Study On The Material Basis And Mechanism Of Zhuofajian In The Treatment Of "Zhuhuang"

BackgroundZhugao Fajian Decoction?Pork lard and human hair decoction,LHD?was first recorded in Treatise on Febrile and Miscellaneous Diseases.LHD had been used in the treatment of jaundice for thousands of years,especially in the Qing Dynasty.However,in modern clinical practice,LHD was not only rarely used in the treatment of jaundice,its pharmacological effects and material basis was not involved in modern research.The main reason for this great contrast is that doubts about the efficacy of LHD limited its clinical application.However,it was found that LHD was worthy of a series of studies through systematic literature review.Besids,from the perspective of drug synthesis,this was the major pioneer in ancient China in drug chemical synthesis.Therefore,LHD had its important value in clinical and research.Charcoal traditional Chinese medicine?charcoal medicine?has a long history and abundant clinical accumulation.Its exact effect has been affirmed by both clinical applications and modern pharmacological researches.However,the material basis of charcoal medicine has not been revealed.Besides,the current Pharmacopoeia of the People's Republic of China?PPRC?even lacks the record of the quality standard of charcoal medicine.The existing research conclusions can not explain the material basis of charcoal medicine reasonably.The classic research model for the material basis of Chinese medicine may not be applicable to the study of charcoal medicine.It may not be applicable to study the mechanism of LHD in the treatment of“various kinds of yellowing”.The results of our previous researches showed that the carbonization process is the key point to study charcoal medicine.Based on the concept and technology of nanoscience,the active components obtained in the carbonization process were analyzed and they were found to be a new kind of components which had not been recognized.According to the structural characteristics and various pharmacological effects of these active components,we named them Nano-components?NCs?.ObjectiveBased on the literatures,the preparation technology of LHD was reproduced and optimized.Guided by pharmacological effects,the effect of LHD in the treatment of“various kinds of yellowing”was confirmed,and the best preparation conditions were screened out.Using the technology of nanoscience,we discovered and characterized the Pork lard and human hair decoction nano-components?LHD-NCs?,and analyzed the correlation between its characteristics and its biological activities.Further,three animal models were used to study the effect and mechanism of LHD-NCs in the treatment of“various kinds of yellowing”.The original record was deeply analyzed and its scientific connotation was interpreted.Methods1.Guided by modern science and technology,the preparation technology of LHD was reproduced in the laboratory.Based on the literatures,relatively reasonable preparation conditions of LHD was preliminarily screened,so as to provide a basis for further research.2.Guided by pharmacological effects,an intrahepatic cholestatic jaundice mice model induced by alpha-Naphthylisothiocyanate?ANIT?,and an acute liver injury mice model induced by Carbon tetrachloride?CCL₄?were used to evaluate the efficacy of LHD prepared under different conditions,and the best preparation condition was screened out.3.The method of extraction was used to separate LHD into water-soluble components?WSC?and oil soluble components?OSC?.The effect of LHD,WSC and OSC were evaluate.The preparation process of LHD was further optimized.4.Based on the methods of nanotechnology and the previous research results of our team,we established a relatively standardized and repeatable preparation process of LHD-NCs.According to the optimized preparation process,LHD-NCs were prepared under four different conditions:300?1h,300?2h,350?0.5h and 350?1h.Their morphological,optical and structural characteristics were characterized in detail.5.The safety of LHD-NCs was evaluated by animal experiments and cytotoxicity experiments,and the safety parameters of LHD-NCs were obtained.6.The effect and mechanism of LHD-NCs on ANIT-induced jaundice model was studied,including the influences on serum bilirubin,urinary bilirubin,serum TNF-?,IL-1?and IL-6,superoxide dismutase?SOD?and malondialdehyde?MDA?in liver homogenate as well as the pathological characteristics and ultrastructure of liver tissue.Then further study on the effect