| Objective:To screen out the Citrus grandis samples which are coincident with Chinese Pharmacopoeia 2015.To establish quantitative analysis of multi-components by single marker(QAMS)which can be appropriated for five flavones in Citrus grandis’Tomentosa’ and Citrus grandis(L.)Osbeck.To reveal the discrepancy of two source of Citrus grandis(Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck).Method:The quality control methods of Citrus grandis which would be determined by QAMS was established at first.The moisture content,ash content and extract content of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck were determined by the methods in Chinese Pharmacopoeia 2015.Micorphological and microscopic identification was used to definite the difference of appearance and microscopic features between Citrus Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck samples.The fingerprint of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck had been established with thin layer chromatography.High performance liquid chromatography.After the qualified samples were screened out.Naringin,neohesperidin,rhoifolin,naringenin,apigenin in Citrus grandis ’Tomentosa’ or Citrus grandis(L.)Osbeck were determinated by quantitative analysis of multi-components by single-marker,and compared with correlation coefficient method.The quality of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck was compared.Results:1 Quality control of Citri Grandis medicinal materials which were used to QAMS1.1 Physiochemical identificationThe result showed that range of moisture content in 31 batches of Citrus grandis’Tomentosa’ was 7.77%～9.81%,and the average content was 8.78%.Range of moisture content in 17 batches of Citrus grandis(L.)Osbeck was 7.68%～10.88%,and the average content was 9.46%.Range of total ash content in 31 batches of Citrus grandis ’Tomentosa’ was 2.77%～4.57%,and the average content was 3.59%.Range of total ash content in 17 batches of Citrus grandis(L.)Osbeck was 3.60%～4.88%,and the average content was 4.19%.The moisture content and total ash content in 48 batches samples were no more than 11%and total ash content were no more than 5%.The samples coincident with Chinese Pharmacopoeia 2015.Range of water-solubility maceration extraction content by method of hot-maceration in Citrus grandis’Tomentosa’ was 36.60%～49.45%,and the average content was 42.77%.Range of water-solubility maceration extraction content in Citrus grandis(L.)Osbeck was 37.91%～48.16%,and the average content was 43.37%.Range of ethanol-solubility maceration extraction content by method of hot-maceration in Citrus grandis’Tomentosa’ was 20.52%～27.12%,and the average content was 23.68%.Range of ethanol-solubility maceration extraction content in Citrus grandis(L.)Osbeck was 18.27%～28.34%,and the average content was 23.85%.1.2 Micorphological and morphological charactersThe surface of 31 batches of Citrus grandis ’Tomentosa’ were yellow and green,densely covered with white tomenta.Mesocarp parenchyma cells,ducts,calcium oxalate column crystals were found in two kinds of samples power.Besides,non-glandular hair was only found in Citrus grandis ’Tomentosa’.The transection of Citrus grandis ’Tomentosa’ had non-glandular hair between epidermis cells,with circular or oval oil cavity which closed to epicarp.Surface of 17 batches of Citrus grandis(L.)Osbeck were sandybrown,smooth,and covered with no tomenta.1.3 Thin Layer ChromatographyThin layer chromatography from Chinese Pharmacopoeia 2015 showed that the Citrus grandis samples displayed band as same palce as naringin reference substance.Thin layer chromatography from the the research,Rf=0.35 and Rf= 0.27,no band was found in in ethanol extracted parts of Citrus grandis ’Tomentosa’,while blue band was found in ethanol extracted parts of all Citrus grandis(L.)Osbeck samples on Rf=0.35.Meanwhile,Rf= 0.27 blue band was found in ethanol extracted parts of some Citrus grandis(L.)Osbeck samples.According to these methods the difference between the two kinds of Citrus