| Objectives:Carbon medicine is a kind of Chinese herbal pieces prepared by the method of stir frying or calcining.There are 26 kinds of carbon drugs in the current Chinese Pharmacopoeia,and more than 70 kinds of carbon drugs are commonly used in local standards and clinical practice.At present,the production process control and product quality evaluation of carbon drugs mainly depend on the artificial observation and judgment results of color change.Most of the index components can not accurately reflect the quality of efficacy,which makes the carbonization degree difficult to control,and the quality of the pieces is very different.Therefore,the establishment of quality indicators which can accurately reflect the quality of efficacy and can be objectively quantified is the common demand of quality assurance of carbon drugs.This study takes rhubarb carbon as an example,under the guidance of "cooling blood and removing blood stasis and hemostasis",explores the mechanism of the three indexes of"shape chemistry biology",the processing and transmission rules,and preliminarily reveals the pharmacodynamic substances of rhubarb carbon,so as to improve the rationality and accuracy of the quality evaluation method of rhubarb charcoal,and provide new ideas and methods for the quality evaluation and quality control research of other carbon drugs.Methods:1.From the perspective of shape,the color and texture of rhubarb charcoal were selected as the shape index according to the standards of "stir frying to black on the surface and brown on the inside" in Chinese Pharmacopoeia(2020 Edition)and "light weight" in Beijing processing standard(2008 Edition).The prepared powder and decoction pieces were weighed before and after heating,and the yield of samples was calculated by the ratio,which indicated the change of texture;Secondly,the color L*,a*,b*were determined to obtain the color quantization values of different samples.2.From the chemical point of view,anthraquinones of rhubarb charcoal specified in Chinese Pharmacopoeia 2020 edition were selected as the chemical indexes,gallic acid related to hemostatic activity of rhubarb charcoal,5-hydroxymethylfurfural(5-HMF)related to color and heating degree of rhubarb charcoal,and sennoside related to purgative effect were included as the chemical indexes of rhubarb charcoal.The contents of free anthraquinone,bound anthraquinone,sennoside gallic acid and 5-HMF in rhubarb powder and Rhubarb from three sources were determined by HPLC under different heating conditions.3.From the biological point of view,the clinical application of rhubarb charcoal in the treatment of inflammatory diseases and the traditional efficacy of" cooling blood and removing blood stasis and hemostasis" were selected.The LPS induced mouse macrophage(raw 264.7),which is widely used in the screening of anti-inflammatory drugs,was used as the inflammatory model,and the LPS induced vascular endothelial cell(HUVEC)was used as the in vitro research model of "removing blood stasis".To study the effect of rhubarb powder water extract and chemical indexes on the release of inflammatory factors tumor necrosis factor-A(TNF-?)and interleukin-1(IL-1?)from two kinds of cells.And nitric oxide(NO)and reactive oxygen species(ROS).In order to explore the key active components of rhubarb charcoal,the effects of the above components on the expression of tight junction protein(ZO-1)and occludin were determined after the HUVEC cells were treated with LPS.4.Rhubarb powder with different heating degree was prepared,and its shape index and chemical index were determined,then the corresponding water extract of rhubarb powder was prepared.The effect of water extract on LPS induced releases from raw 264.7 cells and HUVEC cells was studied,and the relationship between biological index and chemical index of rhubarb carbon was explored.Then,the processed pieces of Rheum tanguticum Maxim.ex Balf.,Rheum palmatum L.and Rheum officinale Baill.with different heating degrees were baked,the processed pieces of Rheum tanguticum Maxim.ex Balf.with different heating degrees were fried,and the carbon monomer compounds of Rheum tanguticum Maxim.ex Balf.were heated to different degrees to explore the correlation mechanism between morphological index and chemical index.5.Principal component analysis(PCA),two-dimensional correlation spectroscopy(2DCOS),JMP 10.0,SPSS,SIMCA 14.1,graphpad prism 8.0.2 and other softwares were used to analyze the correlation between the quantitative values of the morphological,chemical and biological indexes and establish the mathematical model.To explore the correlation between the external performance,chemical composition and biological activity of rhubarb charcoal,and clarify the processing and transfer law of rhubarb charcoal.Results:1.Through literature review and network pharmacology screening,free anthraquinone including rhein,emodin,aloe emodin,chrysophanol and physcion were included,combined with anthraquinone rhein-8-O-?-D-glucoside,emodin-8-O-?-D-glucoside,chrysophanol-8-O?-D-glucoside,aloe emodin-8-O-?-D-glucoside,emodin-8-O-?