| ObjectiveAurantii Fructus decoction pieces used in Guangdong province and Hong Kong followed concocted to "integrated traditional Chinese medicine experience" Aurantii Fructus cut processing methods described steaming,that is to take the raw herbs,pitted and flesh,soaked with water,to be fermented,steamed brown to purple,sliced and dried.This method is different from the processing method of Chinese Pharmacopoeia collection contains,therefore,Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong is a local characteristics decoction pieces.However,it is less by modern research about this by fermentation used in Aurantii Fructus decoction pieces,why in Guangdong and Hong Kong still continue to use fermentation processing methods have been scientific explanation is not obtained.Literature studies have shown that microbes have a strong ability to break down conversion,and can produce a wealth of secondary metabolites,used in traditional Chinese medicine has many advantages.Therefore,this paper uses the same batch of raw materials of Aurantii Fructus for the original ingredients,study on chemical variation between raw and fermentation processed Aurantii Fructus and optimization of processing method,to find its influence on the component change,to explore the material basis and mechanisms of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong,to provide experimental data for the development of quality evaluation method of Aurantii Fructus fermentation pieces,and provides experimental evidence for further development and utilization of fermentation processing technology and scientific evaluation of this feature for future Aurantii Fructus processing method.Methods1.In the same batch of raw Aurantii Fructus herbs as raw materials,refer to Guangdong and Hong Kong's traditional processing methods made the Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong sample,as well as use the traditional processing methods default fermentation step made the Aurantii Fructus control decoction pieces sample.The three samples were analyzed and compared by UPLC,used naringin standard for comparison,find the UPLC salient features peaks of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong,calculate the relative retention times and variable range,to establish this decoction pieces UPLC peaks characteristic.2.The four UPLC characteristic peaks areas and mildew traits were considered as indicators,taking the temperature of 18 ?,24 ?,30 ? three levels,taking the humidity of 70%,80%,90%three levels,time to take 3 d,5 d,7 d three levels,used the L9(34)orthogonal design table designed the orthogonal experiment,to optimize Aurantii Fructus decoction pieces fermentation processing technology by range analysis and variance analysis.3.Using the same batch of raw Aurantii Fructus materials for the original ingredients,refer to "Chinese Pharmacopoeia" 2010 edition of Aurantii Fructus decoction pieces processing requirements made the Aurantii Fructus decoction pieces sample;refer to"Chinese Pharmacopoeia" 2010 edition bran fried processing general and bran fried Aurantii Fructus decoction pieces processing requirements made the bran fried Aurantii Fructus decoction pieces sample;taken the same batch raw herbs of Aurantii Fructus,washed,soaked with water,into the sack,after fermentation by the temperature of 30 ?and humidity of 70%for 7 days,washed out,steamed 3 hours,bored one night,to the interior of purple-brown,removed,sliced and dried to make the Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong sample.With digital zoom microscope to observe the exterior features and micro characteristics on the three samples.Explore appearance and micro characteristics changes before and after processing of Aurantii Fructus.in order to identify Aurantii Fructus concocted decoction pieces.4.Using the same batch of raw Aurantii Fructus materials for the original ingredients,refer to "Chinese Pharmacopoeia" 2010 edition of Aurantii Fructus decoction pieces processing requirements made the Aurantii Fructus decoction pieces sample;refer to"Chinese Pharmacopoeia" 2010 edition bran fried processing general and bran fried Aurantii Fructus decoction pieces processing requirements made the bran fried Aurantii Fructus decoction pieces sample;taken the same batch raw herbs of Aurantii Fructus,washed,soaked with water,steamed 3 hours,bored one night,to the interior of purple-brown,removed,sliced and dried to make the steam control Aurantii Fructus decoction pieces sample;taken the same batch raw herbs of Aurantii Fructus,washed,soaked with water,into the sack,after fermentation by the temperature of 30 ? and humidity of 70%for 7 days,washed out,sliced and dried to make the fermentation control Aurantii Fructus decoction pieces sample;taken the same batch raw herbs of Aurantii Fructus,washed,soaked with water,into the sack,after fermentation by the temperature of 30 ? and humidity of 70%for 7 days,washed out,steamed 3 hours,bored one night,to the interior of purple-brown,removed,sliced and dried to make the Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong sample.The five samples were tested by UPLC-Q-TOF/MS,to analyze chemical variation before and after processing.Identified component structure by mass spectrometry analytical,literature and analyze the impact of the change component of clinical efficacy.Results1.By comparing the UPLC profiles of the test samples,showing that the fermentation processed Aurantii Fructus could produce the peak 3 and peak 4 two new chemical components significantly which could not be found in the raw samples and default fermentation process control samples.Prove the chromatographic peak 3 and peak 4 are the new ingredients of Aurantii Fructus after microbial fermentation,showing that natural microbial fermentation make Aurantii Fructus produce new ingredients,also indicated that Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong