Study On The Quality Markers Of Cinnamomi Ramulus And Persicae Semen In Guizhi Fuling Capsule

Guizhi Fuling Capsule is composed of five traditional Chinese medicines:Cinnamomi Ramulus,Poria,Moutan Cortex,Persicae semen,and Paeoniae Radix Alba.It is a modern dosage form of the classic famous Guizhi Fuling Decoction of Zhang Zhongjing in the Eastern Han Dynasty.Clinically,it is mainly used to treat gynecological diseases such as ovarian cysts,uterine fibroids,dysmenorrhea,chronic pelvic inflammatory disease,and endometriosis.In this paper,from the perspective of quality markers,using Cinnamomi Ramulus and Persicae semen as research objects,the influencing factors of quality markers in the production of medicinal materials are studied.At the same time,the quality of Cinnamomi Ramulus and Persicae semen quality markers in Guizhi Fuling capsule preparations are discussed.The effects of absorption,metabolism,pharmacokinetics,distribution and excretion in rats and primary dysmenorrhea model rats,with a view to laying a material foundation for quality control and in vivo analysis of medicinal materials and compound preparations.Chapter one,Literature researchThis article systematically summarized the research progress of the chemical constituents,pharmacological effects and quality markers of Cinnamomi Ramulus and Persicae semen,and provided the theoretical basis and scientific basis for designing experimental schemes and studying "in vitro-in vivo" quality markers.Chapter two,Study on Influencing Factors of Quality Markers in the Production Process of Cinnamomi Ramulus and Persicae semenSection 1,Study on Influencing Factors of Quality Markers in the Production Process of Cinnamomi Ramulus1.Effect and Evaluation of Harvesting Period on Quality Markers of Cinnamomi RamulusThe content of quality markers will change dynamically with the growth of medicinal plants.The content of quality markers in Cinnamomi Ramulus was compared at 21 harvest times from early April to the end of October.The optimal harvest time of Cinnamomi Ramulus was determined based on the contents of quality markers coumarin,cinnamaldehyde,cinnamic acid,cinnamyl alcohol and o-methoxycinnamaldehyde.The results showed that the content of quality markers of Cinnamomi Ramulus harvested from August to October was higher.2.Influence and Evaluation of Drying on the Quality Markers of Cinnamomi RamulusThe quality of Cinnamomi Ramulus was evaluated by sun-drying,over-drying and three modern processing drying methods.The principal component analysis was performed by SPSS software,and the comprehensive evaluation was performed using the principal component score.The contents of quality markers were significantly different after different drying treatment methods.Among them,microwave drying caused the most loss of active ingredients.The overdried,sun-dried,and low-temperature hot-air drying were beneficial to the retention of active ingredients.The results showed that taking drying time,chemical composition content,appearance properties,odor,etc.as evaluation indicators,it was determined that hot air drying at 50? was a more suitable modern drying processing method for Cinnamomi Ramulus.3.Study on the Influence of Different Producing Areas on the Quality Markers of Cinnamomi RamulusTaking 32 batches of Cinnamomi Ramulus from Guangdong and Guangxi as research objects,a thin-layer identification method for various components of Cinnamomi Ramulus from different regions was established.The results show that the commercial Guizhou medicinal materials from different regions show a certain difference.The Guangdong base of Cinnamomi Ramulus showed high consistency.Establish the UPLC characteristic map of Cinnamomi Ramulus,and use the characteristic map to evaluate the quality of Cinnamomi Ramulus from different producing areas.As a result,it is found that the similarity between all samples and the control map is greater than 0.900.There are good consistency among batches of samples in the Guangdong base.The sales of Cinnamomi Ramulus showed some differences.A method for simultaneous determination of five phenylpropanoids was established using quantitative analysis of multicomponents by single marker(QAMS),and the content of quality markers of Cinnamomi Ramulus from different origins was evaluated.It was found that the content of cinnamaldehyde in all samples from different origins was higher than the limit of 1.0%in the 2015 Pharmacopoeia.Except for cinnamaldehyde,the content of each ingredient in the cinnamon sticks was significantly different,and its content was generally lower.The content of commercially available samples from different producing areas fluctuated greatly,and the samples at the Guangdong base were relatively stable.Section 2,Study on Influencing Factors of Quality Markers in the Production