The modern scientific mystery of traditional Chinese medicine processing--take some common traditional Chinese medicine as examples.

The processing of traditional Chinese medicine (TCM) is a unique traditional pharmaceutical technology in China, which is the most important feature that distinguishes Chinese medicine from natural medicine and plant medicine. Since the record in Huangdi Neijing (Inner Canon of the Yellow Emperor), till now, the processing of TCM has experienced more than 2000 years of inheritance, innovation, and development, which is a combination of TCM theory and clinical practice, and plays an extremely important position in the field of TCM. In recent years, as a clinical prescription of TCM, Chinese herbal pieces have played a significant role in the prevention and control of the COVID-19 and exhibited their unique value, and therefore they have become the highlight of China's clinical treatment protocol and provided Chinese experience and wisdom for the international community in the prevention and control of the COVID-19 epidemic. This paper outlines the research progress in the processing of representative TCM in recent years, reviews the mechanism of the related effects of TCM materials after processing, such as changing the drug efficacy and reducing the toxicity, puts forward the integration and application of a variety of new technologies and methods, so as to reveal the modern scientific mystery of the processing technology of TCM.

16S rDNA sequencing combined with metabolomics profiling with multi-index scoring method reveals the mechanism of salt-processed Semen Cuscuta in Bushen Antai mixture on kidney yang deficiency syndrome.

Kidney yang deficiency syndrome (KYDS) is a classic syndrome of traditional Chinese medicine (TCM). The salt-processed product of Semen Cuscuta (YP) is the monarch drug in Bushen Antai Mixture (BAM), can improve the reproductive dysfunction caused by KYDS, and the effect is better than that of raw products of Semen Cuscuta (SP). However, its mechanism is not completely clear yet. In this study, an integrated strategy combining untargeted metabolomics with microbiology was used to explore the mechanism of YP in the BAM improving KYDS. 16S rDNA gene sequencing showed that BAM containing YP (Y-BAM) had a significantly better regulatory effect on Desulfobacterota and Desulfovibrionaceae_unclassified than BAM containing SP (S-BAM). Untargeted metabolomics studies showed that Y-BAM significantly regulated 4 metabolites and 4 metabolic pathways. In addition, multi-index analysis showed that the effect of Y-BAM on arachidonic acid metabolism, tyrosine metabolism, purine metabolism, fructose and mannose metabolism and total metabolism was closer to that of the control group compared to S-BAM. The analysis of serum biochemical indexes showed that Y-BAM had more significant regulating effect on the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and superoxide dismutase (SOD) in serum of KYDS rats compared to S-BAM. Spearman correlation analysis showed that there was a significant correlation between intestinal microorganisms and metabolites and serum biochemical indexes. For example, Desulfovibrionaceae_unclassified was positively correlated with arachidonic acid, and negatively correlated with SOD and LH. This study suggests that YP may enhance the regulation of intestinal flora and endogenous metabolism of KYDS, so that BAM shows a better therapeutic effect on KYDS, which also reasonably explains why BAM uses Semen Cuscuta stir-baked with salt solution.

A UPLC-Q-TOF-MS-Based Metabolomics Approach to Screen out Active Components in Prepared Rhubarb for Its Activity on Noxious Heat Blood Stasis Syndrome.

