Flavone C-glycosides,orientin (luteolin-8-glucoside) and vitexin for example,are functional ingredients existing in some food and pharmaceutical products such as hawthorn ^[1],bamboo leaf extracts ^[2] and trollflowers ^{[1, 3, 4, 5, 6]}. Flavone C-glycosides are composed of flavone aglycones and glycosides connected with C-C bonds,which are chemically stable and resistant to normal acid hydrolysis,different from flavone O-glycosides ^[7]. Under strong acidic conditions,isomerization of flavone C-glycosides could occur,known as Wessely-Moser rearrangement ^{[8, 9]}. However, detailed investigations of isomerization kinetics of flavone C-glycosides remain obscure. During our investigations of functional ingredients of the flowers of Trollius chinensis Bunge, orientin-200-O-galactopyranoside (OGA),orientin,vitexin and their ester derivatives were isolated and identified ^[10]. Moreover,some refined fractions (TC-1 to TC-3) of this herbal extract and their acid hydrolysates also possessed antioxidant capacities ^[11]. These flavone C-glycosides were known to be responsible for multiple pharmacological functions of trollflowers including antiviral, antimicrobial and anti-inflammation activities ^{[5, 12, 13]}. Therefore, innovative separation technologies are necessary to obtain these ingredients for in-depth food and pharmaceutical applications. The objective of the present study was to investigate the preparation of these flavone C-glycosides and its mechanisms under acidic conditions. The TC-1 fraction,which contains mainly OGA,orientin and vitexin,was selected as a model sample to evaluate the kinetic parameters of OGA in detail. This is the first investigation on the acid hydrolysis kinetics of flavone C-glycosides.

OGA,orientin and vitexin standards were purified and identified by their ESIMS,NMR,and UV adsorption spectra in the authors’ lab ^[10]. The purities of the three compoundswere all higher than 98.0% as determined by HPLC. Isoorientin (JZ20120912B) and isovitexin (JZ20130321B) standards were purchased from Nanjing Jingzhu Bio-technology Co.,Ltd. (Nanjing,Jiangsu,China). Three flavone C-glycosides’ fractions,TC-1 to TC-3,were prepared fromT. chinensis as reported before and the qualitative and quantitative analyses of OGA and other analytes were conducted using the HPLC method ^[11].

: For acid hydrolysis of OGA,orientin,isoorientin,vitexin, isovitexin standards (1.0 mg for each one) and TC-1 fraction (5.0 mg),each sample was dissolved in 2 mol/L hydrochloric acid (5.0 mL) and the mixture was allowed to stir for 6 h. The reaction temperaturewas set at 80 ℃.Thereactionsolutionswereneutralized using 4mol/L NaOH at set time and then analyzed by HPLC. For the kinetic analysis of acid hydrolysis process of the TC-1 fraction,each sample (50mg) was dissolved in 10.0 mL 1.0mol/L sulfuric acid (dissolved with 20% ethanol) in a glass tube,and hydrolyzed at 80 ℃. Sample solutions (0.4mL) at set time (0-24 h) were taken out, neutralizedimmediately using4 mol/LNaOHtopH6-7,diluted with methanol to 1.0mL followed by HPLC analysis. Further hydrolysis tests on TC-1 were conducted to evaluate the effects of different hydrolysis parameters,including substrate concentration (3.0-20.0mg/mL of TC-1),solvent concentration]FID$DT_37[(10%-50% of ethanol),acidity (0.5-3.0 mol/L of H⁺),temperature (60,70 and 80 ℃) and reaction time (0-24 h). Sample solutions were then prepared as mentioned above followed by HPLC analysis.

The TC-1 fraction had been extracted previously from trollflowers using macroporous resin,which was mainly composed of OGA,orientin and vitexin ^[11]. During our investigation of the antioxidant properties of TC-1 and its acid hydrolysates,OGA and other ingredients were observed to convert to some hydrolysates under acidic conditions. To further investigate these phenomena, hydrolysis of TC-1was conducted at 80 ℃ using 2mol/L HCl,and the hydrolysates at set time intervals were analyzed using the HPLC method. By comparing the retention time of the hydrolysates with that of the standards,the transformation ofOGAand otherflavone Cglycosides could be readily monitored as shown in Fig. 1. OGA (1) could be hydrolyzed to orientin (2) slowly and disappeared after 4 h. Orientin (2) isomerized to isoorientin (4) after 6 h of hydrolysis,and finally reached equilibrium with a ratio of about 1:0.8 of 2:4 after 24 h. Vitexin (3) could also undergo a similar isomerization to isovitexin (5) and reached equilibrium(1:0.5 of 3:5) after 24 h under the same hydrolysis conditions. These transformations could also take place when using the OGA,orientin,isoorientin,vitexin or isovitexin standards,respectively (data not shown).

Fig. 1

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Fig. 1. HPLC chromatograms of mixed standards and TC-1 hydrolysates at 0,2,4 and 6 h monitor at 340 nm. (1) OGA,(2) orientin,(3) vitexin,(4) isoorientin,(5) isovitexin.