Process of Persicae semen1.Comparative study on chemical constituents between Prunus persica seeds and Prunus davidiana seedsUltra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF/MS)and gas chromatography-mass spectrometry(GC-MS)were used to explore the chemical composition differences between Prunus persica seeds and Prunus davidiana seeds.The results of multiple samples from different producing areas showed that the chemical composition of Prunus persica seeds and Prunus davidiana seeds did not differ in species,but there were some differences in relative content,such as dodecanoic acid,myristic acid,hexadecenoic acid,palmitic acid,heptadecanoic acid,linoleic acid,oleic acid,decanoic acid,mandelic acid amide-?-gentiobioside,mandelic acid-?-D-glucopyranoside,benzyl-?-gentiobioside,amygdalin,prunasin,and Prupersin B showed a significant difference.The difference is mainly reflected in the content of medicinal ingredients,saturated fatty acids such as lauronic acid and mandelic acid amide-?-gentiobioside,mandelic acid-?-D-glucopyranoside,prunasin,and Prupersin B in Prunus persica seeds were significantly higher than that of Prunus davidiana seeds.The contents of unsaturated fatty acids such as hexadecenoic acid and benzyl-?-gentiobioside,amygdalin Prunus davidiana seeds were significantly higher than that of Prunus persica seeds.2.Effect and Evaluation of Harvesting Period on Quality Markers of Persicae semenSix samples of Persicae semen with different harvest time from July to September were collected,and it was found that the different harvest time would affect the content of the quality markers of Persicae semen.If the fullness of the Persicae semen and the content of the quality marker amygdalin are used as indicators to determine the optimal harvest time,the results show that the measured content of the Persicae semen sample collected in mid-July is higher,and the seed is full.But the variation of the content during the harvest period under review is small.3.Study on the Influence of Different Producing Areas on the Quality Markers of Persicae semenTaking 32 batches of Persicae semen from Shanxi,Inner Mongolia,Ningxia,and Gansu as research objects,a thin-layer identification method of Persicae semen from different places was established.It was found that the thin-layer spots of samples from different places were similar,but the color of the spots was slightly different.Among them,the Persicae semen in the Shanxi base showed a high consistency.UPLC characteristic maps of Persicae semen were established.The fingerprints were used to compare the differences between Persicae semen from different places,and the peaks were identified using UPLC-QTOF/MS.It was found that the similarity between all samples and the control map was greater than 0.900,indicating that the overall chemical composition of the samples of Persicae semen was similar,and there was good consistency between the samples of each batch.The contents of amygdalin and Prunasin in medicinal materials of Persicae semen from different places were determined by ultra performance liquid chromatography.The content of amygdalin in all samples was higher than 2.0%of the limit stipulated in the 2015 Chinese Pharmacopoeia.Analysis of the differences in the content of the Persicae semen from different places of origin showed that the content of Prunasin was generally low.The content of commercially available samples from different regions is relatively low.Chapter three,Research on the Quality Markers of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsule Based on In vivo ProcessSection 1,Establishment and verification of rat primary dysmenorrhea modelEstablish a rat model of primary dysmenorrhea using estradiol benzoate combined with oxytocin.To verify the success of the model,by observing the writhing response,detecting the biochemical analysis of plasma and tissue,and uterine pathology,the results show that the primary The dysmenorrhea rats were successfully modeled and can be used for subsequent in vivo process experiments.Section 2,Intestinal Absorption of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in RatsThe intestinal perfusion model of rats was used to explore the characteristics of intestinal absorption in rats of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsule.The effects of different concentrations on intestinal absorption were investigated.The results showed that in the experimental concentration range,the small intestine absorption of amygdalin,cinnamic acid,and o-methoxycinnamaldehyde appeared as a passive diffusion absorption mechanism;the absorption of cinnamaldehyde and coumarin was saturated Phenomenon,its absorption mechanism may not be completely passive transport.The investigation of different intestinal segments showed that amygdalin was absorbed in