Construction And Analysis On "Activity Fingerprint" Of Citri Reticulatae Pericarpium From Different Cultivars

ObjectiveCitri Reticulatae Pericarpium（CRP,Chenpi in Chinese）,the dried mature pericarp of Citrus reticulata Blanco or its cultivars,can be consumed with tea,food,alcohol,and medicine.CRP which can be divided into "Chenpi"and "Guangchenpi".The dried ripe pericarp of Citrus reticulata‘Chachi’（Guang Chenpi in Chinese）,is regarded as the genuine Chinese medicinal material in Xinhui,Guangdong province based on its excellent quality.CRP is pungent,slightly bitter,warm.The spleen,stomach and lung meridian.It is described as possessing the power to regulate qi,to strengthen the spleen,to dry dampness,and to resolve phlegm.In clinical practice,CRP is primarily applied for digestive and respiratory diseases such as treatment of indigestion,stomach ache,abdominal distension,and cough by combining with other herbs based on the TCM theory.China is abundant in citrus resources of various cultivars.Chemical composition and pharmacological activity of CRP is exact,however,the quality of various citrus peel from different cultivars are significantly uneven.The confused application on these medical herbs might have a negative influence on the pharmacological effect of CRP.Moreover,the knowledge and understanding of the orange peel is insufficient,people throw away a large amount of them resulting in waste and environmental pollution.Although HPLC fingerprint is widely applied for a traditional mean of quality control for herb,there are so many defects such as chemical components remained unknown irrelevant to pharmacological effects.Therefore,the establishment of a more scientific quality assessment on medicinal value for CRP should be quite important.Several studies have reported about quality control of CRP,however,to the best of our knowledge,no such study was yet reported about it.Therefore,the purpose of this paper is to establish a quality evaluation system for various CRP based on the "activity fingerprint".Firstly,in order to explore the pharmacodynamic material of CRP,some systematic separation and analysis methods coupled with LC-MS would be carried out,which based on the pharmacological model of relieving cough and resolving phlegm.Then,on the basis of the full manifestation of the pharmacodynamic material in CRP,analysis and evaluation would be carried out and focused on construction method and elucidation process of "activity fingerprint"by HPLC in eighty-nine batches of peel samples of twenty-one cultivars,and the results may be in favor of revealing the genuineness feature of various CRP in material basis aspect through comparison of the types and the contents of effective constituents and the differences in similarity of chemical constituents.The results of the project would not only play an important role on the quality evaluation and the guidance of identity for various CRP,but also contribute to the protection of the improved cultivars of CRP and the effective development and utilization of citrus resources.MethodsCRP was extracted by methanol,and the extract was divided into three different concentrations including high,medium and low dosage.The murine models of concentrated ammonia spraying were established.Tracheal phenol red method was used to study the pharmacodynamics of cough and phlegm.To evaluate the quality of CRP from China,a simple,and accurate method involving high-performance liquid chromatography with dual wavelength detection was developed for establishing chromatographic fingerprint of CRP and simultaneous determination of five bioactive flavonoids,hesperidin,nobiletin,3,5,6,7,8,3′,4′-heptamethoxyflavone,tangeretin and5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone in 89 batches of CRP samples of twenty-one cultivars collected from different regions in China.The flavonoids were detected at 283 nm and 330 nm by the UV detector using an Agilent 1260 Series HPLC system（Agilent Technologies Inc.,USA）.The compounds were separated on a DIKMA Diamonsil C₁₈ Column（4.6 mm x 250 mm i.d.,5μm）and at a column temperature of 25 ^oC.The solvent system consisted of acetonitrile（B）and water（A）with the following gradient elution program:0-15 min,15-40%acetonitrile;15-35min,40-50%acetonitrile;and 35-40 min,50-75%acetonitrile.By means of high performance liquid chromatography-ion trap-time of flight coupled with mass spectrometry（LC/MS-IT-TOF）method,qualitative analysis of the chemical constituents of the methanol extract from CRP was to analyze the chemical compositions.The LC separation was performed on the same optimal condition.Electrospray ion（ESI）source was applied for the TOF-MS analysis