土炒白术健脾止泻作用增强的机理研究
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摘要
土炒是一种加辅料炒的常用炮制方法,历史悠久,始于晋、唐时期,其后历代均有记载,传统认为土炒可增强药物健脾止泻的作用或降低刺激性,目前常用的土炒饮片有白术、山药、当归等20余种,在临床广泛使用。关于土炒炮制的原理,从已有的文献检索来看,研究多以某单味中药炮制前后化学成分的含量变化为主,尚不能提供足够的科学依据来揭示其内涵,尤其是忽略了辅料在炮制过程中引起药物药效、毒性变化的实质意义,其科学内涵亟待阐明。
白术为菊科植物白术Atractylodes macrocephala Koidz.的干燥根茎,具有补气健脾,燥湿利水,止汗,安胎的功效,《本草真经》中谓“白术为脾脏第一要药也”,为土炒代表药物之一,传统理论认为白术土炒后健脾止泻作用增强,从古至今一直在临床上广泛使用,但关于白术土炒的炮制机理研究目前还未见文献报道。本课题以白术为研究对象,基于土炒传统要求须在药物表面挂土,作汤剂使用时土可与饮片煎剂一同进入体内的特点,提出研究假说：土除了可对炮制药物成分及溶出产生影响外,也可在体内产生作用,从而达到增效的目的。围绕上述假说,采用对比研究的方法,以揭示土在白术炮制前后药效变化中所起的实质作用为关键问题,通过土炒白术与生白术、清炒白术、炒灶心土之间药效,化学成分含量、组成、溶出及其在体内吸收、分布、代谢三方面的比较,首次从体内和体外两方面、多环节对土炒炮制的科学原理进行系统研究,阐明土炒炮制科学性。主要研究内容如下：
1.文献研究
通过查阅大量的古典医籍对土炒的历史沿革,辅料研究现状等进行了系统研究；对白术炮制历史沿革、炮制规范、炮制工艺、炮制原理等研究现状进行了系统的分析和整理；对近10年来关于白术的药材来源、化学成分及药理作用进展进行了系统回顾。通过文献研究,一方面对研究白术土炒的炮制机理奠定了理论基础,也为本课题研究找到了切入点,对研究方案的制定和顺利实施提供了有益的借鉴。
2.实验研究
2.1土炒增强白术健脾止泻作用的药效学实验研究
根据土炒白术健脾止泻增强,多用脾虚泄泻的传统理论,本文选择符合中医证候特点的脾虚泄泻动物模型,采用对脾虚动物腹泻指数的影响,对胃肠运动的影响,对胃肠激素的影响实验,首次对土炒白术健脾止泻作用进行了系统研究。研究结果表明,土炒白术能明显减少番泻叶致脾虚腹泻小鼠稀便率；调节脾虚腹泻小鼠胃肠运动的紊乱；显著升高脾虚小鼠血清胃动素、胃泌素水平；在减少脾虚腹泻小鼠稀便率、抑制脾虚腹泻小鼠胃排空加快指标上,其作用强度与生品(P<0.01)、清炒品(P<0.05)相比有显著差异,证实白术土炒后其健脾止泻作用确实明显增强,这与传统中医药炮制理论相吻合。研究结果还表明白术多糖具有与土炒白术水煎液一致的药效作用,提示白术多糖是白术健脾止泻的一类主要有效成分。研究证实灶心土也能显著减少番泻叶致脾虚腹泻小鼠稀便率,表明辅料本身即有一定的止泻作用。
2.2土炒炮制前后白术化学成分比较研究
中药经过炮制后,化学成分的组成和含量产生的不同程度改变,可能是其药理作用、临床疗效发生相应变化的主要原因之一。因此本研究为了探讨土炒后白术健脾止泻作用增强的原理,采用现代分析技术,首次对土炒炮制前后,白术挥发油、内酯类、多糖等主要化学成分在含量及组成上的变化进行了系统研究,为土炒炮制机理的科学阐释提供依据。
2.2.1挥发油类成分的测定比较采用水蒸气蒸馏法提取白术生品、清炒品、土炒品中挥发油成分,测定总挥发油含量,结果生品挥发油含量为0.79％,清炒品含量为1.24％,土炒品含量为0.59％,表明白术经土炒炮制后挥发油含量有所降低,但清炒后含量却增高。采用GC-MS方法对三种挥发油成分进行分析比较,结果表明三者在成分组成上无差异,与文献报道一致,但土炒品与生品、清炒品相比在主要成分相对含量上有显著差异：土炒品苍术酮、香叶烯B明显降低,Y-榄香烯、棕榈酸、亚油酸等成分明显升高。
2.2.2白术内酯Ⅰ、Ⅱ、Ⅲ的含量比较采用HPLC法测定各批次白术中白术内酯Ⅰ、Ⅱ、Ⅲ的含量,结果表明白术经炒制后白术内酯Ⅱ含量不变,白术内酯Ⅰ、Ⅲ含量显著升高,清炒和土炒对白术内酯类成分含量影响无显著差异,表明白术炮制过程中内酯类成分的转化主要由炒制加热引起,辅料土对内酯类成分变化无实质作用。
2.2.3多糖含量及组成比较采用硫酸苯酚法测定各批次白术中多糖的含量,采用HPLC柱前衍生法分析白术多糖的单糖组成,结果表明：白术经炒制后白术多糖含量显著升高,而清炒和土炒对白术多糖含量影响无显著差异；多糖组成分析表明,白术炒制前后多糖成分均由甘露糖、核糖、鼠李糖、葡萄糖、半乳糖及阿拉伯糖组成,葡萄糖百分含量最高,但各单糖摩尔比无显著性差异。表明白术炮制过程中多糖含量增加主要由炒制引起,辅料土无实质作用。