concocted by the fermentation can produce some new ingredients,preliminary showed the advantage of this characteristic processing method,provide a scientific basis for its processing mechanism,preliminary explored the material basis of this characteristics concocted processing.Used naringin standard for comparison,find the four UPLC salient features peaks of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong,calculate the relative retention times and variable range,to establish this decoction pieces UPLC peaks characteristic,multi-index comprehensive evaluation the quality of Aurantii Fructus decoction pieces,to provide scientific experimental basis for the inherit and carry forward traditional processing techniques.2.The results showed that the temperature factor have significant impact on the five indicators results of the four characteristic peaks area and mildew traits,time factors also have significant impact on the results of the peak 4 area,and humidity have not significant impact on the five indicators results of the four characteristic peaks area and mildew traits.Confirm the humidity of 70%,80%,90%levels have little effect on the results of the fermentation processing,in the fermentation processing process in Aurantii Fructus,first consider the effects of temperature factors,followed consider the effects of the time factor.Finally,overall balance method for multiple indicators for processing,and do verification tests,select Aurantii Fructus optimum conditions for the fermentation processing are met at a temperature of 30 ? and humidity of 70%for 7 days.3.The exterior features and micro characteristics were observed and compared on three samples of the Aurantii Fructus decoction pieces,the bran fried Aurantii Fructus decoction pieces,the Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong.The results showed that micro characteristics changes before and after processing were very obvious on the bran fried Aurantii Fructus decoction pieces and the Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong.Summarized as follows:Viewed from the vertical longitudinal section of the skin,the oil chamber tattered,white discharge scattered in the periphery of the organization for proprietary micro characteristics of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong;Viewed from the vertical longitudinal section of the skin,smooth edge intermediate oil chamber dry,possession of black carbon tip fragments,and mesocarp spongy voids in possession of black charcoal fragments tip for proprietary micro characteristics of bran fried Aurantii Fructus decoction pieces.This proprietary micro characteristics can be used as the basis for identifying bran fried Aurantii Fructus decoction pieces and Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong.Observations indicated that concocted processing have significant impact on the oil chamber shape and structure,showing that change the structure of the oil chamber to alter the content of volatile oil or oil chamber component escaping effect occurs with other components is one way concocted role of Aurantii Fructus.4.Five samples in positive and negative ESI modes are shown mainly concentrated in 4 to 14 minutes peak,For most of the constituents,[M-H]-,[M+H]+ and/or[M+Na]+adductions were observed.Due to the use of formic acid in mobile phase,the[M+46-H]-fragment ions corresponding to[M+HCOOH-H]-were also observed in negative ion mode.The results provided valuable information for confirming accurate molecular weights and the composition of the constituents.Based on the retention behaviors,accurate molecular weight and MSn fragment data,combined with TOF-MS matching the structural formulas may identify,retrieve ChemSpider chemical database and literature data,30 possible compounds were identified,the identified compounds can be classified into four classes including 16 flavanone,six flavonoids,three coumarins and five limonene terpene.The total ion chromatograms detected under positive and negative ion mode,and variable index based on ion peak area ratio before and after processing were compared to seek their differences.The results indicated that new compounds such as eriodictyol-7-glucoside,hesperetin-7-O-glucoside and 5-demethylnobiletin were generated after the fermentation processing step.The fermentation processing step also significantly increased the naringenin and hesperetin level,and increased the Sudachinoid A,Obacunoic acid,Limonin and Nomilinic acid content.At the same time,explored the produced pathway of new compounds and the conversion pathway of naringenin and hesperetin compounds significantly increased by chemical structure analysis.ConclusionThe fermentation processed Aurantii Fructus could make significant changes in the chemical composition and the optimum fermentation processing could improve the quality of Aurantii Fructus decoction pieces prepared in Guangdong and Hong Kong.Preliminary summarized that the processing mechanism of Aurantii Fructus decoction pieces used inGuangdong province and Hong Kong is use of microbial fermentation in the rich natural extracellular intracellular enzymes,prompting some glycosides degradation to aglycone or secondary glycosides,which increase the bioavailability of Aurantii Fructus,at the same time,micro-organisms secreted extracellular enzymes can also destroy the cell wall structure,increasing the gap between the cells,conducive to the diffusion of substances inside and outside cells,in favor of the dissolution of limonoids derivative components,in order to increase the content of limonoids derivative components to increase the biological activity of Aurantii Fructus.Showing Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong is great significance to enhance clinical efficacy by fermented,it is worth further strengthen the protection and promotion. |
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