of LHD-NCs on the model of acute liver injury induced by CCL₄ was managed,and the hepatoprotective effect of LHD-NCs and its mechanism were evaluated.7.A hyperbilirubinemia?HBIL?model was explored and established,the time-effect relationship of LHD-NCs on serum bilirubin and urinary bilirubin were analyzed in HBIL model.Then the liver pathological characteristics and hippocampal pathological characteristics were analyzed and the liver and brain protective effects of LHD-NCs were evaluated.The effect and mechanism of LHD-NCs in reducing bilirubin were further evaluated.Results1.In this study,the preparation technology of LHD was reproduced in the laboratory guided by modern science and technology.Combined with the standard of“melt of human hair and pork lard”and“CunXing”,the reaction temperature and reaction time of the preparation of LHD were studied,and a stable and repeatable preparation process was established.It was found that 300?1h,300?2h,350?0.5h and 350?1h were the optimum preparation conditions.Under the above conditions,the yield of LHD was relatively stable,ranging from75%to 80%.2.By comparing the effect of LHD,WSC and OSC in CCL₄ mice model,the levels of serum aminotransferase and blood lipid were evaluated.It was found that WSC not only had the best efficacy but also had no burden on the blood lipid metabolism.3.In this study,TEM was used to observe WSC,in which abundant and well dispersed spherical particles with an average diameter of 3.47±1.09 nm and range of 1-6 nm were observed.According to the previous research results of our team,we called them“Pork lard and human hair Decoction nano-components”?LHD-NCs?.On this basis,the preparation process and related parameters of LHD-NCs were further optimized,and a more perfect and repeatable preparation process of LHD-NCs was established.4.According to the optimized preparation process,LHD-NCs were prepared under four different conditions:300?1h,300?2h,350?0.5h and 350?1h.Their morphological,optical and structural characteristics were characterized in detail.The results showed that LHD-NCs were spherical particles with a diameter of about 1-6 nm.LHD-NCs prepared at 300?1h and350?0.5h had better dispersion,and LHD-NCs prepared at 300?2h and 350?1h showed bonding and overlapping.LHD-NCs showed bright blue color under ultraviolet light which can be easily observed by naked eyes.The UV-Vis absorption spectra of LHD-NCs prepared under different conditions were similar.The absorption peaks were visible at about 318 nm,which may be related to the electronic transition of C=C bond in LHD-NCs.The maximum excitation wavelength of LHD-NCs was about 328-334 nm,and the maximum emission wavelength was about 396-407 nm.The maximum excitation wavelength and emission wavelength of LHD-NCs prepared at 350?0.5h were 331 nm and 407 nm respectively.LHD-NCs mainly contained five elements:C,O,N,S and P.The contents of C,O and N were 72.28%,16.1%and 11.11%,respectively.The contents of these three elements accounted for 99.49%of all the elements.The fluorescence quantum yield was about 12.74%.The results of infrared spectroscopy analysis of LHD-NCs prepared at 350?0.5h were consistent.It was further proved that LHD-NCs prepared at 350?0.5h had abundant active groups such as hydroxyl,carboxyl,carbonyl and amino groups.5.LHD-NCs could promote cell proliferation at high concentrations,but had almost no effect on cell growth at low concentrations.LHD-NCs had no effect on 12 serum biochemical indexes of normal mice?P?0.05?,which indicated that LHD-NCs was safe.6.The effect and mechanism of LHD-NCs were evaluated in ANIT jaundice model.The results showed that three doses of LHD-NCs could significantly reduce the serum bilirubin content and urinary bilirubin content of model mice,and the effect of low dose group was the best.LHD-NCs could reduce the level of aminotransferase in model mice.The low dose group had the best effect,followed by the high dose group and the medium dose group.Three doses of LHD-NCs could reduce the levels of IL-6 and IL-1?in serum of model mice and alleviate inflammation.All three doses of LHD-NCs could decrease MDA content and increase SOD level of liver tissue of model mice,which indicated good antioxidant effect.In addition,the pathological and ultrastructural features of liver tissue suggested that LHD-NCs had hepatoprotective effects in this model.7.The effect and mechanism of LHD-NCs were evaluated in CCL₄ liver injury model.The results showed that three doses of LHD-NCs could