| Purpose:The precious Chinese herbs "Citri Grandis Exocarpium" is the immature or near-mature dry outer peel of the plant-derived Citrus grandis ’Tomentosa’ or Citrus grandis(L.)of the genus Rutaceae,which has the functions of qi and phlegm.The outer skin of Citrus grandis ’Tomentosa’ is densely covered with fluff,and its medicinal material is called Citrus grandis ’Tomentosa’.It is a famous local medicinal material.Citrus grandis‘Tomentosa’ has a long history of medicinal use and has remarkable curative effect.It has served as a royal phlegm and cough tribute.Because the authentic medicinal materials of medicinal herbs,cough and phlegm have remarkable curative effect and are well-known overseas,the domestic and international pharmaceutical market has a large demand,but its medicinal materials are basically purchased at high prices overseas.The medicinal market has long been rare domestic.This phenomenon is due to the reason that the medicinal material Citrus grandis(L.)is flooding the domestic medicinal market,so the efficacy of medicinal Citri Grandis Exocarpium is significantly reduced.Therefore,it is of great significance to vigorously develop the production of Citri Grandis Exocarpium,especially the high-yield and high-quality Citrus grandis ’Tomentosa’ varieties.After more than a thousand years of cultivation,Citrus grandis‘Tomentosa’ has formed more than a dozen cultivars such as MiYe,HuangRong,FengWei,ZhengMao,and JiaXiYang.The plant morphology,appearance and quality of the medicinal materials have been slightly different.It was found that the new cultivar MiYe has the advantages of high yield and good stress resistance.However,there have been no reports on the analysis of the yield and quality of new varieties of Citrus grandis‘Tomentosa’.Therefore,systematic research on MiYe has laid a research foundation for the promotion,which is of great significance to the development of the production of "Citri Grandis Exocarpium" medicinal materials.For this reason,the authors studied the newly cultivars MiYe and other common cultivars of Citrus grandis ’Tomentosa’ through systematically compared their yield,quality,thin-layer chromatographic behavior,principal component content,fingerprint and phylogenetic relationship,and preliminarily clarify its yield,quality and kinship characteristics of this cultivars,so as to provide research data and scientific basis for the promotion and application of the new cultivar MiYe of Citrus grandis ’Tomentosa’.Methods:1 Study on different cultivars of Citrus grandis ’Tomentosa’ resources1.1 The distribution of different cultivars of Citrus grandis ’ Tomentosa’ was investigated by means of field visits and questionnaires.The main producing areas of Zhongdong Town,Pingding Town,Xin’an Town,Ligang Town,Yangmei Town and Baoyu Town of Huazhou City were investigated and compare the distribution of differnt cultivars.1.2 Comparative study on yield of different cultivars of Citrus grandis ’Tomentosa’ Five different cultivars of MiYe,HuangRong,ZhengMao,Feng Wei and JiaXiYang were used as materials to conduct experiments in Baoyu Town and Pingding Town of Huazhou City using random distribution method.The division of the block was carried out and the agronomic operation and field management in the experiment were carried out in accordance with the "Quality and Technical Requirements" in the "Announcement on Approving the Protection of Geographical Indication Products for Citri Grandis Exocarpium".2 Study on the pharmacognosy of different cultivars of Citrus grandis ’Tomentosa’2.1 Morphological study of different cultivarsTraits of different cultivars of Citrus grandis ’Tomentosa’ were observed and described by trait identification method,and statistical analysis(SPSS 22.0)was used for cluster analysis.2.2 Morphology and quantity of Pollen from different cultivars of Citrus grandis’Tomentosa’The pollen of different cultivars of Citrus grandis ’Tomentosa’ was observed by scanning electron microscopy,and the microscopic morphology was observed.3 The quality of the new varieties of the Citrus grandis ’Tomentosa’ and other common cultivarsChinese Pharmacopoeia(2015 edition)were used to study the general traits,thin layer,moisture,ash and volatile oil content of different cultivars of Citrus grandis ’Tomentosa’;Using the UV to determinate the content of total flavonoids;the contents of naringin,quercetin and apigenin was determined by UPLC.The fingerprint behavior of the samples was analyzed by HPLC and evaluated by the Chromatographic Fingerprint Similarity Evaluation System of Traditional Chinese Medicine.The volatile oil composition and its relative content was determined by GC-MS.4 Genetic information of the new varieties of the citrus red and other mainstream cultivars.After extracting the DNA from different cultivars of the cultivar Citrus grandis’Tomentosa’,the genetic information was analyzed using the Illumina Hiseq X Ten benchtop high-throughput sequencer,and Software treebest(1.9.2 version)was used and Software