Background: Prepared rhubarb was obtained by steaming raw rhubarb with wine. Different from raw rhubarb with a purgative effect, prepared rhubarb shows effects of promoting blood circulation and removing blood stasis. However, the mechanisms of its action through regulating endogenous metabolites remain unclear. Purpose: The purpose of this study was to explore active chemical components in prepared rhubarb for its activity on noxious heat blood stasis syndrome (NHBS) by comprehensive metabolomics profiling. Study design: Plant extracts usually show their activities in a synergistic way; therefore, integrated omics was developed as a rational way for a better understanding of their biological effects and potential active compounds. Methods: The activities of prepared rhubarb were evaluated by biochemical and metabolomic analysis; meanwhile, serum chemical profiles were sought using UHPLC-Q-TOF-MS. Gray correlation analysis (GCA) was used for calculating the underlying correlations between them. Results: The metabolomics profiles of rat plasma from model and control groups were significantly different, with 31 endogenous metabolites changed by NHBS. Then, after the administration of prepared rhubarb, 18 of them were regulated. Multiple metabolic pathways were disturbed after NHBS modeling and restored by prepared rhubarb, among which had a greater impact on sphingolipid metabolism. A total of 28 compounds from prepared rhubarb absorbed into the plasma were identified, including nine prototypes and 19 metabolites. Statistical results suggested that rhein and its metabolites accounted for half of the top 10 active compounds in prepared rhubarb for its biomedical activities. Conclusion: This study presented evidence for the therapeutic effects and active chemicals of prepared rhubarb on NHBS in the way of metabolomics.

LABEL-FREE BIO-AFFINITY MASS SPECTROMETRY FOR SCREENING AND LOCATING BIOACTIVE MOLECULES.

Despite the recent increase in the development of bioactive molecules in the drug industry, the enormous chemical space and lack of productivity are still important issues. Additional alternative approaches to screen and locate bioactive molecules are urgently needed. Label-free bio-affinity mass spectrometry (BA-MS) provides opportunities for the discovery and development of innovative drugs. This review provides a comprehensive portrayal of BA-MS techniques and of their applications in screening and locating bioactive molecules. After introducing the basic principles, alongside some application notes, the current state-of-the-art of BA-MS-assisted drug discovery is discussed, including native MS, size-exclusion chromatography-MS, ultrafiltration-MS, solid-phase micro-extraction-MS, and cell membrane chromatography-MS. Finally, several challenges and limitations of the current methods are summarized, with a view to potential future directions for BA-MS-assisted drug discovery. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Development of liquiritigenin-phospholipid complex with the enhanced oral bioavailability.

In the present study, liquiritigenin-phospholipid complex (LPC) was developed and evaluated to increase the oral bioavailability of liquiritigenin. A single-factor test methodology was applied to optimize the formulation and process for preparing LPC. The effects of solvent, drug concentration, reaction time, temperature and drug-to-phospholipid ratio on encapsulation efficiency were investigated. LPCs were characterized by UV-visible spectroscopy, differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), and powder X-ray diffractometry (PXRD). The apparent solubility and n-octanol/water partition coefficient were tested. The pharmacokinetic characteristics and bioavailability of the LPC were investigated after oral administration in rats in comparison with liquiritigenin alone. An LPC was successfully prepared. The optimum level of various parameters for liquiritigenin-phospholipid complex was obtained at the drug concentration of 8 mg·mL-1, reaction time for 15 min, reaction temperature of 30 ℃, a ratio of 1∶4.5 (W/W) drug-to-phospholipid and anhydrous ethanol as reaction solvent. Compared to liquiritigenin, the AUC0-t of the LPC was increased by 239%. The liquiritigenin-phospholipid complex significantly increase the lipid solubility and bioavailability of liquiritigenin, suggesting that it is an effective formulation for further development and clinical applications.

Development of an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method for comparative pharmacokinetics of six triterpenoids in rat plasma and application to different forms of Phytolacca acinosa.

Phytolacca acinosa is an herb for treatment of ascites and tumor. Two forms of P. acinosa, i.e. raw and vinegar-processed herb, have been used in clinic. However, pharmacokinetic difference between the two forms of P. acinosa has not been fully understood. Herein, a comparative pharmacokinetic method based on liquid chromatography with tandem mass spectrometry was developed for quantification of six bioactive triterpenoids, including esculentoside H, esculentoside T, esculentoside A, esculentoside B, phytolaccagenic acid, and phytolaccagenin in rat plasma after oral administration of different forms of P. acinosa. Separation was performed on an Acquity BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The method was validated over a linear range of 2.0-5000 ng/mL. Intraday and interday bias were within ±5%. Besides, all triterpenoids were stable in plasma during different storage conditions. The described method was successfully applied to a comparative pharmacokinetic study of raw and vinegar-processed P. acinosa in rats. Notably, double peak phenomenon for six triterpenoids of P. acinosa was observed for the first time. AUC0→t and Cmax values of esculentoside H, esculentoside T, phytolaccagenic acid, and phytolaccagenin were significantly lower in vinegar-processed group than that of raw group, indicating the oral bioavailability of the four triterpenoids was decreased after vinegar processing.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar-processed Corydalis turtschaninovii.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar-processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra-day and inter-day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar-processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar-processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar-processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.

