Effects Of Stored Citrus Fruits And Excessive Consumption Of Panax Ginseng On "Shanghuo" And Analysis Of Its Mechanism

“Shang huo” is a common name in traditional Chinese medicine for the symptoms of non-adaptive fever in the body,which mainly manifested as oral ulcer,swelling gum,sore throat,nosebleeds,red eyes,dry mouth,elevated body temperature,and other symptoms.Citrus fruits are rich in nutrients,taste and flavor,and contain a variety of functional components that are beneficial to human health;Panax ginseng is a kind of traditional Chinese medicine with the same origin of medicine and food,which has rich pharmacological components and high medicinal values,and it is often added to various functional foods as a dietary supplement.Citrus fruits and Panax ginseng have been popular with consumers,but excessive consumption of broad-skinned citrus(such as satsumas)or Panax ginseng in daily life can easily cause “shang huo”,which is similar to inflammation in symptoms.In addition,many fresh citrus fruits on the market are more or less stored for a period of time,but the effect of stored citrus fruits on “shang huo” is not clear;The material basis and action mechanism of “shang huo” induced by citrus fruits and Panax ginseng,as well as whether there is an association between them have not yet been elucidated.Based on this,this study mainly used stored citrus fruits and fresh ginseng as materials to explore the material basis and mechanism of “shang huo” effected by stored citrus fruits and Panax ginseng,and tried to find out the common points of the two.The main findings are as follows:1.Effect of stored citrus fruits on “shang huo” and its physiological mechanismVolunteer sensory experiment and in vitro cell experiment were used to evaluate the effects of satsumas stored at low temperature(5.8 ± 0.2 ℃,LT)and room temperature(21 ± 1 ℃,RT)for 0 d,30 d,and 60 d on “shang huo”;The content changes of components(sugars,acids,proteins and flavonoids),antioxidant activities and pro-inflammatory activities of 7 varieties of citrus fruits during storage were determined;And the correlation relationship between components and physiological activities were analyzed.The results showed that satsumas stored after 30 d could reduce “shang huo” compared with satsumas before storage,and the incidence of“shang huo” decreased by 23.3% and 20.1%,respectively;The throat was the site with the highest incidence of “shang huo”,and the incidence decreased by 36.6% and 43.3%,respectively.The result of correlation analysis between components and physiological activities showed that sugar content,acid content,flavonoid(naringin,neohesperidin,eriocitrin and didymin)content,antioxidant capacities(ABTS and FRAP)and proinflammatory activity were negatively correlated,whereas the protein content and proinflammatory activity were positively correlated.In addition,the protein content also decreased significantly after 30-day storage.It indicated that the decrease of protein content in the pulp might be the reason for reducing the stimulation of satsumas to the human body and lead to the decrease of the incidence of “shang huo”.2.The mechanism of Panax ginseng induced inflammation and screening of candidate proteinsThrough step-by-step extraction,the main pro-inflammatory components in Panax ginseng were excavated,and the mouse macrophage cell line RAW 264.7 was used to evaluate the effects of different extracts on the expression of inflammatory cytokines.The results showed that GFC-F1-1 protein component significantly promoted the phosphorylation of JNK and ERK in the MAPKs pathway,and p65 and Ik Ba in the NF-κB pathway,inducing the expression of inflammatory factors.GFC-F1-1 was identified by Q Exactive LC-MS/MS,and 11 candidate proteins were screened,including antioxidant enzymes(SOD,GPx,APx,GR,GDP),heat shock 70 like protein(HSP70L)and heat shock 90 like protein(HSP90L),as well as translationally controlled tumor protein(TCTP)might be the key candidate proteins for ginsenginduced “shang huo”.3.Functional verification of ginseng TCTP-induced inflammationThe Pichia pastoris protein expression system was used to recombinantly express TCTP,and its pro-inflammatory activity was verified by in vitro cell experiment.The results showed that ginseng recombinant protein TCTP(r TCTP)could promote the upregulated expression of various inflammatory factors at both m RNA and protein levels,and had obvious pro-inflammatory activity.The results of transcriptome sequencing and inhibitor experiments showed that r TCTP promoted the transcription and translation of inflammatory factors through activating the TLR4/NF-κB signaling pathway,thereby participating in the regulation of inflammatory response.4.Mining and verification of key shared proteins of “shang huo” induced by satsumas and Panax ginsengFrom the mass spectrometry identification results of the main pro-inflammatory components of satsumas and Panax ginseng,the shared proteins were screened;An overexpression vector was constructed,the Agrobacterium-mediated citrus fruit transient transformation technology was used to infect grapefruit fruit that is not easy to induce “shang huo”,and the pro-inflammatory function of the shared proteins was verified by detecting the expression of inflammatory factors in RAW 264.7macrophages induced by infected fruit juice.The results found that the homologous proteins of heat shock family HSP70 and HSP90 were identified in both satsumas and Panax ginseng;The expression levels of inflammatory genes COX-2 and i NOS in the fruit juice-treated group after the overexpression of HSP70-1 and HSP90 were remarkably higher than that in the control group;It indicated that HSP70 and HSP90 proteins might be the key shared proteins that satsumas and Panax ginseng induced“shang huo”.In conclusion,whether citrus fruits,including satsumas,and Panax ginseng induce“shang huo” was related to the content of related proteins in the fruit tissues;The reduction of “shang huo” induced by stored satsumas was closely related to the decrease of protein content in the fruit.Inflammation induced by the ginseng protein component GFC-F1-1 was closely related to the activation of JNK/ERK-MAPKs and NF-κB signaling pathways;Among GFC-F1-1 components,antioxidant enzymes,HSP70 L and HSP90 L,as well as TCTP might be key candidate proteins for ginseng induced “shang huo”.Further research showed that TCTP could indeed promote the expression of inflammatory factors and induce inflammation by activating the TLR4/NF-κB signaling pathway.HSP70 and HSP90 might be the shared proteins that cause “shang huo” in satsumas and Panax ginseng.This study can provide a research direction for the analysis of “shang huo”,and provide a scientific basis for the quality improvement of citrus fruits and Panax ginseng and dietary recommendations.