| Research BackgroundTrichosanthes kirilowii Maxim is a species of local origin in China,and its cultivation was first seen in the Shennong Ben Cao Jing.Except for its traditional medicinal value,new medicinal,health,edible and ornamental values have been discovered in recent years,and which is one of the most active and promising special economic crops emerging from the current agricultural restructuring,and the scale of cultivation is expanding.However,most of the varieties used in production at present are simply domesticated or selected from wild-type varieties and are still in the primitive breeding stage.The mainstream commercial source strains in the market are the "Hai Shi Gualou" from the Anguo area in Hebei and the "Wangua Series" from Anhui.According to the survey,the Anhui "Wan Gua Series" mainly harvests the seeds of Trichosanthis Pericarpium and produces the edible seeds to generate economic benefits,while the peel produced by taking the seeds is used as piper’s skin for medicinal purposes,while the "Hai Shi Gualou" in the An Guo area of Hebei mainly become whole piper’s medicinal use.There is a lack of systematic research on whether the peel obtained from seed extraction of Trichosanthis Pericarpium has the same efficacy as the traditional medicinal strain.Purpose of the studyThis study takes the peel of different cultivated strains of Trichosanthis Pericarpium as the research object,and based on the traditional efficacy of Trichosanthis Pericarpium in clearing heat and resolving phlegm and relieving qi and broadening the chest,conducts a comparative analysis and effect evaluation of its resourceful substances to provide scientific basis and theoretical support for the comprehensive utilization and value discovery of cultivated strains of peel of Trichosanthis Pericarpium resources.Research methodsⅠ.By collecting and collating relevant literature,the historical evolution of the use of Trichosanthis Pericarpium and its current status of cultivation of the base source plant are summarized.At the same time,a systematic review of the research progress on the material basis of the efficacy of Trichosanthis Pericarpium in clearing heat and resolving phlegm and relieving qi and broadening the chest is conducted,mainly including an overview of the research on the application of Trichosanthis Pericarpium in lung diseases and its progress in chronic obstructive pulmonary diseases,the research progress on the intervention of Trichosanthis Pericarpium in myocardial ischemia models and the research progress of metabolomics in coronary heart diseases,with a view to providing reference for the comprehensive utilization of Trichosanthis Pericarpium The research in this chapter lays the foundation for the design and systematic study of the overall thesis.Ⅱ.Based on the research methods established by the group in the previous stage,multivariate resource-based substance analysis was conducted on Trichosanthis Pericarpium of different cultivated strains,with sugar,flavonoid and triterpenoid,amino acid and nucleoside components as evaluation indicators,and by principal component analysis,in order to comprehensively evaluate the quality of Trichosanthis Pericarpium of each strain in each origin.Ⅲ.Using a mouse model of chronic obstructive pulmonary disease(COPD)induced by cigarette smoke combined with lipopolysaccharide,the efficacy of Trichosanthis Pericarpium in clearing heat and resolving phlegm was comparatively analyzed between seeds and medicinal uses,and its mechanism of action was investigated using network pharmacology and metabolomics.A correlation analysis was conducted to identify and reveal the material basis associated with the efficacy of Trichosanthis Pericarpium in clearing heat and resolving phlegm.Ⅳ.The mechanism of action of Trichosanthis Pericarpium in mice with myocardial ischemia was investigated by using metabolomics,and a partial least squares regression algorithm was used to correlate the efficacy indexes of Trichosanthis Pericarpium in mice with the small molecules found in various types of chemical composition and metabolomics studies.Research resultsⅠ.The compositions of the pericarp of different cultivars of Trichosanthes kirilowii Maxim differ little,but their contents are significantly different.The total amount of resource components in Trichosanthes kirilowii Maxim from different cultivars of traditional medicinal Trichosanthes kirilowii Maxim is higher than that from other cultivars,among which the contents of amino acids and flavonoids differ significantly.Ⅱ.blood,alveolar lavage fluid and lung tissues were collected from COPD model mice at the end of the experiment,and biochemical indexes were measured,histopathological analysis and metabolomics analysis were performed respectively.1.The results of biochemical indexes showed that the levels of IL-6,IL-8 and TNF-α inflammatory factors were significantly higher in the blood and alveolar lavage fluid of the model mice,compared with those of the blank mice.The lung histopathological sections also showed significant pathological changes in the model mice.After the drug intervention,the lung histopathological sections showed improved pathological changes and the levels of IL-6,IL-8 and TNF-α inflammatory factors in the serum and alveolar lavage fluid of the mice were regressed towards the mice in the blank group,with significant differences compared to the model group(P<0.05).Compared with seeded Trichosanthis Pericarpium,medicinal Trichosanthis Pericarpium could significantly reduce the levels of IL-6 