2.2.4指纹图谱比较研究本研究分别建立了白术不同极性部位——正己烷和甲醇提取部位指纹图谱。结果表明不论是正己烷提取部位还是甲醇提取部位,土炒品、清炒品对照指纹图谱与生品指纹图谱相似度均<0.9,清炒品对照指纹图谱与土炒品对照指纹图谱相似度均>0.9。通过对HPLC指纹图谱进一步比较分析后发现,白术在炒制前后主要为色谱峰峰面积的改变,而共有色谱峰的峰数目基本未发生变化,说明白术在炒制后,其化学成分仅仅在含量上产生一定的变化,但清炒和土炒对其影响无显著性差异。
从测定结果可以看出,土炒对白术挥发油、内酯类及多糖类成分的含量均有影响,但通过与清炒组的对比首次明确,土炒对白术化学成分的影响主要是加热炒制引起的,辅料土在白术挥发油成分变化中起实质作用。
2.3辅料土对白术化学成分吸附作用研究
辅料在炮制过程中除了可使药物成分发生变化外,也可对药物成分产生定的吸附作用,从而影响药效。本研究首次以辅料灶心土为研究对象,运用TLC、HPLC方法对其炒制前后的化学成分进行了分析比较。结果表明,在土炒过程中白术多个挥发油成分被土吸附。
2.4土炒炮制前后白术主要化学成分煎出率的比较研究
中药传统主要以汤剂形式入药,中药经过炮制,除对其固有成分含量及组成有影响外,其化学成分的溶出也可能会发生改变,从而影响药效。本研究首次对白术水煎液制备工艺进行了优选研究,并对白术各炮制品标准水煎液中总糖、白术内酯Ⅰ、Ⅱ、Ⅲ的含量及煎出率进行了比较,结果土炒后白术醇浸出物含量、总糖含量、白术内酯Ⅰ、Ⅱ、Ⅲ总含量均明显增高,与生品相比有极显著差异(P<0.01)；在醇浸出物、总糖含量指标上,土炒品明显高于清炒品(P<0.05)；表明与辅料土共同加热炮制后更有利于白术化学成分溶出。
2.5土炒炮制前后白术内酯Ⅲ药动学比较研究
白术饮片经土炒炮制后,一部分土会附着在饮片表面,与饮片水煎液一同进入机体,除直接发挥作用外,也可能影响药物成分的吸收、分布、代谢等从而影响药效。本研究首次采用血药浓度法对白术土炒前后水煎液中活性成分白术内酯Ⅲ在大鼠体内的吸收代谢情况进行了初步比较研究,对各给药组药时曲线下面积(AUC)、最大血药浓度(Cmax)、达峰时间(Tmax)等参数进行了统计比较。结果表明白术内酯Ⅲ为一级吸收的二室模型,在土炒水煎液中其AUC、Cmax有增大趋势,但与生品、清炒品、单体给药组相比无显著差异(P>0.05)；Tmax明显延迟,与其他给药组相比有显著差异(P<0.05)。研究结果提示辅料进入机体后可对有效成分的吸收代谢等产生影响。
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 research
We 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 protocol
2. Experimental study
2.1 Pharmacodynamic research on the effects of strengthening Spleen and relieving diarrhea of soil-fried Bai zhu
According 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 processed
Change 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 oils
Volatile 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,Ⅲ
AtractylenolideⅠ,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 polysaccharides
The 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 spectrum
In 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 zhu
In 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 fried
The 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 processed
Some 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.
