Network pharmacology analysis and experimental verification of the antithrombotic active compounds of trichosanthis pericarpium (Gualoupi) in treating coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichosanthis pericarpium (TP; Gualoupi, pericarps of Trichosanthes kirilowii Maxim) has been used in traditional Chinese medicine (TCM) to reduce heat, resolve phlegm, promote Qi, and clear chest congestion. It is also an essential herbal ingredient in the "Gualou Xiebai" formula first recorded by Zhang Zhongjing (from the Eastern Han Dynasty) in the famous TCM classic "Jin-Guì-Yào-Lüe" for treating chest impediments. According to its traditional description, Gualou Xiebai is indicated for symptoms of chest impediments, which correspond to coronary heart diseases (CHD). AIM OF THE STUDY: This study aimed to identify the antithrombotic compounds in Gualoupi for the treatment of CHD. MATERIALS AND METHODS: A CHD rat model was established with a combination of high-fat diet and isoproterenol hydrochloride (ISO) administration via subcutaneous multi-point injection in the back of the neck. This model was used to evaluate the antithrombotic effect of two mainstream cultivars of TP ("HaiShi GuaLou" and "WanLou") by analyzing the main components and their effects. Network pharmacology, molecular docking-based studies, and a zebrafish (Danio rerio) thrombosis model induced by phenylhydrazine was used to validate the antithrombosis components of TP. RESULTS: TP significantly reduced the body weight of the CHD rats, improved myocardial ischemia, and reduced collagen deposition and fibrosis around the infarcted tissue. It reduced thrombosis in a dose-dependent manner and significantly reduced inflammation and oxidative stress damage. Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as candidate active TP compounds with antithrombotic effects. The key potential targets of TP in thrombosis treatment were initially identified by molecular docking-based analysis, which showed that the candidate active compounds have a strong binding affinity to the potential targets (protein kinase C alpha type [PKCα], protein kinase C beta type [PKCß], von Willebrand factor [vWF], and prostaglandin-endoperoxide synthase 1 [PTGS1], fibrinogen alpha [Fga], fibrinogen beta [Fgb], fibrinogen gamma [Fgg], coagulation factor II [F2], and coagulation factor VII [F7]). In addition, the candidate active compounds reduced thrombosis, improved oxidative stress damage, and down-regulated the expression of thrombosis-related genes (PKCα, PKCß, vWF, PTGS1, Fga, Fgb, Fgg, F2, and F7) in the zebrafish model. CONCLUSION: Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as the active antithrombotic compounds of TP used to treat CHD. Mechanistically, the active compounds were found to be involved in oxidative stress injury, platelet activation pathway, and complement and coagulation cascade pathways.

Trichosanates A-G and cucurbitacins W-Y, anticomplement monoterpenoids and cucurbitane-type triterpenoids from the pericarps of Trichosanthes kirilowii.

The pericarps of Trichosanthes kirilowii are often used to treat cough in traditional Chinese medicine, and its ethanol extract exhibited effective therapeutic effects on acute lung injury (ALI) in vivo caused by H1N1. An anticomplement activity-guided fractionation on the extract resulted in the isolation of ten new terpenoids, including seven monoterpenoids, trichosanates A-G (1-7), and three cucurbitane-type triterpenoids, cucurbitacins W-Y (8-10), as well as eleven known terpenoids (11-21). The new terpenoids' structures were determined by spectroscopic analysis, X-ray crystallographic analysis (1), electronic circular dichroism (ECD) analysis and calculations (2-10). Twelve monoterpenoids (1-7 and 11-15) and five cucurbitane-type triterpenoids (8-10, 18, and 20) exhibited anticomplement activity in vitro. For the monoterpenoids, the long aliphatic chain substituents might enhance their anticomplement activity. Additionally, two representative anticomplement terpenoids, 8 and 11, obviously attenuated H1N1-induced ALI in vivo by inhibiting complement overactivation and reducing inflammatory responses.

The Ethanolic Extract of Trichosanthes Kirilowii Root Exerts anti-Cancer Effects in Human Non-Small Cell Lung Cancer Cells Resistant to EGFR TKI.

