| Rationales:The medicinal properties of traditional Chinese medicine are of great significance in the following four aspects.Clinical application:The medicinal properties of cold and heat are one of the important attributes of traditional Chinese medicine,which is of great significance to the clinical application of traditional Chinese medicine.According to the patient’s constitution,condition and environment and other factors,choosing the appropriate cold and hot drugs can achieve good treatment results.Drug research:The medicinal properties of cold and heat are also one of the important directions of pharmacological research of traditional Chinese medicines.By studying the cold and hot properties of drugs,we can deeply understand the mechanism of action of drugs and provide theoretical basis for the development of new Chinese medicines.Classification of medicinal properties:Cold and hot medicinal properties are a kind of classification of medicinal properties of traditional Chinese medicines,which is of great significance to the classification,sorting and standardization of drugs.In the compatibility and application of traditional Chinese medicines,the classification of medicinal properties is an important reference basis.Theoretical research:The theory of cold and heat balance in the theory of traditional Chinese medicine is an important part of the theory of traditional Chinese medicine,and by studying the medicinal properties of cold and heat,we can deeply explore the mechanism of cold and heat balance in the theory of traditional Chinese medicine,and provide theoretical support for the development of traditional Chinese medicine.However,the reasons for the cold and heat of traditional Chinese medicine have not been well explained.Using scientific means to explain the causes of the cold and heat properties of Chinese medicine is of great significance for improving the scientific nature of Chinese medicine and even the world of Chinese medicine.It is hoped that the causes of the cold and heat properties of traditional Chinese medicine can be explored from the aspects of substances and channels of action on the body.Raw ground yellow has a cool taste and sweetness,and has the effect of nourishing yin and clearing heat and cooling blood and rejuvenation.Ripe ground yellow,slightly warm and sweet,has the effect of nourishing yin and replenishing blood and filling the essence.The highest content in rehmannia is the carbohydrate component,and the carbohydrate composition changes significantly after processing.Previous reports have shown that carbohydrates are related to the cold and heat properties of traditional Chinese medicines,and exploring whether rehmannia polysaccharides are related to the transformation of raw and cooked rehmannia medicinal properties can help reveal the causes of cold and hot medicinal properties.The influence of cold and heat on the body is manifested in body temperature,energy metabolism and other aspects,and the change of these indicators is the basis for determining the drug properties of cold and heat.And there are reports that cold and heat affect the body’s intestinal flora.Studies have found that some traditional Chinese medicines can activate or inhibit temperature-related TRP channels on the surface of mammalian cells,thereby producing a series of reactions and affecting energy metabolism,and TRP channels may be related to cold and heat medicinal properties.For example,capsaicin activates TRPV1 channels to produce a burning sensation,and menthol activates TRPM8 channels to produce a cooling sensation,and this preliminary feeling is also one of the bases for people to determine the medicinal properties of cold and heat.Studies have found that capsaicin can activate TRPV1 channels to increase intracellular ATP content,while intracellular ATP content decreases after using TRPV1 inhibitors.Aconitine in thermogenic drugs can activate TRPV4 channels to increase intracellular temperature,while baicalin inhibits TRPV4 channels to reduce intracellular temperature.These findings coincide with animal experiments that thermal drugs can improve energy metabolism and body temperature in animals,while cold drugs inhibit energy metabolism and reduce body temperature.At present,some TCM compounds have been found to target TRP channels,and it is speculated that the cold and heat properties of TCM may be related to the regulation of TRP channel expression,but this speculation still needs further study.Objective:Firstly,the changes of rehmannia polysaccharides were explored,and then the correlation between rehmannia polysaccharides and cold and heat medicinal properties was explored from the aspects of physical and chemical indexes and TRP channel expression.Method:（1）The traditional Chinese medicine rehmannia was extracted by water extraction alcohol precipitation method,followed by papain enzyme method to remove the protein in the polysaccharide,and then the sugar content was determined by phenol sulfuric acid method and the protein content was determined by Coomassie brilliant blue method.The most degradation conditions for degradation of rehmannia polysaccharides by trifluoroacetic acid and H2O2 were determined by single factor experiments,which mainly investigated the effects of degradation temperature,degradation time and reagent concentration on degradation products,and finally determined the optimal degradation conditions for degrading rehmania polysaccharides.The molecular weight of rehmannia polysaccharides was studied by high performance liquid chromatography,the monosaccharide composition of oligosaccharide fragments was studied by complete acid hydrolysis and derivatization,and the structure of special functional groups present in polysaccharides was determined by Fourier transform infrared spectroscopy（FT-IR）.（2）In vitro activity of rehmannia glutinosa polysaccharides was tested by reducing activity test and NO scavenging test.