Processing Mechanisms Of The Charred Areca Nut's Characteristics Of "Good At Relieving Dyspepsia" And The Suitable Arecoline Content In Charred Areca Nut

Areca nut?AN?,the dried seeds of Areca catechu L.,is commonly used for treating parasites,dyspepsia,abdominal distension,edema and malaria,etc.In Chinese traditional medicinal theory,it's considered that charred areca nut?CAN?,the processed product of AN,is good at relieving dyspepsia compared to the raw AN;however,so far the related researches regarding animal experimental evidences and active substances basis are lacking.In recent years,the toxic effects induced by long-term chawing of betel-nut chewing have aroused comprehensive attention,and it's reported that arecoline is the most important toxic constituent.However,the qualities control of areca nut and its processed products only faucus the low limit of arecoline.Objective:This study aimed to explore the processing mechanisms of CAN regarding its feature of“good at relieving dyspepsia”via investigating the chemical constituents changes between AN and CAN?including monosaccharides,amino acids,maillard reaction compounds and polysaccharides?and investigating spectrum-effect relationships of“HPLC-promoting gastrointestinal motility”.In addition,this research is also designed to explore the optimum arecoline content in CAN using the“target constituent removal combined with bioactivity assay”strategy.Methods:1.Charred areca nut samples were obtained by using the on line non-contact infrared temperature measurement system?NCTS?.2.The promoting effects of AN and charred areca nu CAN on gastrointestinal motility were compared via gastrointestinal smooth muscle contraction test in vivo and gastric emptying and small intestinal transit experiments in vitro.In addition,another gastrointestinal motility disorders mice model was constructed by atropine sulfate,and gastric emptying and small intestinal transit experiments were then carried out.3.HPLC analysis was used to compare the chemical changes between AN and CAN,and then the Prepared HPLC?PHPLC?combined ¹H-NMR&¹³C-NMR technologies were used to isolate and identify the new appeared constituents of CAN compared to AN.Besides,we also investigated other constituents changes between AN and CAN,including monosaccharides,amino acids and polysaccharides.Furthermore,the potential mechanisms of new appeared constituents were discussed.Spectrum-effect relationships between HPLC fingerprints and duodenum contractility of CAN on rats were explored to discuss the possible active constituents of CAN for its promoting effects on gastrointestinal motility based on bivariate correlation analysis using SPSS 17.0.4.Prepared TLC technology was used to prepare the arecoline totally removed CAN water extracts?WAC-100R?and a series of different ratios of arecoline-removed CAN samples?WAC-Rx?.5.The acute toxicity test in mice and MTT assay in HaCaT cells were carried out to evaluate the toxic effects of WAC-Rx?WAC,WAC-10R,WAC-20R,WAC-30R,WAC-40R,WAC-50R,WAC-60R,WAC-70R,WAC-80R,WAC-90R and WAC-100R?.The promoting effects of WAC-Rx?WAC,WAC-30R,WAC-50R and WAC-100R?on gastrointestinal motility were studied via gastrointestinal smooth muscle contraction test in vivo and gastric emptying and small intestinal transit experiments in vitro.6.Nuclear staining with DAPI and flow cytometer staining with FITC conjugated Annexin V/PI assays were used to determine the apoptosis of HaCaT cells.Western blotting assays were used to determine the regulative effects of WAC-Rx?WAC,WAC-30R,WAC-50R,WAC-100R?on proteins expressions of Caspase-3,c-fos,c-jun,COX-2,PGE2and IL-1 in HaCaT cells.Results:1.Our results showed that both the AN and CAN could enhance the smooth muscle contractility of stomach and intestine.Compared to AN,CAN possessed a better ability for enhance the smooth muscle contractility at the concentrations over 10?g/mL.In vivo animal experiments exhibited a similar results as the in vitro experiments,both the AN and CAN could promote the gastrointestinal motility of rats,and the CAN showed a better promoting effects than AN.Besides,experiments regarding the atropine sulfate induced gastrointestinal motility disorders mice model also demonstrated that AN and CAN could enhance the gastrointestinal motility,and CAN has a better promoting effect than that of AN.2.By using PHPLC and NMR technologies,3 new appeared constituents were isolated andidentifiedas2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone?DDMP?,5-Hydroxymethyl-2-furfural?5-HMF?and 3-hydroxy-2-methyl-?