Studies On The Common Materials Of Herbs With "Spleen-stimulated Activity" After Wheat Bran-frying Based On Maillard Reaction

This work were financially supported by Jiangsu Province Key Laboratory Foundation of traditional Chinese medicine processing (ZYPZ009) and Fundamental Research Foundation of Nanjing University of Chinese Medicine (10XJC07).There are totally nine drugs recorded in the 2010 edition of China Pharmacopoeia using bran-frying processing method, including Dioscorea opposita, Atractylodes macrocephala, Rhizoma Atractylodis, Semen Euryales, Fructus Aurantii, Fructus Aurantii Immaturus, Cortex Ailanthi, Semen Coicis and Bombyx Batryticatus. The first eight drugs hold obvious spleen-stimulated activity after bran-frying, wheras Bombyx Batryticatus was bran-fried to mask its stinky taste. In this paper, four commonly used spleen-stimulated herbs were chosen:A. Macrocephala, D. opposita, S. Coicis and Rhizoma Atractylodis as experimental object for study.Based on the guidence of Traditional Chinese Medicine theory and modern scientific research, we put forward a view that "The burnt flavor of bran-frying herbs was formed by Maillard reaction". To prove such a view, we simulated the bran-frying process by synthesizing a series of Maillard reaction products. Then, we compared the GC-MS and LC-MS chramatographic fingerprinting of A. Macrocephala, D. opposita, S. Coicis and Rhizoma Atractylodis before and after the bran-frying discovered that the Maillard reaction products were appeared during the proccess of bran-frying, these products including 2-Formylbutane, 3-Formylbutane,5-Hydroxymethylfurfural, Furfural, Cysteine, Cystine and Cysteine glucoside. Pharmacological studies showed that all of these compounds had spleen stimulation activity. These Maillard reaction products had a certain proportion in the burnt bran after the bran-frying, we used such proportion of products to validate the spleen stimulation activity of bran-frying drugs. The results of this study indicated that certain sugar and amino acid in the bran had Maillard reaction during the traditional bran-frying way to process drugs, the products of such chemical reaction created burnt flavor and stimulated the spleen activity which thought to be the therapeutic basis of Chinese traditional bran-frying method to process drugs. This conclusion may lay a foundation for further basic studies of traditional bran-frying way to process drugs.The main content and results of this study includes following aspects:1. Literature researchRecent studies indicated that bran had remarkable effects to enhance drug effect, moderate medicinal properties and mask the stinky taste. In addition, some of the components in bran were proved to cure and prevent multiple diseases.Maillard reaction, also known as "non-enzymatic browning reaction", mainly refers to the complex reaction between reducing sugars and amino acids(proteins). Most products of Maillard reaction have burnt fragnance, wheras during the bran-frying process the drugs also create strong burnt flavor. Consider bran contains abdundant sugars, proteins and amino acids, we come up the theory that the burnt flavor which stimulate the spleen activity from bran-fried drugs may be caused by the Maillard reaction between sugars, proteins and amino acids in bran during the bran-frying process.2. GC-MS and LC-MS analysis of chemical components in drugs before and after the bran-fryingIn this section, eight Pharmacopoeia stated bran-frying drugs were analyzed using GC-MS, Maillard reaction products were both found in the chromatography of eight bran-fried drugs, including Acetic acid,2-Formylbutane,3-Formylbutane,5-Hydroxymethylfurfural and Furfural. Among them,2-Formylbutane processes coffee aroma,3-Formylbutane with an apple fragnance, Furfural contains the freshly-made bread smell,5-Hydroxymethylfurfural process caramel taste and nuts smell. LC-MS analysis indicated that bran-fried Rhizoma Atractylodis, A. Macrocephala, D. opposita and S. Coicis both contains Cysteine, Cystine and Cysteine glucoside. Among them, Cysteine and Cystine are mainly as a flavouring agent in baking food. We validated the formation of Cysteine and Cystine during the process of bran-frying. The results showed that Cysteine is formed from methionine and serine by heating, the sulfhydryl group of Cysteine was further condensated to disulfide bond to form Cystine. Cysteine can also react with the sugar in bran to form Cysteine glucoside. These compounds were both with obvious burnt flavor.It is generally acknowledged in traditional Chinese Medicine that the burnt smell of processed drugs created by the bran-frying holds the spleen-stimulated activity. This smell has obvious effect to promote appetite and stimulate spleen activity. According the properties of the detected compounds and the methods of the bran-frying, we put forward a hypothesis that "There is a close relationship between spleen stimulated activity of bran-fried drugs and the burnt smell compounds created by Maillard reaction during the process of bran-frying, these compounds created by the sugars and amino acids from the bran and drugs which thought to be the therapeutic basis of Chinese traditional bran-frying method to process drugs.’3. Synthesize Maillard reaction products by simulating the bran-frying conditions and validate their spleen stimulation activityIn this section, we simulated the bran-frying process by synthesizing a series of Maillard reaction products, includine 2-Formvlbutane.3-Formvlbutane.5-Hvdroxvmethvlfurfural. Furfural, Cysteine, Cystine and Cysteine glucoside. We use D-xylose excretion of rats and saliva amylase activity test to evaluate their spleen stimulation activity. The results showed that, the Maillard reaction products created by different frying temperature both can stimulate the saliva amylase activity. By comparison of different bran-frying conditions, the Maillard reaction products at temperature 100 ℃ and 120 ℃ process obvious effect against rat’s spleen deficiency caused by R.officinale. In the D-xylose excretion of rats experiment, the Maillard reaction products at temperature 100℃ and 120℃ also hold strongest effect. The experiments of this section indicated that the