An UPLC - Q - Orbitrap method for pharmacokinetics and tissue distribution of four triterpenoids in rats after oral administration of Poria cocos ethanol extracts.

Poria cocos (Schw.) Wolf, is a fungus that is widely used as medicine and dietary supplement in China. But its action mechanism is still not very clear. In this paper, a rapid, specific and sensitive high performace liquid chromatography coupled with hybrid quadrupole - orbitrap mass sepctrometry (UPLC - Q - Orbitrap MS) method has been developed and validated to simultaneously determine of four triterpenoids including Dehydrotumulosic acid (DTA), Dehydropachymic acid (DPA), Pachymic acid (PA), Dehydrotrametenolic acid (DMA) from Poria cocos in rat plasma and tissues. The analyte was extracted from rat plasma and tissue homogenates by protein precipitation with acetonitrile using glibenclamide as the internal standard (IS). Chromatographic separation was carried out on ACQUITY UPLC BEH - C18 column (2.1 mm × 50 mm, 1.7 µm) with a mobile phase composed of acetonitrile - water (containing 1.0 mmol/L ammonium acetate) using gradient elution at a flow rate of 0.2 mL/min. Electrospray ionization (ESI -) under negative ion mode was used, and its quantization was performed with multiple reaction monitoring (MRM) mode. The method was fully validated and successfully applied to pharmacokinetics and tissue distribution study in rats after oral administration of ethanol extracts of Poria cocos. Compared with that of plasma exporsure, triterpenoids could be detected in various tissues with a relatively high degree of tissue distribution. After oral administration, the concentration orders in seven different tissues were ranked as DTA > PA > DPA > DMA in intestine and stomach, wheras DTA > DMA > PA > DPA in heart, liver, spleen, lung and kidney tissues, which is speculated that DPA, PA may be converted into DMA in vivo. In conclusion, this results may provide a material basis for study of the pharmacological actions of triterpenoids in Poria cocos.

[Studies on quality standards of Poria].

In order to provide scientific recommendations for the revision of the quality standards of Poria in Chinese Pharmacopoeia(Ch. P) 2020 edition, a series of experiments were carried out to improve the quality standards of Poria. TLC methods were established to identify Poria by using pachymic acid, dehydrotumulosic acid and reference herbs as reference substances. The contents of water, total ash, pesticide residues, heavy metals and deleterious element, mycotoxins, sulfur dioxide residues and ethanol-soluble extract of herbal materials and decoction pieces of Poria were determined according to the methods recorded in the volume Ⅳ of Ch. P 2015 edition. An HPLC method was developed for the determination of pachymic acid and dehydropachymic acid. The contents of polysaccharide were determined by spectrophotometry using D-glucose as reference substance. The quality standards were established on the basis of the research results, in which the [assay] were added, and the [identification] and [tests] were revised when compared with Ch. P 2015 edition. The established methods are simple, specific, repeatable, and suitable for the quality evaluation of Poria.

Comparison and quantitative analysis of wild and cultivated Macrohyporia cocos using attenuated total refection-Fourier transform infrared spectroscopy combined with ultra-fast liquid chromatography.

Dried sclerotium of Macrohyporia cocos is a well-known and widely-consumed traditional Chinese medicine and is also used as dietary supplement. According to the differential treatment between cultivation and wild habitats in the market, the comparison and quantitative analysis of wild and cultivated M. cocos were performed using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and ultra-fast liquid chromatography combined with partial least squares discriminant analysis and partial least squares regression (PLSR). 636 samples were used for the spectral scan and chromatographic analysis. Results indicated that contents of dehydrotumulosic acid, poricoic acid A and dehydrotrametenolic acid in cultivated samples were significantly different from wild samples in two medicinal parts. Differences of dehydropachymic acid and pachymic acid just existed in inner part samples (P < 0.05). Wild M. cocos samples could be discriminated with cultivated samples with >95.14% efficiency using spectral data. ATR-FTIR combined with PLSR provided satisfactory performance for content predictions of poricoic acid A and dehydrotrametenolic acid. This study demonstrated that growth patterns could affect the quality of inner part and epidermis of M. cocos, and ATR-FTIR was a promising technique for the identification of wild and cultivated M. cocos and the rapid determination of triterpene acids contents.