The aim of this study was to investigate whether the ethanol extract of the Trichosanthes kirilowii root (ETK), traditionally used to treat lung diseases, exhibits anticancer activity in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells. ETK treatment suppressed the growth of EGFR TKI-resistant NSCLC cells, including H1299, H1975, PC9/ER (erlotinib-resistant PC9) and PC9/GR (gefitinib-resistant PC9) cells, in a concentration- and time-dependent manner. Dose-dependent decline in anchorage-dependent and -independent colony formation was also detected following ETK treatment. We demonstrate that the growth-inhibitory effect of ETK was related to apoptosis induction, based on flow cytometry results showing ETK-induced increase in the percentage of cells with sub-G1 DNA and the population of annexin V-positive cells. Consistently, ETK induced chromatin condensation and cleavage of poly(ADP-ribose) polymerase (PARP). As a molecular mechanism, the phosphorylation level of signal transducer and activator of transcription 3 (STAT3) and Src was decreased by ETK. ETK-induced apoptosis was partially reversed by transfection of constitutively activated STAT3, indicating that STAT3 inactivation mediated ETK-induced apoptosis in EGFR TKI-resistant NSCLC cells. Our results provide basic evidence supporting the role of ETK as a novel therapeutic in EGFR TKI-resistant NSCLC.

Effect of microwave heating on lipid composition, chemical properties and antioxidant activity of oils from Trichosanthes kirilowii seed.

Trichosanthes kirilowii Maxim seed is a primary source of edible vegetable oil and possesses a high nutritional value, making them extremely beneficial to humanity. To promote the extraction process of Trichosanthes kirilowii Maxim seed oil, the effect of microwave heating time (700 W for 0, 2, 4, and 6 min) on lipid composition, chemical properties, and antioxidant activity of oils was studied. The results showed that the oil yield of the seed increased with the microwave heating time. Besides, microwave heating time significantly affects (p < 0.05) DPPH and tocopherols, and the IC50 value of DPPH was highest with microwave heating for 6 min, whatever the shells are reserved. The tocopherol content was highest with microwave heating for 2 min in the seed shell oil, which was 1930.60 mg/kg. The longer microwave heating time could improve the oil yield and antioxidant activity of Trichosanthes kirilowii Maxim seed oil. The seed shell also affects chemical properties, fatty acid composition, antioxidant activity, and tocopherol contents of the Trichosanthes kirilowii Maxim seed oil. The Trichosanthes kirilowii Maxim seed shell oil has higher DPPH and tocopherols contents than seed kernel oil, while seed kernel oils showed higher oil yield and acid value. Our finding is valuable for manufacturers to choose suitable means to produce Trichosanthes kirilowii Maxim seed oil of required qualities and chemical compositions for targeted use.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

[Screening of effective antioxidant components from Trichosanthes extract and assessment of their antioxidant activity].

OBJECTIVE: To screen the effective antioxidant components in Trichosanthes extract based on the mean value of Deng's correlation degree and assess the antioxidant activity of the identified components. METHOD: High-performance liquid chromatography (HPLC) was used to obtain the fingerprints of Trichosanthes extract, and the clearance rates of DPPH · and O2-· by 3, 9 and 27 mg/mL Trichosanthes extract were determined. The antioxidant spectrum effect of Trichosanthes extract was analyzed by calculating the mean value of Deng's correlation degree to screen the effective antioxidant component group. According to the contents of each known components in the antioxidant effective component group, mixed solutions of the components were prepared and tested for their clearance rates of DPPH · and O2-·. RESULTS: The 36 common peaks in HPLC fingerprints of Trichosanthes extract showed different degrees of correlation with DPPH · and O2-· clearance. The common peaks with a correlation degree greater than the median value included peaks 21, 36, 8, 31, 14, 5, 27, 2, 24, 15, 18, 33, 22, 34, 35, 19, 28 and 25. The 5 components, namely kaempferol (peak 36), isoquercitrin (peak 8), luteolin (peak 31), rutin (peak 5) and apigenin (peak 35), were tentatively identified to constitute the effective antioxidant component group with a mass ratio 3∶2∶2∶ 1∶1 in Trichosanthes extract. The prepared mixed solutions of antioxidant effective component group (6.12, 2.04, and 0.68 µg/mL) showed clearance rates of DPPH · of 65.4%, 64.0% and 61.0%, and clearance rates of O2-· of 12.9%, 9.5% and 8.3%, respectively. CONCLUSION: We identified the material basis for the antioxidant activity of Trichosanthes and screened the antioxidant effective component group in Trichosanthes extract.