A biochemometrics strategy for tracing diuretic components of crude and processed Alisma orientale based on quantitative determination and pharmacological evaluation.

We proposed a biochemometrics strategy for tracing diuretic components of herbs based on quantitative determination and pharmacological evaluation. First, a sensitive and robust liquid chromatography coupled with tandem mass spectrometry approach was established for simultaneous quantification of six major triterpenoids in crude and salt-processed Alisma orientale. The separation of triterpenoids was achieved on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid. Six major triterpenoids were detected by multiple reaction monitoring in the negative ion mode. Glycyrrhetinic acid was used as the internal standard. The approach showed good linearity. Intra- and inter-day precisions were all within 2.9%. The recovery rates of each triterpenoid ranged from 97.9% to 103.2%. The approach was then successfully employed for quantitative analysis of six triterpenoids in ten batches of crude and salt-processed A. orientale. Second, the diuretic effects of crude and salt-processed A. orientale were evaluated in mice. Third, principal component analysis and canonical correlation analysis were used to uncover the relationship between the contents of six major triterpenoids and the diuretic effect of different crude and salt-processed samples. Alisol B, alisol F, and alisol A have a close positive correlation with the diuretic effect.

Comparative Study on Pharmacokinetics of Four Active Compounds in Rat Plasma after Oral Administration of Raw and Wine Processed Chuanxiong Rhizoma.

In Chinese medicine, the effect of promoting blood circulation and removing stasis could be enhanced after Chuanxiong Rhizoma is processed by wine. However, the relevant mechanism remains unclear. In this manuscript, a rapid and sensitive quantification method employing ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and validated to simultaneously determine butylidenephthalide, ligustilide, senkyunolide A and ferulic acid in rat plasma after oral administration of raw Chuanxiong Rhizoma (RCR) and wine-processed Chuanxiong Rhizoma (WCR) respectively. All analytes were extracted from plasma by proteins precipitation with methanol. Chromatographic separation was carried out on a Hypersil GOLD C18 column by using a gradient mobile phase system of acetonitrile and water with 0.01% formic acid, the flow rate was 0.3 mL/min. For exact mass detecting, quick switching mode was used, positive and negative ions could be detected in one injection. The pharmacokinetic profiles of four components in the two groups were evaluated and compared. The results showed that, compared to the RCR group, the Vd and AUC0→t values of four active compounds were increased and decreased respectively in WCR group, which revealed the effect of wine processing to Chuanxiong Rhizoma: the stronger the effect, the wider the distribution.

Screening for Potential Active Components of Fangji Huangqi Tang on the Treatment of Nephrotic Syndrome by Using Integrated Metabolomics Based on "Correlations Between Chemical and Metabolic Profiles".

