The Discovery Of Pomegranate Peel Carbon Nano-components And Their Antidiarrheal Effects

Background:Granati Pericarpium Carbonisatum is a kind of traditional Chinese medicine charcoal.It is the dried peel of pomegranate plant Punica granatum L.-the product of pomegranate peel processed at high temperature.The earliest record of Granati Pericarpium Carbonisatum appears in "Elbow Reserve Emergency Recipe" " recorded the method of processing Granati Pericarpium:"burning red is the last." Granati Pericarpium Carbonisatum was widely used in ancient times to treat dysentery,diarrhea,intestinal bleeding,hemorrhoids,galls,chancre,heartache,black beard,gums For diseases such as bleeding and earache,the most common application of Granati Pericarpium Carbonisatum is to treat diarrhea.However,at present,the effective ingredients of Granati Pericarpium Carbonisatum and its mechanism of action are not clear.This study will use modern nanotechnology research methods to deeply explore the antidiarrheal effect of Granati Pericarpium Carbonisatum and its material basis.Objective:1.Separate the nano-components of Granati Pericarpium Carbonisatum from Granati Pericarpium Carbonisatum,and screen and identify the effective components of Granati Pericarpium Carbonisatum.2.Optimize the temperature of Granati Pericarpium Carbonisatum preparation.3.Evaluate the safety of GPC-NCs through cell experiments.4.Establish a mouse diarrhea model to study the antidiarrheal effect of GPC-NCs.5.Establish a rat IBS-D model to explore the antidiarrheal mechanism of Granati Pericarpium Carbonisatum.Method:1.Granati Pericarpium Carbonisatum is fired at 350? by high-temperature calcination method,and an aqueous solution of Granati Pericarpium Carbonisatum is obtained by crushing,water extraction,and dialysis.Obtain the morphological size,particle size distribution and lattice spacing of GPC-NCs;use HPLC to eliminate the interference of small molecules exerting pharmacological effects;use ultraviolet spectroscopy(UV-Vis),fluorescence spectroscopy(FL),infrared spectroscopy(FTIR)),X-ray photoelectron spectroscopy(XPS)and other methods to analyze the chemical group information of GPC-NCs.2.Granati Pericarpium Carbonisatum is fired at 300?,350?,400? by high temperature calcination method,and then the Granati Pericarpium Carbonisatum of these three temperatures is crushed,water extracted and dialyzed to obtain pomegranate at three temperatures.The aqueous solution of skin charcoal uses ultraviolet spectroscopy(UV-Vis),fluorescence spectroscopy(FL),infrared spectroscopy(FTIR)and other methods to analyze the chemical group information of GPC-NCs.3.Through cell experiments,study the effect of GPC-NCs on cell viability and explore its safety.4.Using the diarrhea model of mice induced by senna leaf,explore the antidiarrheal effect of Granati Pericarpium Carbonisatum by measuring the diarrhea index,the incubation period of diarrhea,and the intestinal propulsion rate of the mice.5.Establish a rat diarrheal irritable bowel syndrome model by senna+restraint,and detect the concentration of 5-hydroxytryptamine,substance P,vasoactive intestinal peptide in the rat serum and the concentration of P in the colon by enzyme-linked immunosorbent assay.The concentration of substances and vasoactive intestinal peptides are used to study the antidiarrheal mechanism of GPC-NCs.Results:1.The raw Granati Pericarpium is carbonized by the high-temperature pyrolysis method,and the carbonized product is extracted,filtered,separated and dialyzed to obtain a new water-soluble nano-component,named as the Granati Pericarpium Carbonisatum nano-component(GPC-NCs).The HPLC fingerprint spectrum did not detect the presence of small molecules in the GPC-NCs solution.GPC-NCs was observed by TEM to be spherical and well-dispersed.The particle size distribution of the nano-components was mainly concentrated in 1.2?2.3 nm,with a certain The uniform stability.HRTEM shows that the lattice spacing of GPC-NCs is 0.321 nm.X-ray diffractometer speculates that GPC-NCs are composed of highly amorphous carbon structures.Ultraviolet spectroscopy(UV-Vis),fluorescence spectroscopy(FL),and infrared spectroscopy(FTIR)show that the surface of GPC-NCs contains functional groups such as hydroxyl,carboxyl and amino groups.X-ray photoelectron spectroscopy shows that GPC-NCs contain hydroxyl,carboxyl,and amino groups.The existence of these functional groups