Discovery Of New Nano-components In Guanzhong Carbon And Its Liver-protecting Effect

Background:Traditional Chinese medicine is a treasure of the Chinese nation,charcoal medicine is a kind of medicine with distinctive characteristics in traditional Chinese medicine.Dryopteridis Crassirhizomatis Rhizoma Charcoal（DCC）is a high-temperature carbonized powder from Dryopteridis Crassirhizomatis Rhizoma.It has the functions of treating blood stasis,vomiting blood,traumatic bleeding,uterine bleeding,blood in the stool and other bleeding syndromes.It has wide application and exact curative effect.However,research on the active ingredients and quality standards of DCC has not made substantial progress.The high-temperature carbonization process is the most critical step in the process of carbon formation.The high-temperature carbonization in the preparation of DCC is similar to the process of high-temperature pyrolysis in the preparation of nano-components in materials science.This inspired us to discover new nano-components from the high-temperature carbonization process.The liver is the main organ for detoxification and regulation of glucose metabolism and lipid metabolism.It is also very important and a very vulnerable organ.The damage may be caused by trauma or toxic drugs,chemicals and viral infections.Therefore,it is very meaningful to find a natural medicine that can protect the liver and has no toxic side effects.Our experimental team has studied 18 kinds of charcoal drugs in the early time,and has certain theoretical and practical foundations for the study of the quality and quality standards of traditional Chinese medicine charcoal.In the study of DCC,we introduced nanoscience methods,combined with the foundation of the team’s research for many years,to find out new ways to discover the new ingredients produced by burning the charcoal and to investigate its hepatoprotective effects.Objective:（1）In this study,DCC as the research object,and draws on nanotechnology and research methods,the research on the process of making charcoal and the charcoal produced by DCC can be studied in order to find new nano-components in charcoal.（2）Finely characterize the novel nano-components,study their microscopic morphology,elemental composition,and spectral characteristics,and calculate the fluorescence quantum yield.（3）The safety of the discovered nano-components was studied by cell experiments.（4）To study the protective effects of nano-components on liver and the mechanism in liver protection.Methods:（1）The crude medicine is made into charcoal by muffle furnace.The DCC carbon dialysate is prepared by pulverizing,extracting,filtering,concentrating,and dialysis of the common charcoal of the fired products at different temperatures.We using transmission electron microscopy（TEM）,high-resolution transmission electron microscopy（HR-TEM）,ultraviolet-visible spectrophotometer（UV-vis）,Fourier transform infrared spectroscopy（FT-IR）,fluorescence spectrometer（PL）,etc.to discover novel nano-components in DCC.（2）Characterization of nano-components of DCC:Based on the techniques and methods for the study of nano-components in nanotechnology,the composition of newly discovered in DCC is characterized.The microstructure of the DCC nanocomponents was characterized by transmission electron microscopy（TEM）.X-ray photoelectron spectroscopy（XPS）was used to study the elemental composition of the of the DCC.The UV,IR and fluorescence spectrometers were used to characterize the nanocomponents.The spectral properties of the nanocomponents were investigated and their fluorescence quantum yields were calculated.High-performance liquid chromatography（HPLC）was used to compare the difference in composition between the nano-components of DCC and crude medicine.（3）To evaluate the safety of DCC nanocomponents,cell experiments were conducted to study the effects of nanocomponents on cell viability.