As for traditional Chinese medicine (TCM) prescription, what puzzled researchers most was how to select proper chemical markers to represent the whole pharmacological action system. In this paper, an integrated metabolomic method was presented for a systematic discovery of potential active components in Fangji Huangqi Tang (FHT), a well-known TCM prescription for nephrotic syndrome treatment, based on "correlations between chemical and metabolic profiles." Firstly, a metabolomics study was carried out to select representative biomarkers of nephrotic syndrome. Then, after drug administration, the dynamic process of serum composition was investigated by the ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) technique to detect the prototypes and related metabolites of relative components from FHT. Pearson correlation analysis was finally used to find out the correlations between the endogenous metabolic spectrums and the chemical serum spectrums. As a result, 17 biomarkers for nephrotic syndrome indication were identified, and the main metabolic pathways of their concern included linoleic acid metabolism; cyanoamino acid metabolism; alpha-linolenic acid metabolism; glycine, serine, and threonine metabolism; arachidonic acid metabolism; and glycerophospholipid metabolism. Meanwhile, active components in FHT for nephrotic syndrome treatment were screened out, including (+)-tetrandrine demethylation, fenfangjine G hydrogenation, tetrandrine, N-methylfangchinoline, tetrandrine demethylation, fangchinoline, glycyrrhetic acid, astragaloside II alcohol dehydration, atractylenolide III demethylation + hydrogenation, atractylenolide III demethylation + hydrogenation, and licoricone-N-acetylcysteine conjugation. This study demonstrated a promising way to elucidate the active chemical material basis of TCM prescription.

Pharmacokinetic study of six triterpenoids of raw and processed Alisma plantago-aquatica in rat plasma by using ultra performance liquid chromatography-tandem mass spectrometry approach.

Alisma plantago-aquatica is known to regulate water and fluid balance in cells, and is under testing for the therapy for patients suffering from chronic nephritis. Herein, a UHPLC-MS/MS method was established and validated for the determination of six bioactive triterpenoids of raw and salt-processed Alisma plantago-aquatica in rat plasma. The acquired plasma was subjected to protein precipitation with acetonitrile. Glycyrrhetinic acid was employed as internal standard. The pretreated samples were separated on a reversed phased column with a mobile phase of acetonitrile and water (including 0.1% formic acid). The MRM mode for the six triterpenoids were at m/z 535.4 → 489.4 for alisol A, m/z 517.3 → 471.4 for alisol B, m/z 533.3 → 487.3 for alisol F, m/z 577.4 → 531.4 for alisol A-24-acetate, m/z 559.4 → 495.4 for alisol B-23-acetate, m/z 573.3 → 509.3 for alisol C-23-acetate, and 469.3 → 425.3 for the IS. The accuracy and precision of the method were determined as -2.2%-3.6% and 0.8%-3.0%, respectively. This approach was employed to a pharmacokinetic study of the six bioactive triterpenoids after intragastric administration of raw and processed Alisma plantago-aquatica in rats. The two-phasic pharmacokinetic of alisol B, alisol C-23-acetate and alisol F were reported for the first time, which may be ascribed to enterohepatic recirculation of these triterpenoids.

Development of an UHPLC-MS/MS method for comparative pharmacokinetics of nine anthraquinones in rats and application to dosage conversion between different Semen Cassiae forms.

Semen Cassiae, called Juemingzi in Chinese, is widely used in clinic for alleviating constipation, improving eyesight and preventing hyperlipidemia. It can be used as medicine or food including many application forms, such as traditional pieces and ultrafine granular powder (UGP). In this paper, comparative pharmacokinetics of Semen Cassiae in different forms of traditional pieces and UGP were achieved to research the clinical dosage of UGP. Also, the scientific connotation of brewing way for traditional pieces of Semen Cassiae application in clinic was revealed. To achieve this purpose, a rapid, sensitive and reliable UHPLC-MS/MS method was developed for simultaneous determination of rhein, emodin, aloe-emodin, chrysophanol, physcion, aurantio-obtusin, chryso-obtusin, obtusifolin and obtusin in rat plasma. Multiple reaction monitoring mode via an electrospray ionization was applied for the quantitation of the analytes. The separation was carried out on an Agilent Extend-C18 column (100 mm × 2.1 mm, 1.8 µm) with an 8.0 min gradient elution using ultra-purify water and acetonitrile as mobile phase. The samples were prepared by liquid-liquid extraction with ethyl acetate. The development and validation of bioanalytical method were performed according to the latest "Bioanalytical Method Validation: Guidance for Industry" issued by FDA in 2018. Finally, the clinical dosage of UGP was concluded to be 1/4 of Semen Cassiae traditional pieces in oral administration way by comparing the pharmacokinetic parameters of UGP to that of traditional pieces in the aspect of mathematical statics using plus of AUC values.

