Study On The Improvement Of The Quality Standard Of Rhododendri Mollis Flos

Rhododendri Mollis Flos(RMF)is the dried flower of Rhododendron molle G.Don.It is one of the traditional poisonous Chinese medicinal materials in our country,It was first published in the “Shen Nong’s Materia Medica”,listed as a lower grade.It has the effects of dispelling wind and dampness,dispelling blood stasis and relieving pain.“Chinese Materia Medica Mongolian Medicine Volume” records that RMF is distributed in Jiangsu,Anhui,Zhejiang,Jiangxi,Fujian,Henan,Hubei,Hunan,Guangdong,Guangxi,Sichuan,Guizhou and other places.Modern studies show that chemical components of RMF such as diterpenoids,flavonoids,triterpenoids,Volatile oil,etc.Among them,diterpenoids are toxic ingredients,which have anti-inflammatory,analgesic,hypotensive,anesthetic,antiviral and other pharmacological activities,and also have toxic effects on the nervous system,heart,and liver.RMF is one of the ten most poisonous traditional Chinese medicines and is included in the calendar “Chinese Pharmacopoeiasand” and local “processing standards”.“Chinese Pharmacopoeia” contains “April and May flowers harvested when they first bloom” and “gray-yellow to yellow-brown”,but the definition of“early bloom” is unclear.RMF is a cold-backed medicinal material,and commercial medicinal materials are sold in a unified way,without distinction of grades and flowering periods.“Chinese Pharmacopoeia” 2020 edition only contains traits,identification,and inspection,but lacks the determination of index components.The quality standards need to be improved.In this subject,a systematic literature study on RMF from the textual research of Materia Medica and modern research progress;using HPLC techniques to establish the content determination methods of diterpenoids and flavonoids in RMF;for the first time,gas chromatography-ion mobility spectrometry technology was used to determine volatile components and combined with chemometric analysis to establish a method for quickly distinguishing the different origins and flowering dates of RMF;for the first time to objectively quantify the color of the RMF,explore the correlation between color and composition,provided experimental basis for improving quality standards of RMF.The research content is as follows:1 Sample collection and identificationThrough various channels such as specimen inspection,telephone consultation,market survey,etc.RMF in Hubei,Anhui,Jiangxi,Hunan,Henan and other places.The literature reports mostly focus on wild RMF.This survey found that there are bases of RMF in Anhui,Hubei,Shandong and other places.Liuwei Muxiang Capsules use cultivated RMF as raw materials.Therefore,this study collected both wild and cultivated flowers.The commercial medicinal materials of RMF are cold-backed medicinal materials in the market.They don’t distinguish the flowering period.Therefore,samples of storage for one year and the current year,and samples of different flowering periods are collected this time.In this study,17 batches of samples of different origins and 5 batches of different flowering periods were collected,and 23 batches of medicinal materials were identified by traits,microscopy and thin-layer chromatography,and their moisture and extract contents were determined.It has been identified as the dried flowers of Rhododendron molle G.Don.The moisture content meets the requirements of the Pharmacopoeia.The difference between different batches may be related to the growth environment and flowering period factors,which need to be combined with the next step content determination.2 Analysis and Comparison of the Components of RMF in Different Origin and Flowering Period2.1 Comparison of the content of flavonoids in RMFThe flavonoids of RMF has an expectorant effect.In this study,a spectrophotometric method was used to establish a method for the determination of total flavonoids in RMF with hyperoside as the standard substance.The total flavonoid content in RMF in different producing areas was determined to be 1.66 %-6.71 %,the content of total flavonoids in different blooming periods is4.01 %-6.83 %.It shows that the place of origin and flowering period have a influence on the content of total flavonoids than theA method for the determination of hyperoside and quercetin in RMF by HPLC-DAD was established.The content of hyperoside and quercetin in different origins were determined to be 0.029 %-0.206 % and 0.008 %-0.032 %,the content of hyperoside and quercetin in different harvesting and flowering periods ranged from0.100 %-0.156 % and 0.025 %-0.048 %,and both were the lowest in the half-opening period.The place of origin has a greater influence on the content of hyperoside.The content of the two flavonoids is more affected by the place of origin than the flowering period.The established method has strong stability and good reproducibility,which is suitable for the determination of the content of the index components of the RMF to improve the quality standard.2.2 Comparison of the content of diterpenoids in RMFDiterpenoids are toxic ingredients.The HPLC-CAD method was used for the first time to establish a method for the simultaneous determination of Rhodojaponin II,III,and V in RMF,and the content of Rhodojaponin II in RMF from different producing areas was 0.192 %-0.395 %;Rhodojaponin