-D-glucoside,sennoside A,sennoside B,5-HMF and gallic acid were used as chemical indexes of rhubarb charcoal.2.The shape index of rhubarb powder showed that the color of rhubarb powder deepened to black,and the yield decreased gradually;The results of chemical index showed that the contents of free anthraquinone,gallic acid and 5-HMF increased at first and then decreased,while sennoside and bound anthraquinone decreased all the time.The free anthraquinone of powder and water extract heated for 30 min reached the peak value,and the water extract had strong inhibition on NO and ROS.The preliminary results showed that the heating degree of rhubarb charcoal maintained in a suitable range,that is,when the content of free anthraquinone was higher,the content of bound anthraquinone and sennoside decreased to a certain extent.and the color of sample was moderate,the biological activity of rhubarb charcoal was stronger.3.The chemical indexes of rhubarb charcoal are rhein,emodin and rhein-8-O-?-Dglucoside and emodin-8-O-?-D-glucoside.The anti-inflammatory effects of sennoside,gallic acid and 5-HMF showed that all the components had anti-inflammatory effects,and the effects of free anthraquinone rhein and emodin on TNF-? and IL-1? in the same concentration and not the strongest inhibitory effect.The anti-inflammatory activity of free anthraquinone was stronger than that of other compounds.The ROS results also confirmed this result,that is,the inhibitory effect of Rhein was better than that of other compounds mentioned above.Similarly,Western blot results showed that low concentrations of gallic acid,5-HMF and sennoside a could slightly up regulate ZO-1 and occludin,while rhein and rhein-8-O-?-D-glucoside could regulate the expression of both proteins,and the former had better effect on ZO-1 and occludin than the latter.The above results fully show that free anthraquinones play a key role in the biological activity of rhubarb carbon,which can be considered as the key active components of rhubarb carbon.4.The results of shape index of Rheum tanguticum Maxim.ex Balf.,Rheum palmatum L.and Rheum officinale Baill.and stir fried Rheum tanguticum Maxim.ex Balf.showed that with the increase of heating degree,the yield of Rheum tanguticum Maxim.ex Balf.decreased gradually,and the chromaticity values L*,a*,b*and E*ab decreased continuously.The contents of sennoside and bound anthraquinone showed a single decreasing pattern during heating;The contents of gallic acid,5-HMF and free anthraquinone increased first and then decreased.Correlation analysis showed that the total color value E*ab had a quadratic correlation with free anthraquinone,5-HMF and gallic acid,and a linear correlation with bound anthraquinone and sennoside.Emodin and emodin-8-O-?-D-glucoside.The infrared spectrum of the critical point of the heating process of showed that the combined anthraquinones contained in rhubarb decomposed into free anthraquinones and glucose during the heating process,and the two decomposed to form 5-HMF,which further polymerized to form carbon structure,making rhubarb black.The results show that the color of rhubarb darkens with the decomposition,transformation and condensation of compounds in rhubarb during heating,and there is a stable correlation between color and components.Therefore,the quantitative value of color can be used to predict the content of components in actual production.5.In terms of the processing and transfer law of rhubarb charcoal,the statistics of the contents of free anthraquinone and combined anthraquinone in the samples heated by different sources showed that when the carbonization degree of rhubarb charcoal was moderate,the content of free anthraquinone in the samples heated by different sources was distributed in a certain range,and the corresponding chromaticity value was also distributed in a certain range.No matter what kind of rhubarb raw material is processed into rhubarb charcoal,the content range of free anthraquinone,the key active component of rhubarb charcoal,can be preliminarily determined according to the change range of chromaticity value during the processing of rhubarb charcoal.Meanwhile,the lowest content of free anthraquinone,the key active component of rhubarb charcoal,can be defined as the quality evaluation index in the corresponding indexes,Further judge the processing degree of rhubarb charcoal,and quickly realize the guidance and control of rhubarb charcoal quality.In terms of processing technology,the best processing conditions of the samples were obtained under the experimental conditions,which were stir frying at 200 ? and drying at 220 ? for 20 min respectively.However,further research on the actual production parameters was needed.Conclusions:It was found that free anthraquinone is the key active component of rhubarb carbon,which can realize the quality control and biological activity evaluation of rhubarb carbon;The processing and transferring rules of rhubarb charcoal were explained,and the traditional production experience was comprehensively transformed into quantitative shape index and chemical index,which was beneficial to the quality standard design of rhubarb charcoal,and also provided the basis and guidance for the process design and production of rhubarb charcoal;The correlation mechanism of "shape,chemistry and biology" of rhubarb charcoal was explored,which confirmed the rationality of the overall quality evaluation system,and provided a reference for the clinical application of rhubarb charcoal and the establishment of quality evaluation method of correlation efficacy;This study also provides new scientific ideas and methods for the research of other charcoal drugs. |
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