Establishment of a rapid and sensitive UPLC-MS/MS method for pharmacokinetic determination of nine alkaloids of crude and processed Corydalis turtschaninovii Besser aqueous extracts in rat plasma.

Corydalis turtschaninovii (CT) is a traditional Chinese medicine which is known to have analgesic effects, and is under investigation for the management of chronic pain. Our study aims to establish a UPLC-MS/MS method for pharmacokinetic determination of nine bioactive alkaloids of raw and processed CT in rat plasma. Nitidine chloride was selected as internal standard. After protein precipitation with methanol, the plasma samples were separated on a reversed phased column with a mobile phase of acetonitrile and water (including 0.1% formic acid). The MRM parameters were optimized as follows: m/z 354.0 → 188.1 for protopine, m/z 321.0 → 293.1 for coptisine, m/z 371.1 → 189.1 for allocryptopine, m/z 357.1 → 193.3 for tetrahydropalmatine, m/z 324.1 → 176.1 for tetrahydrocoptisine, m/z 340.1 → 176.2 for tetrahydroberberine, m/z 368.1 → 289.1 for corynoline, m/z 370.5 → 192.1 for corydaline, m/z 367.2 → 351.2 for dehydrocorydaline, and m/z 406.0 → 300.3 for the IS. The linearity, accuracy, precision, stability, recovery and matrix effect of the method were well validated. This method was successfully employed for a pharmacokinetic study of raw and vinegar-processed CT in rats. The absorption of the nine alkaloids was accomplished in an hour. The double peak phenomenon of the nine alkaloids may be ascribed to enterohepatic recirculation. Compared with the raw group, AUC0→t and Cmax of the nine alkaloids were significantly elevated in the vinegar-processed group. Our findings suggest that vinegar-processing could increase the bioavailability of the nine alkaloids of CT in rats. The pharmacokinetic information obtained will provide basis for application of processed CT in future clinical therapy.

Nine components pharmacokinetic study of rat plasma after oral administration raw and prepared Semen Cassiae in normal and acute liver injury rats.

In China, Semen Cassiae has long been used to protect liver, brighten eyes, and relieve constipation. Prepared Semen Cassiae is produced from raw Semen Cassiae by processing, the two forms of Semen Cassiae have different clinical applications. Pathological state is an important factor affecting the efficacy of drugs, the pharmacokinetic behavior of drugs could be significantly changed when people or animal were under different pathological state. To clarify the effect of processing mechanism and pathological state for pharmacokinetic behavior, the pharmacokinetics of nine components of raw and prepared Semen Cassiae under normal and acute liver injury rats were examined. The results showed that the bimodal phenomenon appeared on the plasma concentration-time profiles of obtusin, emodin, chrysophanol, aloe emodin and rhein. The Tmax of aurantio-obtusin, obtusin, chrysoobtusin, emodin, chrysophanol, aloe emodin, physcion in normal groups administrated prepared Semen Cassiae were shorter than those administrated raw Semen Cassiae. For the AUC0-t , aurantio-obtusin, obtusin, chrysoobtusin, chrysophanol, aloe emodin and physcione in model groups administrated prepared Semen Cassiae were significantly higher than other groups, unlike above components, rhein had poor absorption in model groups. The study would be useful for further studies on pharmacokinetics and clinical application of raw and prepared Semen Cassiae.

Determination of major components from Radix Achyranthes bidentate using ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry and an evaluation of their anti-osteoporosis effect in vitro.