products of Maillard reaction can treated the spleen deficiency. The most suitable temperature for Maillard reaction is between 100℃ and 120℃, which consistant with the traditional bran-frying temperature.4. Determination of four Maillard reaction products in bran-frying drugs(1) Determination of 2-Formylbutane,3-Formylbutane in bran-fried drugs(Rhizoma Atractylodis, A. Macrocephala, D. opposita and S. Coicis) using GC-MSWe chose four traditional bran-fried drugs in Chinese Pharmacopeia as research subjects, using GC-MS method to analyze the components in the crude drugs,fried drugs, bran-fried drugs and surface scraping of bran-fried drugs hoping to find the variation tendency of Maillard reaction products 2-Formylbutane and 3-Formylbutane. The results showed that the contents of 2-Formylbutane and 3-Formylbutane in bran-fried A. Macrocephala, D. opposita and S. Coicis increased dramatically, wheras the contents of these two compounds in fried Rhizoma Atractylodis have changed in no obvious rules. Conclusion:The contents of 2-Formylbutane and 3-Formylbutane in A. Macrocephala, D. opposita and S. Coicis were increased after the bran-frying. It is worth mentioning that these two bioactive compounds have the highest amount in the surface scraping of bran-fried drugs, which means these components created by bran-frying can be absorbed on the surface of the processed drug.(2) Determiantion of 5-Hydroxymethylfurfural and Furfural in bran-fried drugs(Rhizoma Atractylodis, A. Macrocephala, D. opposita and S. Coicis) using UPLC-DADFurfural is also called the bran-aldehyde, which contains burnt flavor. In this paper, we descibed a UPLC-DAD method to detrermine the contents of 5-Hydroxymethylfurfural and Furfural in the crude drugs,fried drugs and bran-fried drugs for the first time. The results showed that the contents of 5-Hydroxymethylfurfural and Furfural in fried drugs were increased dramatically, which means frying can promote the generation of 5-Hydroxymethylfurfural and Furfural. The bran-fried drugs contains the highest amount of 5-Hydroxymethylfurfural and Furfural, which indicated that both heating and bran-frying can increased the generation of these two comnponds. It is worth mentioning that these two bioactive compounds can be found 20 times higher in the surface scraping of bran-fried drugs, further proves that the components created by Maillard reaction can be absorbed on the surface of the processed drug, created a burnt flavor, which consistent with the Traditional Chinese Medicine drug-process theory.(3) Determiantion of Cysteine, Cystine and Cysteine glucoside in bran-fried drugs(Rhizoma Atractylodis, A. Macrocephala, D. opposita and S. Coicis) using UFLC-MSThe chromatographic separation was achieved on a Shim-Pack NH2 column (3.0 mm×100 mm,5μm) at 40℃. A binary mobile phase system consisted of Acetonile and 0.1% formic acid in water (75:25) at flow rate of 0.6mL/min. All analytes were monitered under positive ionization conditions and quantified by MRM mode. The results showed that the contents of Cysteine, Cystine and Cysteine glucoside in bran-fried drugs were higher than the fried drugs. And these bioactive components can not be determined in the crude drugs.The surface scraping of bran-fried drugs have the highest amount of Cysteine, Cystine and Cysteine glucoside, which further indicated that the components created by bran-frying can be absorbed on the surface of the processed drug.5. Use different pharmacological model to verify the spleen stimulation activity of Maillard reaction products during the bran-frying processOn the basis of content determination, we prepared different combination of Maillard reaction products for pharmacological studies(according the certain proportion in the drug before and after the bran-frying), including crude drug group, bran-fried group, Cysteine group and Cystine group. The results showed that all drug groups stimulate the saliva amylase activity, wheras the bran-fried group, Cysteine condensation group process the most obvious effect against rat’s spleen deficiency caused by R.officinale. In the D-xylose excretion of rats experiment, the bran-fried group, Cysteine+Glucose group and Cysteine condensation group have the strongest effects.Experimental results of isolated rabbit ileum test indicated that different Maillard reaction products have different intensity on the isolated rabbit ileum contraction, the intensity order was as follows:5-Hydroxymethylfurfural, furfural, Cysteine condensation,2-Formylbutane and 3-Formylbutane. Among them, the effect of 5-Hydroxymethylfurfural was particularly obvious.Conclusion:The common spleen-stimulated material in bran-fried drugs are the Maillard reaction products which originated from the sugars, amino acids and proteins in the bran or drugs(2-Formylbutane,3-Formylbutane,5-Hydroxy methylfurfural, Furfural, Cysteine, Cystine and Cysteine glucoside).Innovation points:1. This paper presented the first time that the common spleen-stimulated constituents in bran-fried drugs were Maillard reactions products between sugar, amino acids and proteins from bran or drugs.2. This paper sucessfully simulate the bran-frying conditions to synthesize the Maillard reaction products for the first time. We use D-xylose excretion of rats and saliva amylase activity test to evaluate spleen-stimulated activity of these products. The results confirmed that the Maillard reaction products have obvious spleen-stimulated activity.3. We discovered four Aldehyde substances in bran-fried drugs using GC-MS analysis. The content determination experiment revealed that the contents of all four Maillard reaction products incresed dramatically and have a stationay ratio in the bran-fried drugs.4. We discovered Maillard reaction products in the bran-fried drugs using LC-MS analysis, these products including Cysteine, Cystine and Cysteine glucoside, which proved to be spleen-stimulated components in pharmacological experiments.5.In this study, we discovered for the first time that 2-Formylbutane,3-Formylbutane, 5-Hydroxymethylfurfural, Furfural, Cysteine, Cystine and Cysteine glucoside, the spleen-stimulated components created by Maillard reaction are the common materials in the bran-fried drugs.