Simultaneous quantitative analysis of four triterpenes in Poria Cocos from different habitats by QAMS / 中草药

Objective To establish a new method and validate its feasibilities by the simultaneous quantitative assay of four triterpenes in Poria cocos. Methods A new quality evaluation method of multi-components by single marker (QAMS) was established and validated for P. cocos. Pachymic acid (PA), dehydropachymic acid (DPA), dehydrotumulosic acid (DTA), and dehydrotrametenolic acid (DMA) were selected as analytes while pachymic acid was chosen as internal reference substance to evaluate the quality. The relative correction factor (RCF) of pachymic acid to the other three triterpenes were calculated. The method was evaluated by the comparison of quantity between external standard method and QAMS method. Results The contents of four triterpenes in 17 batches of P. cocos from QAMS method were not significantly different from those from external standard method. Conclusion The method with a single marker is accurate and feasible to determine PA, DPA, DTA, and DMA when some authentic standard substance are unavailable.

Dehydropachymic acid decreases bafilomycin A1 induced ß-Amyloid accumulation in PC12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuling, the sclerotium of Poria cocos, was frequently used in traditional Chinese medicine (TCM) formulae for Alzheimer's disease (AD) intervention over the past 10 centuries. And its extracts exhibited significant effects in both cellular and animal models of AD in previous studies. However, its mechanisms on prevention and treatment of AD have not been well elucidated yet. AIM OF THE STUDY: To investigate the effect and corresponding mechanisms of dehydropachymic acid, which is one of the major triterpenes in P. cocos, on the clearance of ß-amyloid accumulation in bafilomycin A1 induced PC12 cells. MATERIALS AND METHODS: MTT assay was used to examine the DPA effect on the viability of PC12 cells stable transfected with pCB6-APP (PC12-APP). PC12-APP cells were treated with DPA at the concentration of 6.25, 12.5, 25µg/mL for 4h, and then co-treated with 50nmol/L bafilomycin A1 for 48h except the controls. The Aß1-42 content in culture medium was determined by ELISA. The intracellular amount of APP, Aß1-42, LC3, cathepsin D was measured by Western blotting and normalized to GAPDH loading control. The PC12 cells stable transfected with pSelect-LC3-GFP (PC12-LC3-GFP) was used in the fluorescence microscopy estimation of autophagosomes accumulation. The internal pH in lysosome was detected by LysoTracker Red staining. RESULTS: DPA had no significant effect on the cell viability but could significantly decrease Aß1-42 content in culture medium and eliminate the intracellular accumulation of APP and Aß1-42 in bafilomycin A1 induced PC12-APP cells. Furthermore, DPA lowered the LC3-II/LC3-I ratio and reduced the GFP-labeled LC3 puncta which were elevated by bafilomycin A1. And the increase in internal pH of lysosome and decrease in mCatD amount in Bafilomycin A1 induced PC12-APP cells were restored by DPA treatment. These results indicated that DPA could restore the lysosomal acidification and recover the autophgic flux which is impaired by bafilomycin A1. CONCLUSIONS: DPA could effectively clear the accumulation of Aß1-42 in bafilomycin A1 impaired PC12 cells through restoring the lysosomal acidification and recovering the autophgic flux. And these results highlight its therapeutic potential for AD treatment.