Ecdysterone and saponins are the most characteristic components of Radix Achyranthes bidentate, which acts on the human body to promote collagen synthesis and stimulates cell growth. However, the relationship between these components and the differentiation of MC3T3-E1 osteoblastic cells is unknown. We developed a rapid ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry method for direct determination of one ecdysterone and four saponins in crude and salt-processed Radix Achyranthes bidentate. The method was interrogated in terms of linearity, intra- and inter-day precision, repeatability, stability and recovery. The method was linear within the concentration ranges of 0.003-336 µg/mL for ß-ecdysterone, 0.0035-130 µg/mL for 25S-inokosterone, 0.004-423 µg/mL for ginsenoside Ro, 0.0036-66 µg/mL for chikusetsusaponin IV and 0.0044-111 µg/mL for chikusetsusaponin IVa. The intra- and inter-day precisions were all within 2.7%. The standard addition method determined recovery rates for each component (98.7-102.5%). The method was successfully applied to simultaneously quantify five components in ten batches of crude and salt-processed Radix Achyranthes bidentate. Subsequently, the examination of these extracts on the differentiation of MC3T3-E1 osteoblastic cells were carried out. Finally, the relationships between the contents of five components and their anti-osteoporosis effect were investigated by using canonical correlation analysis.

A reliable LC-MS/MS method for the quantification of five bioactive saponins of crude and processed Bupleurum scorzonerifolium in rat plasma and its application to a pharmacokinetic study.

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) method was developed for simultaneous determination of saikosaponin A, saikosaponin B1 , saikosaponin C, saikosaponin D and saikosaponin F in rat plasma using glycyrrhetinic acid as an internal standard (IS). The separation was operated on a Waters BEH C18 column. The mobile phases of gradient elution consisted of acetonitrile (A) and 0.1% aqueous acetic acid (B). The mass spectrometric detection was accomplished in multiple reaction monitoring mode. The five saponins displayed good linearity (r2 > 0.9996). The lower limits of quantitation of saikosaponin A, saikosaponin B1 , saikosaponin C, saikosaponin D and saikosaponin F were determined to be 2.9, 2.3, 3.5, 2.9 and 3.1 ng/mL, respectively. Moreover, the intra- and inter-day precisions of the five saponins showed an RSD within 2.96%, whereas the accuracy (RE) ranged from -2.28 to 2.78%. Finally, the developed method was fully validated and applied to a comparative pharmacokinetic study of the five bioactive saponins in rats following oral administration of crude and vinegar-processed Bupleurum scorzonerifolium.

Simultaneous Quantitation of Five Bioactive Ingredients in Raw and Processed Fallopia multiflora by Employing UHPLC-Q-TOF-MS.

Fallopia multiflora is used for treatment of premature graying hair and blood deficiency. In this study, a quantitative method was developed for determination of five bioactive components (emodin, 2,3,5,4'-tetrahydroxy-stilbene- 2-Ο-ß-d-glucoside, emodin-8-O-ß-d-glucopyranoside, ω-hydroxyemodin and kaempferol) in raw and processed F. multiflora by using ultra-high performance liquid chromatography (UHPLC)-quadrupole time-of-flight mass spectrometry-based method. The sample handling procedure was optimized. Chromatographic separation was carried out on a Thermo Syncronis AQ-C18 UHPLC column with mobile phase consisting of 0.01% aqueous formic acid and acetonitrile. The method was interrogated in terms of linearity, precision, stability and recovery tests. All calibration curves displayed good linearity (R2 > 0.9992). The limit of detection and limit of quantification of these components ranged from 0.01 to 0.03 µg/mL and from 0.03 to 0.07 µg/mL, respectively. The average recoveries of these components were from 98.2 to 102.9% with relative standard deviation values from 0.8 to 2.9% for F. multiflora. The developed method can be applied to quality control of raw and processed F. multiflora.

A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols in rat plasma: Application to a pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs.

This study established a rapid and reliable approach using liquid chromatography-tandem mass spectrometry for the simultaneous determination of cinnamic acid, vanillic acid and protocatechuic acid in rat plasma. This is the first report on a comparative pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs in rats. After liquid-liquid extraction by ethyl acetate, the plasma samples were subjected to LC-MS/MS for multiple reaction monitoring. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.0-16000 ng/mL. The intra- and inter-day accuracy and precision were found to be within 15% of the nominal concentration. The recoveries of the three phenolics ranged from 88.7 to 105.7%. Finally, this approach was successfully applied to pharmacokinetic analysis of the three phenolics after oral administration of standard decoction and dispensing granules of C. cassia twigs in rats. Although the values of AUC0-t of vanillic acid and protocatechuic acid in standard decoction group were larger than those of the dispensing granule group, no significant difference was observed for the two groups. Of note, the elimination rates of vanillic acid were slower in the standard decoction group than the dispensing granule group.