（4）Study on the hepatoprotective effect of nano-components in DCC.The classical mouse liver hemorrhage model and the acute liver injury model induced by carbon tetrachloride were used to study the protective effect of the nano-components on the liver of mice and its possible mechanism.The protective effects of DCC-NCs on CCl₄-induced acute liver injury in mice were studied by observing the surface morphology of liver,observing liver histopathological sections and calculating liver index.Study on the hepatoprotective mechanism of DCC nano-components:analysis of pathological characteristics of mouse liver,detection of alanine aminotransferase（ALT）and aspartate transferase（AST）,total bile acid（TBA）,total bilirubin（TBIL）,and triglyceride（TG）in mouse serum.Determination of Malondialdehyde（MDA）and Superoxide dismutase（SOD）in mouse liver homogenate.Preliminary study on the mechanism of hepatoprotective action of nano-components.Results:（1）Through the study of the core burning process of DCC,the common charcoal of different temperatures was successfully fired.After a series of preparation processes such as pulverization,extraction,filtration,concentration,and dialysis,the microscopic components in the DCC dialysate were observed by transmission electron microscopy.The burnt carbon at 350℃has clear carbon particles,good dispersion and uniformity,and the particle size range is between 5-10 nm,which is consistent with the characteristics of carbon nano-components.The newly discovered nano-components were named as DCC nano-components（DCC-NCs）.（2）Characterization of DCC-NCs:TEM and HR-TEM results show that nano-components are contained in charcoal,and the particle size distribution is between 1and 7 nm,which is consistent with the particle size distribution characteristics of nano-components.The lattices are neatly arranged with a pitch of 0.278 nm.The XPS analysis shows that the carbon-based nano-components contain C,O,N and other elements,among which carbon is dominant.After XPS peak and infrared spectrum,DCC-NCs can be found to be rich in surface functional groups.These functional groups may affect their chemistry.The UV spectra of DCC-NCs indicate that there is a conjugated structure,which may be the n-π*transition caused by the-C=O carbonyl group.The fluorescence spectrum shows that the maximum excitation wavelength is 360 nm and the maximum emission wavelength is 460 nm.The two spectrum have good mirror symmetry.The HPLC analysis of the chemical composition differences between the medicinal materials of crude medicine and DCC-NCs.The results showed that a large number of small molecular components contained in the genus were completely removed after firing,filtration and dialysis.（3）Study on the hepatoprotective effect of DCC-NCs charcoal:Through the study of the administration of mouse hepatic hemorrhage model,the results showed that DCC-NCs have the effect of inhibiting hepatic hemorrhage in mice.In the mouse model of acute liver injury caused by carbon tetrachloride,DCC-NCs can reduce cell necrosis and nuclear fragmentation,relieve liver swelling,and show good liver protection.（4）DCC-NCs can reduce ALT and AST in mice with acute liver injury caused by CCl₄,and can alleviate the increase of TBA and TBIL,which can significantly reduce MDA and increase SOD level.It is indicated that DCC-NCs can improve the body’s ability to scavenge oxygen free radicals,inhibit free radical lipid peroxidation,prevent hepatocyte lipid peroxidation,and avoid liver cell damage.This may be that DCC-NCs play a role in liver protection.mechanism.Conclusion:Through a series of preparation conditions research,microstructure observation,and spectral characteristics investigation,the nano-components similar to carbon dots in DCC were successfully found in the processed charcoal after the processing,and named as DCC-NCs.The optimum firing temperature is 350℃,and the DCC-NCs are uniformly stable,which conforms to the microstructure and optical properties of the nano-components,the surface active groups are rich in variety,and the photochemical stability is good.The HPLC method proved that there was no small molecule component in the isolated DCC-NCs.It is further proved by detailed characterization that the components separated in DCC is the DCC-NCs.The liver hepatic injury model induced by mouse liver hemorrhage model and carbon tetrachloride was used to verify the protective effect of DCC-NCs on the liver,and the liver protection effect was good.The changes of total bile acids and total bilirubin and the changes of superoxide dismutase and malondialdehyde in the serum of mice after DCC-NCs treatment,DCC-NCs on liver injury The protective mechanism may be to improve the body’s ability to scavenge oxygen free radicals,prevent lipid peroxidation of hepatocytes and regulate the metabolism of bile acids and bilirubin,thereby preventing and improving liver cell damage.DCC-NCs have a good enzyme-lowering effect.It may become a green,safe and convenient drug for liver damage diseases.