can be attributed to the multiphoton activity process of various oxygen-containing functional groups.2.Spectral analysis results of the nano-components of Granati Pericarpium Carbonisatum(GPC-NCs)prepared under the conditions of 300?,350?,and 400?.Infrared-visible spectroscopy(FT-IR)analysis shows that the surface groups of the nano-components of Granati Pericarpium Carbonisatum(GPC-NCs)prepared at 350? are more abundant and diverse.UV-Vis spectroscopy(UV-Vis)analysis shows that the peak shapes of the UV spectra of GPC-NCs prepared at 300? and 350? are similar,but the GPC-NCs prepared at 400? have double peaks at 260-280 nm.This may be caused by the different groups on the surface of the particle size.Fluorescence spectroscopy(FL)analysis showed that the maximum emission wavelength and maximum excitation wavelength of GPC-NCs did not change much as the temperature of the preparation conditions increased,which may be related to the size of the surface particle size.The effect of different temperature GPC-NCs solutions on the intestinal transport function of mice.The experimental results show that the GPC-NCs prepared at three temperatures have a good effect on the mouse diarrhea model caused by senna decoction.However,the treatment effect of Granati Pericarpium Carbonisatum at 350? is more obvious in the treatment of mouse diarrhea model,which can indicate that 3 50? is a more suitable temperature for preparing Granati Pericarpium Carbonisatum.3.Observe the effects of different concentrations of GPC-NCs on the viability of RAW264.7 cells through CCK-8 cytotoxicity experiments.GPC-NCs has low toxicity,and the experimental safe concentration range is 93.75 ?g/mL.When the concentration of GPC-NCs drops to 23.44?g/mL,the phenomenon of promoting cell proliferation has appeared.4.In the diarrhea model of mice induced by senna,high,medium and low concentrations of the nano-components of Granati Pericarpium Carbonisatum(GPC-NCs)can reduce the diarrhea index of mice.Among them,the diarrhea index is lower at high and low doses;high,medium and low concentrations of GPC-NCs can significantly prolong the diarrhea index.The incubation period of diarrhea in mice;high,medium and low concentrations of GPC-NCs can reduce the intestinal propulsion rate of mice.The high-dose GPC-NCs group and the low-dose GPC-NCs group have a significant inhibitory effect on diarrhea5.GPC-NCs can significantly reduce the concentration of serotonin,substance P(SP)and vasoactive intestinal peptide(VIP)in rat serum;GPC-NCs can significantly reduce P in mouse colon Substance(SP)concentration,vasoactive intestinal peptide(VIP)concentration.It shows that the antidiarrheal mechanism of GPC-NCs is related to the regulation of neurotransmitters.Conclusion:In this study,new types of nano-components were discovered and separated from the aqueous solution of Granati Pericarpium Carbonisatum.GPC-NCs were characterized by TEM,HRTEM,FTIR,UV Vis and fluorescence spectroscopy.It is studied by HPLC method.The characterization results confirmed that GPC-NCs is a kind of spherical particles with a fluorescence quantum yield of 0.45%and a diameter of 1.2?2.3 nm.There are active groups such as hydroxyl,carboxyl,and amino on the surface,so that it has good solubility.And biological activity.The optimal temperature conditions for preparing Granati Pericarpium Carbonisatum were selected by optical identification and pharmacological and pharmacodynamic experiments.It was found that the Granati Pericarpium Carbonisatum at 350?had the most abundant surface active groups and had the best antidiarrheal effect.The results of pharmacological experiments show that GPC-NCs has a significant antidiarrheal effect,and its antidiarrheal mechanism may be related to the reduction of serotonin concentration,substance P(SP)concentration,and vasoactive intestinal peptide(VIP)concentration in rat serum.In this experiment,the discovery of new nano-components in Granati Pericarpium Carbonisatum and the proof of its antidiarrheal activity provide a new theoretical basis for the research of Granati Pericarpium Carbonisatum and other charcoal materials.This not only provides a new idea for the study of the material basis of Granati Pericarpium Carbonisatum,but also provides new insights for the potential biomedical and healthcare applications of GPC-NCs in the field of antidiarrheal,and lays a solid foundation for future drug discovery.