grandis could be distinguished.1.4 HPLC FingerprintMatching the peaks of 31 batches of Citrus grandis ’Tomentosa’ and 17 batches of Citrus grandis(L.)Osbeck,HPLC fingerprint showed that 24 peaks owned by both samples.Including 21 common peaks.No.8 and No.7 peak were characteristic peaks of Citrus grandis ’Tomentosa’.No.2 peak was characteristic peaks of Citrus grandis(L.)Osbeck.The same source samples’HPLC fingerprint ware between 0.954～0.995 and 0.905～0.999,the HPLC fingerprint similar degrees were more than 0.90.2 The establish research of QAMSRCFs of Neohesperidin,Rhoifolin,Naringenin,Apigenin were 0.898,1.519,0.313,0.406,and repeatability was good in different experimental conditions(RSD<3.0%).Five flavones of Citrus grandis were determinated by QAMS method,and compared with correlation coefficient method.The result showed that there were no significant differences between the quantitative analysis results of QAMS and external standard method.3 Quality evaluation of Citrus grandi ’Tomentosa’ and Citrus grandis(L.)Osbeck by QAMSDetermination result of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck samples displayed that range of the content of naringin,neohesperidin,rhoifolin,naringenin and apigenin in Citrus grandis ’Tomentosa’ were 56.321 mg·g-1～274.573 mg·g-1;0.032 mg·g-1～0.163 mg·g-1;1.307 mg·g-1～14.726 mg·g-1;0.116 mg·g-1～1.162 mg·g-1;0.018 mg·g-1～0.094 mg·g-1.Average content were 127.138 mg·g-1;0.085 mg·g-1;7.079 mg·g-1;0.394 mg·g-1;0.052 mg·g-1.The content of five fluvone in Citrus grandis(L.)Osbeck were 28.387 mg·g-1～229.015 mg·g-1;0.040 mg·g-1～0.143 mg·g-1;0.857 mg·g-1～5.675 mg·g-1;0.083 mg·g-1～0.373 mg·g-1;0.017 mg·g-1～0.124 mg·g-1.Average content were 76.085 mg·g-1;0.078 mg·g-1;2.488 mg·g-1;0.194 mg·g-1;0.052 mg·g-1.The result of Independent t test showed that the content of naringin,rhoifolin,naringenin in two source of samples were remarkable different(P<0.05).Conclusion:1 Quality control of Citri Grandis medicinal materials which were used to QAMSThe moisture content,total ash content,micorphological and morphological characters and TCL of Citrus grandis samples are coincident with Chinese Pharmacopoeia 2015.Preliminarily drawn up water-solubility maceration extraction content of both Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck are not less than 33%,ethanol-solubility maceration extraction content are not less than 18%.TLC from the research is clear and intuitive,can be used for the differentiation of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck.Feature peaks of the section of Citrus grandis samples from HPLC fingerprint can be used to differentiate the source of Citrus grandis samples.The inspection of Citrus grandis ’Tomentosa’ samples are Citrus grandis ’Tomentosa’ and the inspection of Citrus grandis(L.)Osbeck are Citrus grandis(L.)Osbeck.2 The result of QAMS establish researchThe RCFs created by QAMS is accurate and reliable,can be used to determine the content of five kinds of flavone in Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck.QAMS is more convenient and economize than conventional approaches,even under the condition of the component reference for the shortage.The RCFs of Neohesperidin,Rhoifolin,Naringenin,Apigenin as reference to Naringin are accurate and feasible,can reduce the experiment cost.3 Quality evaluation of Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck by QAMSEffective components of Citrus grandis ’Tomentosa’ samples are significant differences with Citrus grandis(L.)Osbeck.The content of naringin is nearly doubled of Citrus grandis(L.)Osbeck.The content of rhoifolin and naringenin is Several times more than Citrus grandis(L.)Osbeck.The content of naringin,neohesperidin,rhoifolin,naringenin,apigenin in Citrus grandis ’Tomentosa’ are remarkable better than Citrus grandis(L.)Osbeck.To differentiate Citrus grandis ’Tomentosa’ and Citrus grandis(L.)Osbeck is good for enhance the total quality of Citrus grandis medicinal materials. |
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