Integrated metallomic and metabolomic profiling of plasma and tissues provides deep insights into the protective effect of raw and salt-processed Achyranthes bidentata Blume extract in ovariectomia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Achyranthes bidentata Blume (AB) is a well-known traditional Chinese medicine for treating osteoporosis and bone fracture. In the current, researches on pharmacological mechanism of AB mostly focused on molecular pathways, knowledge about its metabolic signatures is largely unclear. This study aims to develop an integrative metabolomics and metallomic approach for deciphering the biochemical basis of anti-osteoporosis effects of raw and salt-processed AB. METHOD: Gas chromatography-mass spectrometry (GC-MS) and inductively coupled plasma mass spectrometry (ICP-MS) were combined for metabolomic and metallomic profiling of rats serum, liver and kidney derived from the sham group, model group, E2, raw and salt-processed AB treated groups. Meanwhile, micro-CT and biomechanical analysis were carried out to ensure the success of the osteoporosis model and to validate the anti-osteoporosis effect of raw and salt-processed AB. Partial least squares discriminant analysis (PLS-DA) was employed to screen potential biomarkers and the MetaboAnalyst and KEGG PATHWAY Database were used to investigate the metabolic pathway. RESULTS: Raw and salt-processed AB protected the rats against osteoporosis, as evidenced by the restoration of the alkaline phosphatase activity, osteocalcin concentration, urine calcium/creatinine ratio and urine phosphorus/creatinine ratio. The combination of PCA and PLS-DA revealed deviations in ninety-four differential biomarkers between raw AB treated group and model group. The identified biomarkers were primarily engaged in the metabolic pathways including galactose metabolism, urea cycle, arginine and proline metabolism, alanine metabolism, lactose degradation, ammonia recycling and glycine and serine metabolism. The levels of these biomarkers showed significant alterations and a tendency to be restored to normal values in raw and salt-processed AB treated osteoporosis rats. Of note, the levels of trace elements, such as Zn, Se, Mn, Cu and Fe, were elevated after raw and salt-processed AB treatment. Finally, a correlation network diagram was constructed to show the biomarkers perturbed by raw and salt-processed AB. CONCLUSION: Our findings indicate that raw and salt-processed AB has positive effects on osteoporosis rats. Meanwhile, metabolomic and metallomic method coupled with metabolites enrichment analysis and pattern recognition serves as a useful tool for revealing the action mechanism of traditional Chinese medicine.

A liquid chromatography-tandem mass spectrometry approach for study the tissue distributions of five components of crude and salt-processed Radix Achyranthes in rats.

This study developed a robust and reliable approach using liquid chromatography- tandem mass spectrometry for the simultaneous determination of five saponins in rat tissues: ß-ecdysterone, chikusetsusaponin IV, ginsenoside Ro, 25S-inokosterone and chikusetsusaponin IVa. This is the first report on a comparative tissue distribution study of crude and salt-processed Radix Achyranthes in rats. After one-step protein precipitation by acetonitrile, the tissue samples were sent to LC-MS/MS for multiple reaction monitoring. The retention times of the five saponins and internal standard were 1.77, 3.14, 3.01, 1.83, 3.26 and 4.77 min. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.3-1562.5 ng/mL. The intra- and inter-day accuracy and precision were within 15% of the nominal concentration. The recoveries of the five saponins were 92.0-99.9%. Finally, this approach was successfully applied to tissue distribution analysis of the five saponins after oral administration of crude and salt-processed Radix Achyranthes in rats. The largest concentration of the five saponins was observed in kidney after salt-processing, which indicated that processing could enhance the bioavailability.