the whole intestine of rats,and there was no specific absorption site;the main absorption sites of cinnamaldehyde,coumarin,o-methoxycinnamaldehyde,and cinnamic acid were in ten Duodenum and jejunum.The effects of P-gp inhibitors on the absorption of various components were investigated.It was found that cinnamic acid and coumarin may be substrates of P-gp.Adding P-gp inhibitors can promote their absorption and improve bioavailability.Comparing the difference in absorption between normal rats and primary dysmenorrhea model rats,it was found that compared with normal rats,it can significantly increase the absorption parameters of cinnamaldehyde in the jejunum and ileum of primary dysmenorrhea rats;it can significantly increase the cinnamic acid in the primary Absorption parameters of the four intestinal segments of rats with dysmenorrhea;can significantly increase the absorption of o-methoxycinnamaldehyde in the colon of rats with primary dysmenorrhea;the rest are not significantly different.Section 3,Study on the Metabolites of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in RatsA method of ultra performance liquid chromatography-linear ion trap-electrostatic orbit trap tandem mass spectrometry(UHPLC-LTQ-Orbitrap-MS)was established to administer Guizhi Fuling Capsules to the plasma,bile,urine and feces in rats.The metabolites of cinnamic acid,coumarin and amygdalin were analyzed,and normal rats were compared with primary dysmenorrhea model rats.Finally,25 metabolites were identified,of which 25,18,16,and 8 metabolites were identified in plasma,bile,urine,and feces,respectively.There is no significant difference in species.Section 4,Study on the pharmacokinetics of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in RatsBased on pharmacokinetic experiments,the pharmacokinetic differences of amygdalin,cinnamic acid and coumarin in normal rats and primary dysmenorrhea rats were studied.The results showed that in the pathological state of the primary dysmenorrhea model,T1/2 of amygdalin and CL of cinnamic acid were significantly higher than those of normal rats,and AUC0-t and Cmax of cinnamic acid were significantly lower than those of normal rats.Rats can increase the bioavailability of cinnamic acid and slow its elimination.At the same time,the changes of metabolites were analyzed.Each metabolite showed a consistent blood concentration-time curve in normal rats and primary dysmenorrhea model rats,and most amygdalin was metabolized to Prunasin in the body.Most of cinnamic acid is converted into hippuric acid in the body.At the same time,it was found that the peak time of each metabolite was delayed compared to the prototype compound.Section 5,Study on the distribution of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in RatsThe difference between the distribution of prototype components in normal rats and primary dysmenorrhea model rats was found,and only the amygdalin and coumarin were detected.Among them,amygdalin is mainly distributed in the lung.Compared with the normal group,the distribution of amygdalin in the lung and uterus of the model group increased.The distribution of coumarin in various tissues has no obvious tissue targeting.At the same time,the tissue distribution of each metabolite was analyzed.In the organ uterus directly related to the primary dysmenorrhea,the distribution of hippuric acid in the uterus of the model group was significantly increased in the model group compared with the normal group.Therefore,the dysmenorrhea state may directly affect the distribution and exchange of drugs in the body.Prunasin is mainly distributed in kidney,liver and lung in various tissues.Compared with the normal group,the distribution of Prunasin in the uterus of the model group increased and the distribution in other tissues decreased.Section 6,Study on the excretion of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in ratsStudies on the excretion differences between the prototype components of Cinnamomi Ramulus and Persicae semen in Guizhi Fuling Capsules in normal rats and primary rats.It was found that only prototype amygdalin was detected in bile,urine and feces,and other prototype components were low.On detection line.The amygdalin prototype component is mainly eliminated in urine.Compared with normal rats,in the pathological state of the primary dysmenorrhea model,the cumulative excretion and excretion rate of amygdalin in urine were significantly increased,and there was no significant difference in bile and feces.At the same time,the excretion law of metabolites was analyzed.Humaric acid and Prunasin were mainly excreted from urine.The excretion amount in primary dysmenorrhea model rats was higher than that in normal rats.Other metabolites also show some differences between normal rats and primary dysmenorrhea model rats.