白术为菊科植物白术Atractylodes macrocephala Koidz.的干燥根茎,具有补气健脾,燥湿利水,止汗,安胎的功效,《本草真经》中谓“白术为脾脏第一要药也”,为土炒代表药物之一,传统理论认为白术土炒后健脾止泻作用增强,从古至今一直在临床上广泛使用,但关于白术土炒的炮制机理研究目前还未见文献报道。本课题以白术为研究对象,基于土炒传统要求须在药物表面挂土,作汤剂使用时土可与饮片煎剂一同进入体内的特点,提出研究假说：土除了可对炮制药物成分及溶出产生影响外,也可在体内产生作用,从而达到增效的目的。围绕上述假说,采用对比研究的方法,以揭示土在白术炮制前后药效变化中所起的实质作用为关键问题,通过土炒白术与生白术、清炒白术、炒灶心土之间药效,化学成分含量、组成、溶出及其在体内吸收、分布、代谢三方面的比较,首次从体内和体外两方面、多环节对土炒炮制的科学原理进行系统研究,阐明土炒炮制科学性。主要研究内容如下：
1.文献研究
通过查阅大量的古典医籍对土炒的历史沿革,辅料研究现状等进行了系统研究；对白术炮制历史沿革、炮制规范、炮制工艺、炮制原理等研究现状进行了系统的分析和整理；对近10年来关于白术的药材来源、化学成分及药理作用进展进行了系统回顾。通过文献研究,一方面对研究白术土炒的炮制机理奠定了理论基础,也为本课题研究找到了切入点,对研究方案的制定和顺利实施提供了有益的借鉴。
2.实验研究
2.1土炒增强白术健脾止泻作用的药效学实验研究
根据土炒白术健脾止泻增强,多用脾虚泄泻的传统理论,本文选择符合中医证候特点的脾虚泄泻动物模型,采用对脾虚动物腹泻指数的影响,对胃肠运动的影响,对胃肠激素的影响实验,首次对土炒白术健脾止泻作用进行了系统研究。研究结果表明,土炒白术能明显减少番泻叶致脾虚腹泻小鼠稀便率；调节脾虚腹泻小鼠胃肠运动的紊乱；显著升高脾虚小鼠血清胃动素、胃泌素水平；在减少脾虚腹泻小鼠稀便率、抑制脾虚腹泻小鼠胃排空加快指标上,其作用强度与生品(P<0.01)、清炒品(P<0.05)相比有显著差异,证实白术土炒后其健脾止泻作用确实明显增强,这与传统中医药炮制理论相吻合。研究结果还表明白术多糖具有与土炒白术水煎液一致的药效作用,提示白术多糖是白术健脾止泻的一类主要有效成分。研究证实灶心土也能显著减少番泻叶致脾虚腹泻小鼠稀便率,表明辅料本身即有一定的止泻作用。
2.2土炒炮制前后白术化学成分比较研究
中药经过炮制后,化学成分的组成和含量产生的不同程度改变,可能是其药理作用、临床疗效发生相应变化的主要原因之一。因此本研究为了探讨土炒后白术健脾止泻作用增强的原理,采用现代分析技术,首次对土炒炮制前后,白术挥发油、内酯类、多糖等主要化学成分在含量及组成上的变化进行了系统研究,为土炒炮制机理的科学阐释提供依据。
2.2.1挥发油类成分的测定比较采用水蒸气蒸馏法提取白术生品、清炒品、土炒品中挥发油成分,测定总挥发油含量,结果生品挥发油含量为0.79％,清炒品含量为1.24％,土炒品含量为0.59％,表明白术经土炒炮制后挥发油含量有所降低,但清炒后含量却增高。采用GC-MS方法对三种挥发油成分进行分析比较,结果表明三者在成分组成上无差异,与文献报道一致,但土炒品与生品、清炒品相比在主要成分相对含量上有显著差异：土炒品苍术酮、香叶烯B明显降低,Y-榄香烯、棕榈酸、亚油酸等成分明显升高。
2.2.2白术内酯Ⅰ、Ⅱ、Ⅲ的含量比较采用HPLC法测定各批次白术中白术内酯Ⅰ、Ⅱ、Ⅲ的含量,结果表明白术经炒制后白术内酯Ⅱ含量不变,白术内酯Ⅰ、Ⅲ含量显著升高,清炒和土炒对白术内酯类成分含量影响无显著差异,表明白术炮制过程中内酯类成分的转化主要由炒制加热引起,辅料土对内酯类成分变化无实质作用。
2.2.3多糖含量及组成比较采用硫酸苯酚法测定各批次白术中多糖的含量,采用HPLC柱前衍生法分析白术多糖的单糖组成,结果表明：白术经炒制后白术多糖含量显著升高,而清炒和土炒对白术多糖含量影响无显著差异；多糖组成分析表明,白术炒制前后多糖成分均由甘露糖、核糖、鼠李糖、葡萄糖、半乳糖及阿拉伯糖组成,葡萄糖百分含量最高,但各单糖摩尔比无显著性差异。表明白术炮制过程中多糖含量增加主要由炒制引起,辅料土无实质作用。