It is well known that flavone O-glycosides can be hydrolyzed to the corresponding aglycones and sugar moieties under acidic conditions. Flavone C-glycosides,however,are chemically stable and resistant to normal acid hydrolysis. Under higher temperatures and acidity,isomerizations of flavone C-glycosides could occur following a reversible Wessely-Moser rearrangement reaction ^{[8, 9]}. Accordingly,the terminal O-glucosyl fragment of OGA (1) could be hydrolyzed to produce orientin (2) initially,as depicted in Fig. 2. Under stronger acid conditions or longer time as shown in Fig. 1,orientin and vitexin could then isomerize to corresponding isoorientin or isovitexin,and reach equilibrium finally. This process was also confirmed by the acid hydrolysis of isovitexin and other flavone C-glycosides as mentioned above. It should be noted that other derivatives of flavone C-glycoside ester, including 6'''-(3-hydroxy-3-methylglutaroyl)-2''-O-β-D-galactopyranosyl orientin,7-methoxyl-2''-O-(2'''-methylbutyryl) orientin, 7-methoxyl-2''-O-(3''',4'''-dimethoxybenzoyl) vitexin etc. ^[10] could be hydrolyzed to orientin or vitexin in a similar way,as observed in the TC-2 fraction ^[11].

Fig. 2

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Fig. 2. Acid hydrolysis reaction of OGA (1),orientin (2) and vitexin (3) in TC-1 fraction. OGA could be hydrolyzed to orientin (2); orientin (2) and vitexin (3) could be hydrolyzed to isoorientin (4) or isovetexin (5),respectively via a chaltone intermediate; 4/5 could be hydrolyzed to 2/3 either and finally reach in equilibrium.

Since the hydrolysis process of flavone C-glycosides was not sufficiently investigated before,the kinetics of the OGA acid hydrolysis was further analyzed using the TC-1 fraction as a model sample. It was also of concern to investigate whether these reactions could be carried out under proper control,so that the isomerizations between flavone C-glycosides were inhibited to maintain their natural characters as expected.

As for the acid hydrolysis of TC-1,some reaction conditions were investigated initially. The concentration of TC-1 was set at 5 mg/mL in this case. The addition of organic solvents,such as methanol or ethanol [76TD$DIF](10%-50%),was necessary to improve the solubility of the substrates,and 20% ethanol was found to be optimal. Sulfuric acid was applied instead of the volatile hydrochloride acid. Suitable acidities (0.5-3.0 mol/L) and temperatures (60-80 ℃) were then investigated to achieve satisfied reaction rate and product purity. Finally,the reaction rate was measured in detail under three different acidities (1.0,1.5 and 2.0 mol/L of H+) and temperatures (60,70 and 80 ℃) (Table 1).

Table 1

Table 1 Regression equations and rate constants for the acid hydrolysis of OGA in TC-1 fraction at different temperature and acidity.

Table 1 Regression equations and rate constants for the acid hydrolysis of OGA in TC-1 fraction at different temperature and acidity.

Under these circumstances,OGA was hydrolyzed to orientin smoothly in 7 h. Orientin would not isomerize to isoorientin during the 7 h hydrolysis period unless at 80 ℃ and with 1.5 mol/L H+,in which case isoorientin concentration accounted for 5% of the mixture and 18% of the OGA remained. As our aim was to minimize the isomerization of orientin in this survey,no longer reaction time or stronger hydrolysis condition was conducted in the following tests.

To quantitatively measure acid hydrolysis kinetics of OGA in TC-1,concentrations of OGA in different hydrolysates were determined using the HPLC method. As shown in Fig. 3(A),the hydrolysis process was affected significantly by the reaction temperature. Reaction rate was rather slow at 60 ℃,faster at 70 ℃, and quite rapid at 80 ℃. Acidity could also affect the reaction rate to some content.

Fig. 3

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Fig. 3. Contents of OGA in TC-1 fraction during different acid hydrolysis processes (A) and simulated first-order reaction kinetics for OGA at different temperatures and acidities (B).

The reaction follows first-order reaction kinetics and a plot of ln C vs. t produces a straight line according to Eq. (1):

Furthermore,the temperature dependency of the rate constant k can be described by the Arrhenius equation (2):

Fig. 4

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Fig. 4. Arrhenius plot of OGA in TC-1 fraction at different temperatures and rate constants.

Finally,the optimal hydrolysis conditions were set at 80 ℃, 1.0 mol/L H⁺ and 7 h,and about 77% OGA could be converted to orientin with no detectable isoorientin in this case. Preparations of sample hydrolysates were thus carried out on the basis of the conditions optimized above. As a result,TC-1h to TC-3h was successively obtained from TC-1 to TC-3,respectively. The process ran with satisfaction. For example,by comparison with that of TC-1,the content of orientin in TC-1 h (671.3 mg/mg) increased 1.38-fold with little OGA left.

Transformation mechanisms of flavone C-glycosides of T. chinensis were studied under acidic conditions. OGA could be hydrolyzed to orientin quantitatively,followed by an isomerizing to isoorientin via the Wessely-Moser rearrangement reaction under stronger acidic conditions. Acid hydrolysis kinetics of OGA in TC-1 fitted first-order model. At optimal conditions set at 80 ℃, 1.0 mol/L H+ and 7 h,about 77% OGA was hydrolyzed to orientin with no detectable isoorientin. The results could be helpful for better understanding of the acid hydrolysis kinetics of flavone glycosides and the preparation of these valuable ingredients under controlled acid hydrolysis conditions.

This work was supported by National Natural Science Foundation of China (No. 81274190),Jiangsu Provincial Science and Technology Commission (No. BE2012649),and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).