GCTA(version 1.25.2)for phylogenetic tree and principal component analysis.Results:1 Study on different cultivars of Citrus grandis ’Tomentosa’ resourcesAccording to the investigation,the main cultivars of Citrus grandis ’Tomentosa’ are MiYe,ZhengMao and HuangRong.The original mainstream variety of JiaXiYang,FuMao,JinQianDu and FengWei are gradually replaced by MiYe,ZhengMao and HuangRong.The yield of fresh fruit per cultivar of Citrus grandis ’Tomentosa’ is quite different.Among them,the yield of MiYe is the highest,the yield of fresh fruit per acre can reach 625.33±25.00 kg,followed by JiaXiYang,the yield per mu is 433.33±28.87 kg,followed by HuangRong,and the yield per mu is 413.67±32.15 kg,followed by ZhengMao,the yield per mu is 333.33±57.74 kg,and finally the FengWei,the yield per mu is 170.00±17.32 kg.2 Study on the pharmacy of different cultivars of Citrus grandis‘Tomentosa’2.1 Phenotypic traits studiesThrough the analysis of the traits of the MiYe and most of the cultivar Citrus grandis’Tomentosa’,it showed that there were abundant and different phenotypic variation among different cultivars.Among the five phenotypic traits,the leaf length variation was the largest,and the coefficient of variation was 19.65%;the second was leaf width,the coefficient of variation was 19.09%;the leaf length to width ratio was the smallest,8.66%.Among the phenotypic traits of fruit,the weight variation of dried fruit was the largest,the coefficient of variation was 15.58%,the variation of fresh fruit weight was the smallest,and the coefficient of variation was 8.25%.The observation of 14 morphological characteristics also showed that there were significant differences in plant organs and their plant morphology among different cultivars.For the 7 cultivars selected in this study,when the European square distance is 7.0,the 7 different cultivars can be divided into two categories,the first type is JinQianDu,the second type is Mi Ye,ZhengMao,HuangRong,JiaXiYang,FengWei and FuMao;when the European square distance is 2.5,7 common cultivars can be divided into three categories,namely,the first type is JinQianDu,and the fruit umbilical has a ring similar to the size of copper coins,II The genus is FengWei,and its umbilical style does not fall off.The third category includes MiYe,ZhengMao,HuangRong,JiaXiYang and FuMao.2.2 Study on the number and morphology of pollen grainsThe pollen morphology of different cultivars is relatively single,all of which are single-grain,symmetrical,long-spherical,with obvious long ditch.Among them,the MiYe’s holes are deep and clear,and the distribution is dense;the JiaXiYang is deep and clear,and the distribution is sparse,and the holes are not clear and the distribution is dense,FengWei hole is not clear and densely distributed.In addition,the average values of the polar,eccentric and phoenix axes,equatorial axes and P/E values of the cultivars(MiYe,JiaXiYang and FengWei)of Huazhou pomelo were 27.90 μm,26.54 μm and 26.46 μm respectively;20.26 μm,16.34 μm and 16.38 μm respectively;1.39,1.64 and 1.62 respectively.3 Study on the quality of different cultivars of Citrus grandis‘Tomentosa’3.1 Identification of general traitsThe traits of the traits showed that the samples of each cultivar were similar in morphology.It can be seen from the thin-layer chromatographic behavior that each test product has coloration at the same position of the naringin reference substance.The volatile oil in the sample was higher than the requirement of "Guangdong Provincial Local Standard-Geographical Indication Productized Citri Grandis Exocarpium"(volatile oil content=0.5%).However,the volatile oil content of different cultivars varied greatly,ranging from 0.58 to 1.65%mL/g.Among them,the average content of volatile oil of JinQianDu reached 1.44%mL/g,the highest;followed by HuangRong,the average content was 1.23%mL/g;followed by ZhengMao,MiYe,JiaXiYang,FengWei,the average content was 1.20%mL/g,1.17%mL/g,0.92%mL/g and 0.89%mL/g;the last is the FuMao,the average content is 0.63%mL/g.The moisture content of the MiYe cultivar of the citrus cultivar is between 7.09 and 9.86%,the average content is 8.67%,the total ash content is 3.33～5.89%,and the average content is 4.29%;The moisture content of HuangRong,ZhengMao,JiaXiYang,FengWei,JinQianDu and FuMao was 8.97～10.33%,6.82～11.06%,7.87～10.71%,8.20～9.47%,7.31～7.96%,6.95～9.26%,respectively,and the average content was 9.36%and 8.36%,8.62%,8.83%,7.55%,7.85%,respectively;The total average content was 4.57%,4.79%,4.50%,4.18%,4.05%,and 3.69%.3.2 Comparison of total flavonoids content in different cultivars of Citrus grandis‘Tomentosa’The total flavonoid content of the new cultivar MiYe was between 10.37 and 15.45%,with an average content of 12.91%.The total flavonoids content of other mainstream cultivars were ZhengMao,HuangRong,JiaXiYang,FengWei,JinQianDu and FuMao were 10.21～16.53%,10.19～16.20%,8.51～14.71%,18.15～19.00%,11.15～16.07%,8.07～9.68%,respectively,and the average contents were 13.94%,13.20%,10.74%,18.60%,13.16%and 