2.2.4指纹图谱比较研究本研究分别建立了白术不同极性部位——正己烷和甲醇提取部位指纹图谱。结果表明不论是正己烷提取部位还是甲醇提取部位,土炒品、清炒品对照指纹图谱与生品指纹图谱相似度均<0.9,清炒品对照指纹图谱与土炒品对照指纹图谱相似度均>0.9。通过对HPLC指纹图谱进一步比较分析后发现,白术在炒制前后主要为色谱峰峰面积的改变,而共有色谱峰的峰数目基本未发生变化,说明白术在炒制后,其化学成分仅仅在含量上产生一定的变化,但清炒和土炒对其影响无显著性差异。
从测定结果可以看出,土炒对白术挥发油、内酯类及多糖类成分的含量均有影响,但通过与清炒组的对比首次明确,土炒对白术化学成分的影响主要是加热炒制引起的,辅料土在白术挥发油成分变化中起实质作用。
2.3辅料土对白术化学成分吸附作用研究
辅料在炮制过程中除了可使药物成分发生变化外,也可对药物成分产生定的吸附作用,从而影响药效。本研究首次以辅料灶心土为研究对象,运用TLC、HPLC方法对其炒制前后的化学成分进行了分析比较。结果表明,在土炒过程中白术多个挥发油成分被土吸附。
2.4土炒炮制前后白术主要化学成分煎出率的比较研究
中药传统主要以汤剂形式入药,中药经过炮制,除对其固有成分含量及组成有影响外,其化学成分的溶出也可能会发生改变,从而影响药效。本研究首次对白术水煎液制备工艺进行了优选研究,并对白术各炮制品标准水煎液中总糖、白术内酯Ⅰ、Ⅱ、Ⅲ的含量及煎出率进行了比较,结果土炒后白术醇浸出物含量、总糖含量、白术内酯Ⅰ、Ⅱ、Ⅲ总含量均明显增高,与生品相比有极显著差异(P<0.01)；在醇浸出物、总糖含量指标上,土炒品明显高于清炒品(P<0.05)；表明与辅料土共同加热炮制后更有利于白术化学成分溶出。
2.5土炒炮制前后白术内酯Ⅲ药动学比较研究
白术饮片经土炒炮制后,一部分土会附着在饮片表面,与饮片水煎液一同进入机体,除直接发挥作用外,也可能影响药物成分的吸收、分布、代谢等从而影响药效。本研究首次采用血药浓度法对白术土炒前后水煎液中活性成分白术内酯Ⅲ在大鼠体内的吸收代谢情况进行了初步比较研究,对各给药组药时曲线下面积(AUC)、最大血药浓度(Cmax)、达峰时间(Tmax)等参数进行了统计比较。结果表明白术内酯Ⅲ为一级吸收的二室模型,在土炒水煎液中其AUC、Cmax有增大趋势,但与生品、清炒品、单体给药组相比无显著差异(P>0.05)；Tmax明显延迟,与其他给药组相比有显著差异(P<0.05)。研究结果提示辅料进入机体后可对有效成分的吸收代谢等产生影响。
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 research
We 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 protocol
2. Experimental study
2.1 Pharmacodynamic research on the effects of strengthening Spleen and relieving diarrhea of soil-fried Bai zhu
According 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 processed
Change 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 oils
Volatile 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,Ⅲ
AtractylenolideⅠ,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 polysaccharides
The 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 spectrum
In 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 zhu
In 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 fried
The 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 processed
Some 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.
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