Study On The Mechanism Of Atractylodes Fried By Soil To Strengthen Spleen And Relieve Diarrhea

Stir-baking with soil is an herb processing method with a long history. It has been recorded since Jin-Tang Dynasty. The traditional belief is that the processing of frying with soil can enhance the herbs'effects of stimulating appetite and stopping diarrhea, or reduce the side effects. There are over 20 herbs that are fried with soil, such as Bai zhu, Shan yao and Dang gui. Those processed herbs are widely used in the clinical practice. The research from the existing literature was mainly performed on the change of chemical ingredients'content of single herb before and after being fried with soil. There is not enough evidence to reveal the essence of processing mechanism yet. Especially the substantial role of excipients used in the process on the change of efficacy and toxicity has been overlooked.Rhizoma Atractylodis Macrocephalae (Bai zhu) is the dry rhizome from Atractylodes macrocephala Koidz.. It has the effects of tonifying Qi and strengthening Spleen, drying dampness and promoting diuresis, arresting sweating, and preventing miscarriage. It was recorded in the "Ben Cao Zhen Jing" that Bai zhu is the most important herb of treating the spleen. It is the representative herb that is used with being stir-baked with soil. Traditional theory holds that Bai-zhu fried with soil has stronger effect of strengthening Spleen and relieving diarrhea. It has been widely used in clinical practice for ages. However, the research on the processing mechanism of soil-fried Baizhu has not been reported yet. Based on the facts that the traditional soil-frying process requires the soil adhering to the surface of the herbs and the herbs as well as the soil are decocted and administrated together, we brought up a hypothesis that to increase the efficacy, the soil affects not only the chemical composition and dissolution of the processed herbs, but also in vivo. To test this hypothesis, we performed the comparative study to reveal the essential effect of soil on the efficacy of Bai zhu before and after being processed. We compared the efficacy of raw Bai zhu, fried Bai zhu, soil-fried Bai Zhu and kitchen-subsoil-fried Bai zhu. We also compared the content, composition and dissolution of the chemical ingredients and their absorption, distribution and metabolism in vivo. This systematic research explained the mechanism of herbs processed with soil from multiple aspects both in vitro and in vivo for the first time. The main study contents are as follows: 1. Literature researchWe searched a big amount of medical classics from ancient China and performed systematic research on the history of soil-frying process and current research status of the excipients. We also analyzed and sorted the processing history, regulations, process method and mechanism of Bai zhu, and systematically reviewed the research development in the recent 10 years on the original plants, chemical composition and pharmacology of Bai zhu. Literature research helped us build the theoretic foundation, as well as finding the entry point of our research. It provided useful reference for developing and implementing the protocol2. Experimental study2.1 Pharmacodynamic research on the effects of strengthening Spleen and relieving diarrhea of soil-fried Bai zhuAccording to the traditional theory that soil-fried Baizhu has stronger effect on strengthening Spleen and relieving diarrhea due to Spleen deficiency, we chose the animal model with diarrhea due to Spleen deficiency which fits the symptom character in Chinese medicine. Recording the effect on diarrhea index, gastro-intestinal movement and gastro-intestinal hormones of animals with Spleen deficiency, we performed a systematic research on the effects of strengthening Spleen and relieving diarrhea of soil-fried Bai zhu for the first time. The results showed that soil-fried Bai zhu can significantly reduce the rate of loose bowels induced by Fan xie ye, regulate dysfunctional gastro-intestinal movement, and significantly increase the levels of serum motilin and gatrin in mice with Spleen deficiency. There was significant difference on relieving the loose bowels and inhibiting gastric emptying in mice with diarrhea due to Spleen deficiency between soil-fried Bai zhu and raw Bai zhu (P＜0.01), or fried Bai zhu (P＜0.05). It verified that soil-fried Bai zhu has significantly increased effect on relieving diarrhea and is consistent with the traditional herbal process theory. The results also showed that polysaccharides of Bai zhu perform the similar effects with the water extract of soil-fried Bai zhu. It indicates that polysaccharides of Bai zhu are the active ingredients in relieving diarrhea. The results confirmed that kitchen subsoil can significantly reduce the loose bowels in mice with diarrhea due to Spleen deficiency which demonstrated that the excipients themselves have some effect of relieving diarrhea.2.2 Comparison of soil-fried Bai zhu before and after being processedChange of the chemical composition and content of herbs after being processed could be one of the main factors that cause the change of the pharmacologic and clinic effects. To explore the mechanism of stronger effects on relieving diarrhea and strengthening Spleen of soil-fried Bai zhu, we analyzed systematically the change of the content and composition of the main chemical components (such as volatile oils, lactones and polysaccharides) in Bai zhu before and after being soil-fried for the first time. It provided the basis of explaining the mechanism of herbal process of frying with soil.2.2.1 Comparison of volatile oilsVolatile oils were extracted from raw Bai zhu, fried Bai zhu and soil-fried Bai zhu by steam distillation. The