and IL-8 factors in serum and alveolar lavage fluid of COPD model mice(P<0.05),and seeded Trichosanthis Pericarpium could significantly reduce the levels of TNF-α factors in serum and alveolar lavage fluid of COPD model mice compared with medicinal Trichosanthis Pericarpium(P<0.05).2.The network pharmacology results showed that a total of 81 significantly different KEGG pathways were obtained,among which the phosphatidylinositol 3-kinase/protein kinase B signaling pathway(PI3K-Akt)signaling pathway(hsa04151)and the tumour necrosis factor(TNF)signaling pathway(hsa04668)were significantly enriched,and the pathway with the highest number of enriched targets was the PI3K-Akt signaling pathway.The pathway with the highest number of targets was the PI3K-Akt signaling pathway,and all the above pathways involved IL-6 gene.The results suggest that Trichosanthis Pericarpium may interfere with COPD through antibacterial,anti-inflammatory and immunomodulatory pathways.3.The results of the metabolomic analysis showed that a total of 43 potential biomarkers(31 in serum and 12 in lung tissue),including LysoPC(18:1(9Z)/0:0),LysoPC(0:0/16:0)and 15(S)-Hydroxyeicosatrienoic acid,were identified in the serum and lung tissue samples.The metabolic pathways involved mainly included arachidonic acid metabolism,α-linolenic acid metabolism,glycerophospholipid metabolism,and retinol metabolism,with arachidonic acid metabolism considered to be the most important pathway for Trichosanthis Pericarpium to intervene in COPD model mice,with an effect value of 0.41.After drug regulation,each group had different degree of callback.Seed and medicinal Trichosanthis peel have significant differences in the regulation of(4E)-1-(3,4-dihydroxyphenyl)tetradec-4-en-3-one、LysoPC(0:0/16:0)、LysoPC(18:1(9Z)/0:0)、15(S)-Hydroxyeicosatrienoic acid and other metabolites4.Correlation analysis of the effect of Trichosanthis Pericarpium on Qi and chest relaxation-components,it was found that the serum IL-6,IL-8 and TNF-α indices of seeded Trichosanthis Pericarpium intervention COPD model mice had higher correlation coefficients with flavonoid components(lignan,quercetin,rutin,isoquercitrin,etc.)and amino acid components(citrulline,phenylalanine,tyrosine,etc.),indicating that when the intake of flavonoid components and amino acid components increased,seeded Trichosanthis Pericarpium The better the improvement effect of the intervention in COPD model mice.The correlation coefficients of serum IL-6,IL-8 and TNF-α in mice with COPD model were mainly higher with amino acid components,such as phenylalanine,proline,tyrosine,leucine,etc.,indicating that the improvement effect of medicinal Trichosanthis Pericarpium intervention in COPD model mice was better when the intake of amino acid components was increased,10Z,13Z,16Z,19Z)/0:0)),PM5(LysoPE(0:0/24:6(6Z,9Z,12Z,15Z,18Z,21Z))),PM36(Arachidonic acid),PM32(LysoPC(17:0/0:0)),and amino acid components(arginine,adenosine-3’,5’-cyclic monophosphate,threonine,etc.)had higher correlation coefficients,indicating that amino acid components can affect the lysophospholipid content in mice,thus acting to reduce lung injury in COPD model mice.From the above experimental results,it can be found that both seed and medicinal Trichosanthis Pericarpium can better intervene in the treatment of COPD model mice and can better protect lung tissues.The components that affect the difference between the two effects are mainly flavonoid components and amino acid components,among which the content of amino acid components differs greatly,mainly phenylalanine,proline,tyrosine,etc.These components may be the main reason for the difference between the effects of seed and medicinal Trichosanthis Pericarpium.These components may be the most important reason for the difference in effect between seed and medicinal Trichosanthis Pericarpium.Ⅲ.blood and heart tissues of mice in the myocardial ischemia model were collected at the end of the experiment and subjected to routine indexes,biochemical indexes,histopathological analysis and metabolomic analysis respectively.1.Trichosanthis Pericarpium was administered for intervention.After modelling,it was found that the body weight of the mice did not change significantly during the experiment,but there were significant differences in the general appearance of the heart and cardiac index.The heart to body weight ratio,i.e.the cardiac index,was higher in the model group of mice,which was significantly higher than that of the blank control group.Both seed and medicinal Trichosanthis Pericarpium significantly reduced the cardiac index and improved the degree of cardiac oedema in the model mice(P<0.05).The results of biochemical indexes showed that the levels of creatine kinase(CK),lactate dehydrogenase(LDH),glutamate transaminase(AST)were significantly higher in the model mice compared to the normal mice.Histopathological sections of the lungs also showed significant pathological changes in the model mice.The levels of creatine kinase(CK),lactate dehydrogenase(LDH),glutamate transaminase(AST)in serum and heart tissues of the treated mice were back-regulated towards the mice in the blank group,with differences compared to the model group.Among them,medicinal Trichosanthis Pericarpium significantly reduced myocardial damage enzymes and better protected myocardial ischemic tissue than seed Trichosanthis Pericarpium(P<0.05).2.To investigate the protective effect of seeded and medicinal Trichosanthis Pericarpium on thrombosis through a zebrafish thrombosis model induced by phenylhydrazine,and