3.791 and 3.522 mmol,respectively.Hence,the presence of unsaturated bonds and saturation level of acyl donors may influence the antioxidant ability[4].In addition,the potassium ferricyanide reduction method showed an absorbance peak of hexanoyl-troxerutin(A=0.543)at 700 nm,which was the highest among all employed esters.On the other hand,the absorbance of troxerutin fatty acid esters decreased from A=0.543 to A=0.355 as chain lengths of troxerutin fatty acid esters rose from C6 to C18.The absorbance of troxerutin unsaturated fatty acid esters were also slightly higher than those of troxerutin saturated fatty acid esters for the same chain length (Table 1).Hence,introduction of double bonds in products may increase the electronic supply capability.

Table 1 Antioxidant capacity and oil-water distribution coefficients.

3.4.Oil-water distribution coefficients(P)

The oil-water distribution coefficient is an important parameter to evaluate the lipophilic properties of compounds and their transmission through cell membrane[28,40,41].The logP value of troxerutin was previously estimated as-2.12,indicating that water solubility of troxerutin was too strong to cross the lipid membrane[28].Table 1 also showed that logP values of troxerutin fatty acid esters were larger than that of troxerutin(logP=-2.12),meaning improved lipophilicity of troxerutin fatty acid esters.The increase in troxerutin fatty acid ester chain lengths from C6 to C18 raised the logP values of troxerutin fatty acid esters from 0.15 to 1.94.On the other hand,logP value of oleoyl-troxerutin was estimated to 1.04,which was higher than those of linoleyl-troxerutin (0.63) and alphalinoleyl-troxerutin(0.37).This may be associated with the effect of double bonds on lipid solubility.Although troxerutin fatty acid esters showed lower antioxidant abilities than that of troxerutin,they were more lipophilic.Thus,more efficient in hydrophobic media with increased bioavailability [14].It has been reported that suitable oil-water distribution coefficients may enhance the ability of compounds to pass through cell membrane[28].This may provide support for bioavailability of troxerutin derivatives.

4.Conclusions

Troxerutin fatty acid esters were synthesized through transesterification of troxerutin with saturated fatty acid vinyl esters and unsaturated fatty acid in pyridine as solvent and in presence of alkaline protease enzyme.The structures of obtained products were confirmed by IR,HRMS and NMR analyses.Water content of pyridine ＜1%,molar ratio of troxerutin to fatty acid vinyl ester of 1:3 and amount of alkaline protease of 30 mg/mL induced the highest reaction yield(58%).The reaction yield decreased from 58%to 17%as carbon chain length of acyl donors rose from C6 to C18.The antioxidant capacity of troxerutin fatty acid esters was lower than that of troxerutin.The increase in chain length of troxerutin fatty acid esters from C6 to C18 gradually reduced the antioxidant abilities of troxerutin fatty acid esters.The lipophilicity of troxerutin fatty acid esters improved by transesterification and oil-water partition coefficient of these compounds was better than that of troxerutin.This,in turn,increased the bioavailability of troxerutin and would provide reference for further development of troxerutin.

Conflicts of interest

The authors declare that there is no conflicts of interest.

Acknowledgements

This work was financially supported by Science and Technology Department of Henan Province(No.132102310028)and the Program for Innovative Research Team from Zhengzhou(No.131PCXTD605).