content of total volatile oils was determined:0.79% in raw Bai zhu,1.24% in fried Bai zhu and 0.59% in soil-fried Bai zhu. The amount of volatile oils decreased after being fried with soil, however increased after being fried alone. We analyzed the composition of the volatile oils from the three types of Bai zhu by GC-MS and the results showed that there was no difference. This result consists with what's reported in literature. However, the content of the main ingredients in soil-fried Bai zhu is significantly different from those in raw Bai zhu and fried Bai zhu: atractylone and myrcene B significantly decreased whileγ-elemene, palmitic acid and linoleic acid increased in soil-fried Bai zhu.2.2.2 Comparison on the content of AtractylenolideⅠ,Ⅱ,ⅢAtractylenolideⅠ,Ⅱ,Ⅲwere determined by HPLC. The results showed that the content of AtractylenolideⅡdidn't change after being processed while AtractylenolideⅠandⅢsignificantly increased. There was no significant difference in lactones between fried Bai zhu and soil-fried Bai zhu. It means that the transformation of lactones was caused by the heat during the process and the excipients didn't play an essential role.2.2.3 Comparison on the content and composition of polysaccharidesThe polysaccharides of each type of Bai zhu were determined by phenol sulfuric acid method. The composition of monosaccharide was measured by pre-column derivatization HPLC analysis. The results showed that the content of polysaccharides significantly increased in processed Bai zhu. There is no significant difference in polysaccharides between fried Bai zhu and soil-fried Bai zhu. The composition analysis of polysaccharides showed that mannose, ribose, rhamnose, glucose, galactose and arabinose are the common components in Bai zhu both before and after being processed. Glucose is the highest content. There is no significant difference in molar ratio of monosaccharide. The results showed that the increased polysaccharides were caused by the process of fry and the excipients didn't play an essential role.2.2.4 Comparison of HPLC fingerprint spectrumIn this study, we established the fingerprint spectrum of Bai zhu from its hexane extract and methanol extract. The results showed that the similarity of raw Bai zhu and processed Bai zhu (fried and soil-fried) was<0.9, while that of fried Bai zhu and soil-fried Bai zhu was>0.9 from both methanol and hexane extracts. By comparing the HPLC fingerprint spectrum, we also found that the major changes were the area of chromatographic peaks after being processed, while the amount of the chromatographic peaks did not change. It indicates that only the content of the chemical ingredients changed. There is no significant difference between fried and soil-fried Bai zhu.According to the results, soil-frying process can affect the content of volatile oils, esters and polysaccharide of Bai zhu. By comparing the soil-fried Bai zhu and fried Bai zhu, we came to the conclusion for the first time that the changes of the chemical composition were caused mainly by heat. However, soil played an essential role in the composition change of volatile oils.2.3 Study on the adsorption effect of soil on chemical composition of Bai zhuIn addition to the effect on chemical composition of herbs, soil can adsorb some chemical ingredients of the herbs to affect the efficacy. We researched on kitchen subsoil for the first time and compared the chemical ingredients before and after being processed with TLC and HPLC. The results showed that several volatile oils were adsorbed by soil during the soil-frying process.2.4 Comparison of the extracting rate of Bai zhu before and after being soil friedThe traditional dosage form of Chinese medicinal herbs is decoction. The process affects the content and composition of the chemical components and may also affect the efficacy by changing the extracting/dissolution rate. It was the first research to study the optimization of the preparation technology of Bai zhu decoction and compare the content and extracting rate of total sugar and AtractylenolideⅠ,Ⅱ,Ⅲin the standard decoction of each processed Bai zhu. The results showed that extract content, total sugar and AtractylenolideⅠ,Ⅱ,Ⅲwere significantly increased in the processed Bai zhu. There is significant difference between the raw Bai zhu and process Bai zhu (P＜0.01). Ethanol extract and total sugar content in soil-fried Bai zhu were significantly higher than that in fried Bai zhu (P<0.05). It means that being heated with soil can benefit the extract rate more.2.5 Comparison of pharmacokinetics of Atractylenolide III of Bai zhu before and after being processedSome soil would adhere to the surface of soil-fried Bai zhu and enter the body together with the herbal decoction. In addition to its direct effects, soil can also affect the herb efficacy by changing the absorption, distribution and metabolism of the chemical ingredients in herbs. It was the first time that we adopted plasma concentration to compare the absorption and metabolism of the active ingredients atractylenolide III of Bai zhu before and after being fried with soil, and statistically analyzed parameters including the area under the curve (AUC), peak concentration (Cmax) and peak time (Tmax) in each trial group of rats. The results showed that Atractylenolide III is of a two-compartment model with first order absorption. AUC and Cmax increased in the group of soil-fried Bai zhu but with no significant difference than that of raw Bai zhu, fried Bai zhu or Atractylenolide III (P>0.05). Tmax in the group of soil-fried Bai zhu was obviously delayed and there is significant difference compared to the other groups (P<0.05). Results showed that soil can affect the absorption and metabolism of active ingredients in vivo.