（3）The body temperature and water intake of mice were judged by cold and heat indicators.Energy metabolism is judged by measuring the ATP content of liver tissue.By exploring the spatial distribution and expression of TRPV1,TRPV4 and TRPM8,the correlation between cold and heat medicinal properties and TRP channel expression was explored,and the cold and heat properties of raw and cooked rehmannia polysaccharides were judged.Through the analysis of fecal flora of mice,the relationship between cold and heat medicinal properties and fecal microbiota was explored.Results:1.Fresh,raw and cooked rehmannia polysaccharides have molecular weights mainly of 200KDa and 4KDa,and the content of large molecular weight polysaccharides increases after processing,and the content of small molecule polysaccharides decreases.Rehmannia polysaccharides in fresh rehmannia polysaccharide the most variety,is mannose,glucuronic acid,fucosamine,rhamnose,glucose,galactose,xylose,arabinose and galacuronic acid 9 kinds of monosaccharides composition,raw rehmania polysaccharide is mannose,fucose,rhamnose,glucose,galactose,xylose,galactouronic acid 7 kinds of monosaccharides,cooked rehmania polysaccharide is composed of mannose,fucose,glucose,galactose,xylose,galactouronic acid 6 kinds of monosaccharides.The functional groups of fresh,raw and cooked Rehmannia glutinosa polysaccharides all showed similar signal peaks,that is,the characteristic signal of polysaccharides 3350-3400cm-1:hydroxyl stretching vibration;2950-3000cm-1:C-H stretching vibration of CH2group;1600-1650 cm-1:telescopic vibration of CHO and C=0keys;1375-1425cm-1:C-H key vibration;950-1150cm-1:C-O-C and C-O-H bond absorption.The optimal conditions for hydrolysis of rehmannia trifluoroacetic acid were hydrolysis temperature of 80°C,concentration of trifluoroacetic acid of 1.0 mol/L,and hydrolysis time of 1 h.Hydrogen peroxide hydrolysis found that the hydrolysis temperature,hydrogen peroxide concentration or hydrolysis time were changed,and the hydrolysis results remained basically unchanged.However,compared with the hydrolysis of trifluoroacetic acid,hydrogen peroxide hydrolysis has more oligosaccharide fragments,higher content and simple hydrolysis method,which lays a foundation for the subsequent development of rehmannia oligosaccharides.2.With the increase of concentration,the reducibility of rehmannia glutinosa polysaccharide and water extract gradually increased;Reducibility:cooked rehmannia glutinosa polysaccharide>raw rehmannia glutinosa polysaccharide>fresh rehmannia glutinosa polysaccharide,cooked rehmannia glutinosa water extract>raw rehmannia glutinosa water extract>fresh rehmannia glutinosa water extract;The water extracts of fresh,raw and cooked Rehmannia glutinosa have stronger reducibility than polysaccharides.With the increase of concentration,the NO scavenging rate of rehmannia glutinosa polysaccharide and water extract gradually increased;NO clearance rate:cooked rehmannia glutinosa polysaccharides>raw rehmannia glutinosa polysaccharides>fresh rehmannia glutinosa polysaccharides,cooked rehmannia glutinosa aqueous extract>raw rehmannia glutinosa aqueous extract>fresh rehmannia glutinosa aqueous extract;The NO removal rate of water extract is stronger than that of polysaccharide.3.Through the cold and heat evaluation of body temperature and water intake,it was found that the changes of the rehmannia polysaccharide group were similar to those in the water extraction group,that is,the raw rehmannia water extract and raw rehmannia polysaccharide showed coldness,and cooked rehmannia water extract and cooked rehmannia polysaccharide showed heat.Through DSS scores,it was found that raw and cooked rehmannia polysaccharides and water extract had improved effects on DSS-induced colitis.Liver ATP expression amount,cooked rehmannia aqueous extract group>normal group>raw rehmannia water extract group,cooked rehmannia polysaccharide group>normal group>raw rehmannia polysaccharide group.TRP channel immunohistochemistry found that rehmannia aqueous extract and polysaccharides could reduce the expression of TRPV1 in the colonic mucosal layer of mice and increase the expression of TRPM8.On the contrary,cooked rehmannia aqueous extract and polysaccharide increased the expression of TRPV1 in the colonic mucosal layer of mice and decreased the expression of TRPM8.There were no significant differences in the expression of TRPV4 between the control group,raw and cooked rehmannia aqueous extract and polysaccharide group.The fecal microbiota diversity analysis showed that compared with the control group,rehmannia water extract and polysaccharides increased the diversity of fecal flora,and the fecal flora diversity of polysaccharides was richer.Raw and cooked rehmania water extract and polysaccharides did not show obvious effects of cold and heat on fecal flora,and the reason may be related to insufficient action time,which needs to be further explored.Conclusion:In this study,raw and cooked polysaccharides were taken as the research objects.Through the analysis of molecular weight,functional group and monosaccharide composition of raw and cooked polysaccharides,the molecular conformation changes of raw and cooked polysaccharides were preliminarily studied.After that,the antioxidant activity in vitro of hydrolyzed and cooked polysaccharides and water extracts was tested.It was found that the antioxidant activity in vitro was:cooked rehmannia glutinosa polysaccharide>raw rehmannia glutinosa polysaccharide>fresh rehmannia glutinosa polysaccharide,cooked rehmannia glutinosa water extract>raw rehmannia glutinosa water extract>fresh rehmannia glutinosa water extract;The antioxidant activity of water extract is stronger than that of polysaccharide.Finally,the experiments in vivo showed that the cold and hot properties of the polysaccharides from raw and cooked Rehmannia glutinosa were consistent with those of the water extracts from raw and cooked Rehmannia glutinosa.This study lays the groundwork for further exploration of the relationship between cold and hot drug properties and carbohydrates,and also provided research ideas for the study of cold and hot drug properties. |
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