significantly reduce the levels of serum transaminase and total bile acid in model mice.Three doses of LHD-NCs could reduce the levels of IL-6 and IL-1?in serum and alleviate inflammation.All three doses of LHD-NCs could decrease MDA content and increase SOD level of liver tissue of model mice,which indicated good antioxidant effect.In addition,the pathological and ultrastructural features of liver tissue suggested that LHD-NCs had hepatoprotective effects in this model.8.The model of hyperbilirubinemia?HBIL?was managed successfully,and the time-effect relationship of LHD-NCs on the model of HBIL was evaluated.It was found that the content of bilirubin in the serum of model mice increased significantly in 30 minutes.Although the content of bilirubin in the serum of model mice decreased from 30 minutes to 180 minutes,it was still significantly higher than that of control group.From 30 min to 180 min,the LHD-NCs group could reduce the serum bilirubin content of the model mice.At 180 min,the LHD-NCs group could significantly reduce the serum TBIL content,DBIL content and IBIL content of the model mice.LHD-NCs group could not only reduce the content of bilirubin in serum,but also reduce the content of bilirubin in urine.The corresponding pathological features of liver and hippocampus were analyzed,suggesting that LHD-NCs have protective effects on liver and brain in HBIL model.Conclusions1.Guided by modern science and technology,the preparation process of LHD was reproduced and optimized in the laboratory.Animal experiments have proved the role of LHD in the treatment of“various kinds of yellowing”.The LHD can not only reduce the serum bilirubin level but also decrease the level of transaminase in the model mice.The preparation of LHD prepared at 350?0.5h had the best effect,and can be used as the best preparation condition for LHD.2.This study confirmed that the LHD had the effect of treating“various kinds of yellowing”,and it was found that the pork lard could influence the lipid metabolism and cause hyperlipidemia.However,under the background of Eastern Han Dynasty with frequent wars,the large amount of pork lard used in LHD was reasonable,but under the background of modern diet structure,it was very important to optimize the preparation process of LHD in order to reduce the effect on blood lipid metabolism and improve the curative effect.In this study,the LHD was separated into WSC and OSC by extraction.It was found that the WSC not only had the best efficacy,but also had no effect on blood lipid metabolism.Therefore,extraction and separation should be an important step in the process of reproducing and optimizing LHD preparation based on modern science and technology.3.Based on the methods of nanotechnology,the preparation process and related parameters of LHD-NCs were found and optimized,and a relatively perfect and repeatable preparation process of LHD-NCs was established.Four different conditions of LHD-NCs were prepared according to the optimized process,and their morphological,optical and structural characteristics were characterized in detail.It was found that LHD-NCs prepared under different conditions showed similarities and differences in structural characteristics,physicochemical properties and biological activities.LHD was made from pork lard and human hair,which contained abundant elements such as C,O,N and S.It can not only serve as carbon source of LHD-NCs,but also provide elements for its abundant surface groups.The surface of LHD-NCs had hydroxyl,carboxyl,carbonyl,amino and other active groups,which may lead to abundant biological activities.4.LHD-NCs showed high safety in both cytotoxicity and animal experiments.The safety evaluation of LHD-NCs provided a basis for further study of the biological activities of LHD-NCs,and provided a basis for clinical application of LHD.5.By using ANIT jaundice model,CCL₄ liver injury model and HBIL model,the results proved that LHD-NCs can reduce serum bilirubin content,serum aminotransferase level.Besides,the results showed anti-inflammatory effect,antioxidant effect and liver protective effects of LHD-NCs.LHD-NCs can alleviate the damage of bilirubin to hippocampal neurons and showed brain protective effect.6.In this study,modern technology was used to analyze the classic record deeply.This study proved that LHD-NCs might be the material basis of LHD for the treatment of“various kinds of yellowing”,therefore the scientific connotation of LHD was explained and the application of LHD was expanded.