8.83%respectively.3.3 Comparison of flavonoids content in different cultivars of Citrus grandis ’Tomentosa’The naringin content of different cultivars of Citrus grandis ’Tomentosa’,MiYe,HuangRong,ZhengMao,JiaXiYang,FengWei,JinQianDu and FuMao,were 75.74 mg/g,75.41 mg/g,79.08 mg/g,52.91 mg/g,117.67 mg/g,99.53 mg/g and 35.91 mg/g,respectively;the contents of rhoifolin were 4.88 mg/g,5.86 mg/g,7.41 mg/g,2.35 mg/g,5.76 mg/g,3.14 mg/g,and 3.45 mg/g,respectively;the contents of apigenin 0.31 mg/g,0.30 mg/g,0.29 mg/g,0.34 mg/g,0.33 mg/g,and 0.30 mg/g,among the cultivars,the content of naringin from FengWei was the highest and the content of rhoifolin from ZhengMao was the highest.3.4 Comparison of fingerprints of different cultivars of Citrus grandis ’Tomentosa’The peak of the Citrus grandis ’Tomentosa’ cultivar JiaXiYang and JinQianDu in the position of the rhoifolin is not the second largest peak,and the rhoifolin peak of the Citrus grandis(L.)is not the second largest peak,which suggests the fingerprints of the two cultivars of the f JiaXiYang and JinQianDu are more similar to the fingerprints of the Citrus grandis(L.),indicating that the primordial plant is closer to the Citrus grandis(L.).In addition,according to the similarity evaluation results,the similarity of different cultivars of Citrus grandis ’Tomentosa’ medicinal materials was between 0.245 and 0.803,which showed a large difference,indicating that the cultivars were of similar degree,and different cultivars were MiYe,HuangRong,ZhengMao and JinQianDu.The similarities between the FengWei,JiaXiYang and FuMao are similar.Through cluster analysis of 36 batches of different cultivated Citrus grandis ’Tomentosa’ medicinal materials,it can be seen that when the European square distance is 7.50,different cultivars of Citrus grandis’Tomentosa’ medicinal materials are divided into three categories,the first type is MiYe,and the second type is ZhengMao and HuangRong and JiaXiYang,the third category is FengWei,JinQianDu and FuMao.3.5 Comparison of Volatile Components and Relative Contents of Different Cultivars of Citrus grandis ’Tomentosa’The cultivars of different cultivars were slightly different,and the main chemical components were basically the same,all of which were terpenes,β-pinene,myrcene,P-cymene,limonene,γ-terpinene,linalool,4-decenol,α-terpineol and germacrene D.When the relative percentages of the volatile components of the new cultivar MiYe and other cultivars were analyzed,it was found that the relative contents of the main volatile components of different cultivars of Citrus grandis‘Tomentosa’ had a certain degree of difference.High-quality strains such as MiYe and ZhengMao and HuangRong are grouped together.4 Comparative study on genetic information of different cultivars of Citrus grandis’Tomentosa’The newly cultivated cultivar MiYe and other cultivars,such as HuangRong,JiaXiYang,FengWei,JinQianDu and FuMao,were completely separated,but were carried out with Zhengmao.When compared,it was found that the genetic information was similar and it was difficult to completely separate.Conclusion:1 The comparative study of small area per mu showed that the fresh fruit per cultivar of different varieties of Citrus grandis ’Tomentosa’ is quite different.Among them,the yield of fresh fruit of MiYe can reach 625.33 kg/mu,which is the highest yield of Citrus grandis’Tomentosa’ cultivar.2 The abundant phenotypic variation among different cultivars of Citrus grandis‘Tomentosa’ is rich.In the cluster analysis,it can be found that MiYe,ZhengMao,HuangRong,JiaXiYang and FuMao were classified together.The significant differences in morphology of pollen grains among different cultivars,MiYe hole is deep,clear and concentrated,can be considered for the identification of the cultivars.3 The quality of different cultivars of Citrus grandis‘Tomentosa’ showed that the difference of TLC behavior,moisture and total ash between different cultivars was small,and the contents of volatile oil,total flavonoids and flavonoids were quite different,but all of all met the relevant requirements of "Guangdong Province Local StandardsGeographical Indication Products-Citri Grandis Exocarpium"(referred to as "Guangdong Province Landmark").The fingerprints are lower-similar in behavior,and the volatile components and their relative percentages are basically the same.Among the seven cultivars of Citrus grandis ’Tomentosa’ evaluated,the content of total flavonoids,naringin and rhoifolin in the main indicator of the new cultivar Mi Ye was at a medium-high level.4 There is a significant difference in the genetic information between the new cultivar and the most cultivar of the Citrus grandis ’Tomentosa’,which can be completely separated.In summary,the new cultivar MiYe of Citrus grandis ’Tomentosa’ have the characteristics of high yield and good quality in line with GuangDong’s landmark requirements,and has stable genetic information,which are worthy of promotion and application!... |
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