UPLC fingerprint of triterpenoids in Guizhi Fuling Capsule / 中草药

Objective: To establish the UPLC fingerprint of triterpenoids in Guizhi Fuling Capsule (GFC) and give a new method for quality control. Methods: UPLC was used on an Agilent Zorbox Eclipse Plus HD C18 (100 mm × 2.1 mm, 1.8 μm) column with the gradient elution solvent system composed of acetonitrile-0.1% H3PO4 water solution as mobile phase, the flow rate was 0.2 mL/min, the column temperature was 30℃, and the detection wavelength was 210 nm. The common peaks were identified by Q-TOF/MS. Results: The UPLC fingerprints of triterpenoids in GFC were established. Totally 20 common peaks were selected as the fingerprint peaks of GFC, of which a total of 13 mutual peaks (1-7, 10, 12, and 14-17) from Poria, the peaks of number 8 and 11 were from Cinnamomi Ramulus, the peak of number 9 was from Paeoniae Radix Alba and Moutan Cortex, and the peak of number 13 was from Persicae Semen, Paeoniae Radix Alba, Moutan Cortex, and Cinnamomi Ramulus. The similarity among the fingerprint of 10 batches of GFC samples were 0.90. Fifteen chemical components were identified by UPLC-Q-TOF/MS, which were 1-hederagenin, 2-dehydrotumulosic acid, 3-tumulosil acid, 4-polyporenic acid C, 6-3-epidehydrotumulosic acid, 7-poricoic acid D, 9-α-linolenic acid, 10-dehydropachymic acid, 11-oleanolic acid, 12-pachymic acid, 13-linolic acid, 15-methylcis-9-hexadecenoate, 16-palmitic acid, 17-palmitic acid ethyl ester, and 18-daucosterol. Conclusion: The established UPLC fingerprint has desirable precision, reproducibility, and stability, and could be applied to the quality control of GFC.

Determination of four triterpenoid acids from Guizhi Fuling Capsules by HPLC / 中草药

Objective: To establish an HPLC method for the determination of four triterpene constituents (dehydrotumulosic acid, polyporenic acid C, 3-epi-dehydropachymic acid, and dehydropachymic acid) in Guizhi Fuling Capsules (GFC). Methods: Chromatography conditions were Diamonsic C18 column (250 mm × 4.6 mm, 5 μm), system mobile phase was composed of acetonitrile (A)-0.2% HCOOH aqueous solution (C) in a linear gradient elution mode (0-70 min: 50%-85% A; 70-80 min: 85%-100% A; 80-90 min: 100% A), detective wavelength was set at 242 nm, and column temperature was 40℃. Results: The calibration curve was linear within 0.408-2.04 μg/mL (r = 0.999 9), 0.192-0.96 μg/mL (r = 0.999 5), 0.078-0.39 μg/mL (r = 0.999 5), and 0.075 6-0.378 μg/mL (r = 0.999 5) for dehydrotumulosic acid, polyporenic acid C, 3-epi-dehydropachymic acid, and dehydropachymic acid, respectively. The average recoveries were 97.5% (RSD = 1.4%, n = 5), 98.5% (RSD = 1.6%, n = 5), 97.2% (RSD = 1.2%, n = 5), and 102.3% (RSD = 1.8%, n = 5). Six batches of GFC sample were determined, The average contents of dehydrotumulosic acid, polyporenic acid C, 3-epi-dehydropachymic acid, and dehydropachymic acid were 0.070, 0.015, 0.030, and 0.061 mg/capsule, separately. Conclusion: The method is simple, accurate, and can be used as a quality control method for GFC.

UPLC determination of determine dehydrotumulosic acid etc. six active components in poria / 中国药学杂志

OBJECTIVE: To establish an analytical method to determine dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid and pachymic acid in poria quickly and accurately. METHODS: An UPLC method was established on an HSS T3 Column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of water (containing 0.05% phosphoric acid) and acetonitrile in gradient elution mode, and the detection wavelength was set at 210 nm. RESULTS: The standard curves of dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid, and pachymic acid showed good linearity in 5.400-108.0, 2.040-40.80, 5.020-100.4, 2.120-42.40, 5.060-101.2 and 5.100-102.0 μg · mL-1, respectively and the average recoveries were 98.0% with RSD of 2. 9% for dehydrotumulosic acid, 99. 0% with RSD of 2.8% for tumulosic acid, 101.5% with RSD of 2.5% for polyporenic acid C, 97.1% with RSD of 2.7% for 3-epi-dehydrotumulosic acid, 101.5% with RSD of 2.1% for dehydropachymic acid and 99.6% with RSD of 1.1% for pachymic acid, respectively. CONCLUSION: The method is quick, accurate, and can be used to determine multiple triterpenoid acids in Poria simultaneously.