-pyrone?Maltol?.Our results of A₄₂₀ and pH value determination also revealed that the maillard reactions were happened in the processing of AN.After processing,the contents of reducing saccharides?fructose and glucose?and free amino acids?arginine>lysine>proline>serined?were significantly decreased in the stir-baked CAN compared with AN.Thus,we guess that these three new produced constituents were producted in the maillard reaction.3.This study optimized the CAN polysaccharide extraction by using response surface methodology,and our study also compared the contents of polysaccharide,monosaccharide composition and molecular weight distribution?MWD?between AN and CAN.The results showed that polysaccharide contents in CAN were higher than that of AN,and no difference was found in monosaccharide species between AN and CAN however the ratio of monosaccharide compositions are changed.4.Spectrum-effect relationships between HPLC fingerprints and duodenum contractility of CAN on rats were analyzed.Our results indicated that 5 common peaks in 16CAN samples have positive correlation to the activities,espeacially the 3 maillard reaction products,DDMP?correlation coefficient:0.872,p<0.01?,5-HMF?correlation coefficient:0.843,p<0.01?and Maltol?correlation coefficient:0.782,p<0.01?.5.Based on PTLC method,we prepared CAN lacking all arecoline?WAC-100R?and a series of different ratios of arecoline-removed CAN sample?WAC-Rx?.6.WAC has toxicity on the HaCaT cells with an IC₅₀ value of 107.73?g/mL,and also has a LD₅₀ value of 15.302 g/kg on mice,and decreasing arecoline content by 50%decreased CAN toxicity significantly.Animal results revealed arecoline contents over 50%could guarantee the beneficial effects of CAN on gastrointestinal tract.7.Decreasing arecoline content in CAN by 50%decreased its pro-apoptotic effects significantly.Furthermore,decreasing 50%arecoline content in CAN down-regulated the expressions of Cleaved-Caspase-3,c-jun,c-fos,COX-2,PGE2,and IL-1?.8.Our results revealed that CAN with 50%arecoline content?WAC-50R?has similar beneficial effects on the gastrointestinal tract to CAN,whereas its toxicity was decreased significantly.Our results found that the arecoline contents in CAN is approximate 0.239%,thus we speculate that the optimum arecoline contents is 0.12%.Conclusion:In conclusion,this work systemically verified the characteristic of CAN regarding“good at treating dyspepsia and abdominal distension”,and also reveald its possible processing mechanisms might be related to the following 3 points:?1?arecoline is M cholinergic receptor agonists,and exhibits significant activity with a reasonable concentration/dosage.The processing could make the arecoline contents more reasonable in CAN.?2?Some maillard reaction products could be produced in the processing of areca nut such as DDMP,5-HMF and Maltol,and these maillard products might be also possible active constituents of relieving dyspepsia.?3?Polysaccharides of areca nut might be possible active constituents of relieving dyspepsia,process of areca nut could inhance the dissolution rate of Polysaccharides.The present study investigated the optimum arecoline content in CAN using the“target constituent removal combined with a bioactivity assay”strategy,and the results indicated that the optimum content of arecoline in CAN is approximately 0.12%.Innovations:1.This study investigated the spectrum-effect relationships between HPLC fingerprints and duodenum contractility for the first time;and our results indicated that the maillard products,such as DDMP,5-HMF and Maltol,have closely correlation with the“good at relieving dyspepsia”of charred areca nut.2.This study preliminarily clarified the possible proce processing mechanisms of the charred areca nut's characteristics of“good at relieving dyspepsia”.3.For the first time,the present study investigated the optimum arecoline content in CAN using the“target constituent removal combined with a bioactivity assay”strategy,and the results indicated that the optimum content of arecoline in CAN is approximately 0.12%.