III is 0.106 %-0.296 %;Rhodojaponin V is 0.017 %-0.160 %;in different blooming periods,Rhodojaponin II and III reached the highest at the end of blooming.Rhodojaponin V was the highest in the half-blooming period.The total content of the three diterpenoids was the lowest at the bud stage and the highest at the end of blooming.The place of production has a greater influence on the content of diterpenoids,especially the most obvious of Rhodojaponin V.The cluster analysis showed that the medicinal materials of RMF from different origins can be grouped into three categories.The first type(Yongzhou,Chenzhou,Yueyang,Hubei,Huangshi,and Rizhao,Shandong)of Rhodojaponin V content was significantly lower than that of other regions,and the second type(Jiangxi Shangrao,Fuzhou,and two batches purchased from Hehuachi,one batch of Suizhou,Hubei)had a lower total content of diterpenoids than the other two types,and the third group(Suizhou,Hubei and Yuexi,Huoshan,Anhui)had higher content of three components.In this study,a CAD detector with higher sensitivity was used,and the established method has strong stability and good reproducibility,which is suitable for the determination of the content of the index components of the RMF to improve the quality standard.2.3 Analysis of volatile components of RMFFor the first time,gas-phase ion mobility spectroscopy(GC-IMS)combined with chemometric analysis was used to analyze the volatile components of various batches of RMF.As a result,GC-IMS samples from different origins detected a total of 83 volatile components,a total of 78 components,62 types of deterministic components.Cluster analysis,PCA and OPLS-DA results both show that the volatile organic compounds of RMF are related to its source.The samples from the same origin are similar in composition and clustered together,and the quality characteristics of Hunan and other origins are quite different.70 volatile components in RMF at different flowering periods in the same base,67 of which are common components,and 47 are identified,mainly alcohols,esters,and aldehydes.The content of α-terpineol was not detected in the blooming period and the end blooming period.Carvol is a unique component in the late blooming period.The GC-IMS spectrum,Cluster analysis,PCA and OPLS-DA results showed that the different flowering period samples were clearly distinguished.The five medicinal materials with different harvesting periods are divided into three types: the bud period,the first blooming period,the half blooming period,and the full blooming period and the end blooming period.It shows that the established method can be applied to the rapid identification of RMF in different production areas and different flowering periods.3 Correlation analysis between the color and its components“Pharmacology and Clinical Application of Toxic Chinese Medicine” describes RMF “it is best used as medicine with dry yellow-gray color and no impurities”.In this paper,for the first time,a spectrophotometer is used to objectively quantify the color of the medicinal materials of the RMF.SPSS 23.0 is used to analyze the correlation between the chromaticity value of the RMF with the content of the index components,and to explore whether the color and the composition of the RMF are inherently related.The results showed that the L* value extremely significantly positively correlated with the flavonoids,hyperoside,quercetin,and alcohol-soluble extracts of RMF;the a* value was extremely significantly negatively correlated with the Quercetin and was significantly negatively correlated with rhodojaponin III.The a* value is extremely significantly positively correlated;the b* value was extremely significantly positively correlated with the alcohol-soluble extract.It shows that the color of RMF is related to the quality of medicinal materials.By measuring the chromaticity value,the content of RMF ingredients can be predicted,and the established method can quickly and non-destructively judge the quality of RMF.4 Comparison of different flowering periodsThe “Chinese Pharmacopoeia” stipulates that RMF should be harvested when the flowers first bloom,but the definition of “first bloom” is unclear.At present,the medicinal materials of RMF on the market have a variety of flowering periods such as half-blooming,blooming,and end-blooming,and they are sold in general.In this experiment,the fixed base and plant growth year were collected in Suizhou,Hubei,from the bud stage at the end of March to the end of blooming in late April,a total of5 blooming periods,covering all the flowering periods of the plant.Systematic comparisons are made from traits,identification,inspection,and ingredient content.The results showed that in terms of traits,except for the bud period,the other four flowering periods were in line with the description of the pharmacopoeia.From the perspective of identification,compared with the control medicinal material,the five main spots at the flowering period were consistent with the reference spots.From the perspective of composition,the content of extract,flavonoids,diterpenoids,and volatile components in the flower of RMF are relatively high from the initial period to the blooming period.To sum up,the flowering period of the RMF is suitable to be harvested from the initial period period to the full bloom period.