to observe the effect of seeded and medicinal Trichosanthis Pericarpium on the intensity of cardiac red blood cells in a zebrafish thrombosis model,the results showed that the therapeutic effects of seeded and medicinal Trichosanthis Pericarpium were 25.94%and 36.80%,respectively,and the EC50 was 0.761 mg/g and 0.587 mg/g,respectively,and The medicinal use of Trichosanthis Pericarpium significantly increased the intensity of red blood cells in the hearts of juvenile zebrafish(P<0.05),indicating that the medicinal use of Trichosanthis Pericarpium had a better antithrombotic effect than the seed use.3.The results of metabolomic analysis showed that a total of 44 potential biomarkers(21 in serum and 23 in lung tissue),including LysoPC(18:2(9Z,12Z)/0:0),LysoPC(0:0/16:0)and 16-Hydroxyhexadecanoic acid,were identified in serum and heart tissue samples.The main metabolic pathways involved include the biosynthesis of phenylalanine,tyrosine and tryptophan,the metabolism of glycine,serine and threonine,the metabolism of phenylalanine,and the metabolism of beta-alanine.Of these,the biosynthesis of phenylalanine,tyrosine and tryptophan was considered to be the most important pathway for Trichosanthis Pericarpium to intervene in mice in a myocardial ischaemia model with an effect value of 1.00.After drug regulation,each group had different degree of callback.Seed and medicinal Trichosanthis peel have significant differences in the regulation of LysoPC(18:2(9Z,12Z)/0:0)、LysoPC(0:0/16:0)、PC(16:0/18:1(11Z))and other metabolites.4.Correlation analysis of the effect of Trichosanthis Pericarpium on clearing heat and resolving phlegm-components,it was found that the index of heart quality of mice with intervention of medicinal Trichosanthis Pericarpium in myocardial ischemia model was mainly correlated with higher coefficients of nucleoside components such as thymidine,uridine,xanthine and thymine,indicating that the better the improvement of heart quality of mice with myocardial ischemia model by medicinal Trichosanthis Pericarpium when the intake of nucleoside components increased.The higher correlation coefficients of serum cardiac enzyme indices in mice with myocardial ischemia model intervened by medicinal Trichosanthis Pericarpium mainly correlated with amino acid components mainly tyrosine and valine,indicating that when the intake of amino acid components increased,the better the effect of medicinal Trichosanthis Pericarpium on the improvement of cardiac injury in mice with myocardial ischemia model.PM30(LysoPE(20:0/0:0)),PM26(LysoPE(20:1(11Z)/0:0)),PM29(LysoPC(18:0/0:0))and PM44(12-Hydroxystearic acid)had higher correlation coefficients with nucleoside components(thymidine,uridine,xanthine,guanosine,thymine,etc.),indicating that amino acid components can affect the lysophospholipid content in mice,thus serving to reduce myocardial ischemia.The results showed that the amino acid components could affect the lysophospholipid content in mice,thus reducing myocardial injury in mice.From the above experimental results,it can be found that both seed and medicinal Trichosanthis Pericarpium can better intervene in the treatment of mice with myocardial ischaemia model and can better protect myocardial tissue.Compared with seed Trichosanthis Pericarpium,medicinal Trichosanthis Pericarpium had a better effect on myocardial ischemia in mice model,which might be related to the higher content of amino acids in medicinal Trichosanthis Pericarpium.ConclusionFirst,the types of multi-type resource substances contained in the peels of different cultivated strains of Trichosanthis Pericarpium communis are basically the same,but from the overall analysis and evaluation,the content of each type of resource components varies greatly,and the total amount of resource components and the content of medicinal ingredients in the peels of some strains of traditional medicinal Trichosanthis Pericarpium communis produced in the production area are higher compared with the strains of other production areas,and the main difference components are the medicinal ingredients amino acids and lignocerebroside.The results of the study can provide reference for the selection of clinical use of Trichosanthis Pericarpium and the comprehensive utilization of the resources of different cultivated strains of Trichosanthis PericarpiumSecond,based on the efficacy of Trichosanthis Pericarpium in clearing heat,resolving phlegm and relieving qi to broaden the chest,the anti-inflammatory and anti-thrombotic effects of different cultivated strains of Trichosanthis Pericarpium were evaluated by using COPD mouse model,zebrafish thrombosis model and myocardial ischemia model,and it was clear that the seed used Trichosanthis Pericarpium has common trends with traditional medicinal strains of Trichosanthis Pericarpium in clearing heat,resolving phlegm and relieving qi to broaden the chest.(1)In the mouse model of COPD,which can manifest the effect of clearing heat and resolving phlegm,the effect of Trichosanthis Pericarpium for seeds was equivalent to that of 0.87 times the dosage(6-10 g)of the Chinese Pharmacopoeia;(2)In the mouse model of myocardial ischemia,which can manifest the effect of relieving qi and broadening the chest,the effect of Trichosanthis Pericarpium for seeds was equivalent to that of 0.67 times the dosage(6-10 g)of the Chinese Pharmacopoeia.The results of this study provide scientific basis and theoretical support for the comprehensive utilization and value discovery of cultivated strains of rarion peel resources. |