under the positive ion mode,the mass scanning range was between m/z 100-1000,and the running time was 50 min.By using target compound screening method in the mass spectrometry analysis software,the structures of monitored chemical compositions were identified and speculated by the secondary mass spectra fragment of MS,exact relative molecular weight,retention time and reference substance.Ultraviolet-visible spectrophotometry（UV）was adopted in the content determination of polymethoxylated flavones（PMFs）in CRP from eighty-nine batches of twenty-one cultivars,nobiletin was taken as a reference substance,and the detection wavelength was set at 330 nm.Results1.In the concentrated ammonia spraying model,CRP methanol extract with the high,medium and low dosage group could prolong the incubation period of cough and reduce the frequency of cough ammonia-induced within 3 minutes on mouse.In the experiment of phlegm,the concentration of phenol red was increased in the high,medium and low dosage group that it has a promoting effect.Particularly,CRP methanol extract has better antitussive and expectorant effects with the high dose group.2.Establishing the fingerprint of CRP from Xinhui district Guangdong province,18 common peaks were found in the HPLC fingerprints from different sources,compared with reference compounds to get the peaks belonging of three bioactive flavonoids,namely hesperidin,nobiletin,and tangeretin,using the chromatographic fingerprint similarity evaluation software（2012A version）is to establish fingerprints of twenty remaining different varieties of "Chenpi".3.To evaluate the similarity of in CRP from eighty-nine batches of twenty-one cultivars,the quality of tangerine peels was systematically investigated.The results indicated the similarity of Citrus reticulata‘Chachi’from Guangdong province above 0.994 and Citrus reticulata‘Chachi’from different origins has a high similarity.However,there are certain differences in the similarities of the medicinal materials from different varieties.4.Under certain chromatographic conditions,a total of seventeen chemical compounds were separated and identified completely from the methanol extraction of Citrus reticulata‘Chachi’based on reference substances mass fragment ion information,retain time and related references,including four flavonoid glycosides and thirteen polymethoxyflavones.5.There are significant differences among cultivars in the contents of five bioactive flavonoids above among samples from different cultivar origins.Among them,the content of hesperidin was higher than that others.Nobiletin and tangerine have high content,and the difference is obvious between different varieties.6.The quantitative analyses of five bioactive flavonoids were successfully performed by hierarchical cluster analysis（HCA）and principal component analysis（PCA）in order to control the quality of CRP.The results of HCA and PCA were consistent and were divided into four major categories.7.The results indicated that PMFs showed good linearity in the ranges of 3.012-9.036μg·mL^-1（R²=0.9993）,whose average recovery was 102.173%with the RSD of 1.424%.The content of PMFs varied significantly among the different cultivar origins,which were relatively high from Citrus reticulata‘Chachi’,Citrus reticulata‘Dahongpao’,Citrus reticulata‘Tangerina’,Citrus reticulata‘Ponkan’,Citrus reticulata‘Kinokuni’,Citrus reticulata‘Shiyueju’,Citrus reticulata ‘Szehuikan’,Citrus reticulata‘Bayueju’and Citrus reticulata‘Suavissima’,ConclusionThis project established a traditional animal model of cough and phlegm-removal efficacy successfully.The results showed that methanol extracts of CRP with the high-dose group had better antitussive and phlegm-removing effects.Then,establishing the "activity fingerprint"in CRP of eighty-nine batches from twenty-one different varieties,systematic analysis and comprehensive evaluation were carried out.By analyzing the differences of flavonoids in CRP between the different sources of varieties,which supplied the scientific basis to become available for the development and utilization of citrus resources.Multi-indicator ingredients with hesperidin,nobiletin and tangeretin for comprehensive evaluation of tangerine peels quality are more scientific and comprehensive.There are considered as effective varieties of resources for tangerine peel application,namly Citrus reticulata‘Shiyueju^’,Citrus reticulata‘Ponkan’,Citrus reticulata‘Szehuikan’,Citrus reticulata‘Bayueju’.And they were traditionally not used as CRP.This simple and accurate UV method can effectively eliminate the interference of another flavonoids,which is suitable for the content determination of PMFs in CRP.