| Poria is the dry sclerotia of Poria cocos(Schw.) Wolf of fungus of a branch of multiaperture mushroom. Different medicinal parts have different functions: Poriae Cutis is the dry skin and has been mainly used to promote urination in traditional Chinese medicine clinics; Rubra Poria is the pink part near the skin and has the effects of inducing diuresis, clearing heat, eliminating dampness, supplementing heart and moistening lungs; White Poria is the white part after removal of Rubra Poria and has been used for its diuretic, tonic and sedative effects and invigorating the effect of calming nerves; Poria cum Radix Pini is the white central part with pine root naturally and has the biological activities such as tranquilizing mind by nourishing the heart, inducing diuresis to alleviate edema and tonifying efficacy to treat fatigue. Triterpene compounds of Poria are one of the main effective components in Poria and have anti-tumor, anti-inflammatory, diuretic, immune regulation effects and so on. Current researches of Poria are limited to one or two medicinal parts, which is difficult to comparative study of different medical parts of Poria comprehensively. In the present study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS/MS) method was first developed for the simultaneous qualitative and quantitative determination of triterpene compounds of Poria in four different medicinal parts of Poria combining with principal component analysis(PCA) and t-test to compare the differences among four different medicinal parts. Quality standard of Rubra Poria was also studied and drafted out. These results provided scientific and effective references for quality control of four different medicinal parts of Poria. Part one Simultaneous identification and determination of triterpene compounds by UPLC-QTOF-MS/MS and its application in constituents comparison of four medicinal parts of PoriaObjective: To establish an UPLC-QTOF-MS/MS method for simultaneous qualitative and quantitative analysis of triterpene compounds in Poria and compare differences of components in four medicinal parts including Poria cum Radix Pini, White Poria, Rubra Poria and Poriae Cutis.Method: Electrospray ion source was used and 8 standard store solutions of triterpene compounds of Poria were pumped to the mass spectrometer in positive and negative ion modes with different CE to analyze ion fragments of [M+H]~+ and [M-H]~- and summarize the rules of mass fragmentations. Phenomenex-C18(150 mm × 2 mm, 3 μm) column was applied with acetonitrile and water-0.1% formic acid-2 mmol/L ammonium acetate as mobile phase with gradient elution and the flow rate was 0.3 m L/min. Peak View 2.1 and Multi Quant 3.0 softwares were used for qualitative and quantitative analysis based on data, respectively. Furthermore, the results were used to cluster analysis and t test by using the Markerview 1.2.1 software.Results: Fragmentation rules of 8 triterpene compounds(1 lanosta-8-ene type triterpene, 4 lanosta-7,9(11)-diene type triterpenes, 3 3,4-seco-lanosta-7,9(11)-diene type triterpenes) were summarized and fragmentation rules of 3,4-seco-lanosta-8-ene type triterpenes were referred according to the summarized fragmentation rules. A total of 51 triterpene compounds of Poria were identified including 44 compounds in Poria cum Radix Pini, 46 compounds in White Poria, 40 compounds in Rubra Poria and 39 compounds in Poriae Cutis. The sum of 49 triterpene compounds and single triterpene compound were variuos in different medicinal parts: Poriae Cutis was higher than Rubra Poria, which was higher than Poria cum Radix Pini and White Poria was the lowest; PCA method was used to find different compounds of poricoic acid D, trametenolic acid among Poria cum Radix Pini, White Poria, Rubra Poria, Poriae Cutis. T-test was used to find different compounds of 26-hydroxyporicoic acid G, 16α-acetoxy-lanosta-8,24-dien-21-oic acid, lanosta-7,9(11),24-trien-21- oic acid between Poria cum Radix Pini and other three parts, poricoic acid B, lanosta-7,9(11),24-trien-21-oic acid between Rubra Poria and other three parts,trametenolic acid and poricoic acid B between Poriae Cutis and other three parts and there were no different compounds between White Poria and other three parts. The contents of 3,4-open loop type triterpene compounds increased in the morphological order from inside to outside: Poria cum Radix Pini, White Poria, Rubra Poria and Poriae Cutis. While the contents of closed-loop type triterpene compounds decreased in the same order.Conclusion: A simple, sensitive and specific analytical method was first developed for the simultaneous identification and quantification of triterpene compounds based on UPLC-QTOF-MS/MS method in four medicinal parts including Poria cum Radix Pini, White Poria, Rubra Poria and Poriae Cutis. Simultaneous qualitative and quantitative analysis was realized only by single sample injection analysis. The differences of contents of triterpene compounds in different medical parts of Poria were compared which further provides the theory basis for the illustration of their pharmacological mechanism. Part two Study on quality standard of Rubra PoriaObjective: To establish quality standard of Rubra Poria and control effectively the quality of Rubra Poria.Method: On the basis of the four general principles in the "Chinese pharmacopoeia"(2015 version), 12 batches of Rubra Poria were analyzed. Herbalogical study of Rubra Poria was performed. Morphological characteristics, microscopic features, thin layer chromatography method were used for identification. The moisture, total ash and acid-insoluble ash were determined. The alcohol-soluble extracts by hot dipping method were also quantified.Results: The original of Rubra Poria was confirmed. The morphological characteristics were summarized as follows: cubic blocks or block and thick slices with different sizes are light brown or brown; they are solid and have light smell, adhering teeth when chewing. The powder is light brown or brown. The powder shows the following diagnostic characters: irregular granular or branched clumps are reddish brown; light brown, brown or dark brown myceliums are thin and slightly curved or branched about 2 to 8 μm or somtimes to 11 μm in diameter. TLC identification showed six main spots of the four different medicinal parts were clear; the separating degrees met the requirements, especially Poria and Rubra Poria were distinguished obviously; the separating conditions were little affected by temperature and humidity. The contents of moisture were 9.0% to 17.2% and the average was 14.0%; the contents of total ash were 0.5% to 1.4% and the average was 0.9%; the contents of acid-insoluble ash were 0.1% to 0.9% and the average was 0.4%; the contents of alcohol-soluble extracts were 6.6% to 18.6% and the average was 11.6%.Conclusion: The morphological characteristics of Rubra Poria and microscopic diagnostic characters of powder were summarized; TLC identification method was established and had good specificity and durability; the limits of contents of moisture, total ash, acid-insoluble ash and alcohol-soluble extracts were formulated; finally quality standard of Rubra Poria was established, which provided the basis for quality control of Rubra Poria. |
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