Screening of effective antioxidant components from Trichosanthes extract and assessment of their antioxidant activity / 南方医科大学学报

OBJECTIVE@#To screen the effective antioxidant components in Trichosanthes extract based on the mean value of Deng's correlation degree and assess the antioxidant activity of the identified components.@*METHOD@#High-performance liquid chromatography (HPLC) was used to obtain the fingerprints of Trichosanthes extract, and the clearance rates of DPPH · and O2-· by 3, 9 and 27 mg/mL Trichosanthes extract were determined. The antioxidant spectrum effect of Trichosanthes extract was analyzed by calculating the mean value of Deng's correlation degree to screen the effective antioxidant component group. According to the contents of each known components in the antioxidant effective component group, mixed solutions of the components were prepared and tested for their clearance rates of DPPH · and O2-·.@*RESULTS@#The 36 common peaks in HPLC fingerprints of Trichosanthes extract showed different degrees of correlation with DPPH · and O2-· clearance. The common peaks with a correlation degree greater than the median value included peaks 21, 36, 8, 31, 14, 5, 27, 2, 24, 15, 18, 33, 22, 34, 35, 19, 28 and 25. The 5 components, namely kaempferol (peak 36), isoquercitrin (peak 8), luteolin (peak 31), rutin (peak 5) and apigenin (peak 35), were tentatively identified to constitute the effective antioxidant component group with a mass ratio 3∶2∶2∶ 1∶1 in Trichosanthes extract. The prepared mixed solutions of antioxidant effective component group (6.12, 2.04, and 0.68 μg/mL) showed clearance rates of DPPH · of 65.4%, 64.0% and 61.0%, and clearance rates of O2-· of 12.9%, 9.5% and 8.3%, respectively.@*CONCLUSION@#We identified the material basis for the antioxidant activity of Trichosanthes and screened the antioxidant effective component group in Trichosanthes extract.

The immune-enhancing effects of a mixture of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) or its active constituent nodakenin.

ETHNOPHARMACOLOGICAL RELEVANCE: A mixture (SH003) of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) has beneficial effects against several carcinomas. There have been few reports on an immune-enhancing activity of SH003 and its active constituent nodakenin. AIM OF THE STUDY: This study aimed at identifying the immune-enhancing effect of SH003 and nodakenin. MATERIALS AND METHODS: The immune-enhancing effect was evaluated using RAW264.7 macrophages, mouse primary splenocytes, and a cyclophosphamide (CP)-induced immunosuppression murine model. RESULTS: The results show that SH003 or nodakenin stimulated the production levels of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α, and nitric oxide (NO) and the expression levels of iNOS in RAW264.7 macrophages. SH003 or nodakenin also enhanced NF-κB p65 activation in RAW264.7 macrophages. SH003 or nodakenin stimulated the production levels of IFN-γ, IL-12, IL-2, TNF-α, and NO and the expression levels of iNOS in splenocytes. SH003 or nodakenin increased the splenic lymphocyte proliferation and splenic NK cell activity. In addition, SH003 or nodakenin increased the levels of IFN-γ, IL-12, IL-2, IL-6, and TNF-α in the serum and spleen of CP-treated mice, alleviating CP-induced immunosuppression. CONCLUSION: Taken together, the results of this study show that SH003 improved immunosuppression through the activation of macrophages, splenocytes, and NK cells. These findings suggest that SH003 could be applied as a potential immunostimulatory agent for a variety of diseases caused or exacerbated by immunodeficiency.

Trichosanthis Semen Suppresses Lipopolysaccharide-Induced Neuroinflammation by Regulating the NF-κB Signaling Pathway and HO-1 Expression in Microglia.

Neuroinflammation, which is mediated by microglia that release various inflammatory cytokines, is a typical feature of neurodegenerative diseases (NDDs), such as Alzheimer's disease and Parkinson's disease. Hence, alleviating neuroinflammation by downregulating pro-inflammatory action, and upregulating anti-inflammatory action of microglia is an efficient therapeutic target for NDDs. In this study, we evaluated whether trichosanthis semen (TS), a dried ripe seed of Trichosanthes kirilowii Maximowicz, reduces lipopolysaccharide (LPS)-induced neuroinflammation by regulating microglial responses in vitro and in vivo. Our results presented that TS reduced the release of pro-inflammatory mediators, such as nitric oxide (NO), inducible NO synthase, tumor necrosis factor-α, interleukin-1ß, and interleukin-6 via inhibition of the nuclear factor kappa B (NF-κB) signaling pathway in LPS-treated BV2 microglial cells. Moreover, TS induced anti-inflammatory mediators, such as interleukin-10, found in inflammatory zone 1, and chitinase 3-like 3 by the upregulation of heme oxygenase 1 (HO-1). We further confirmed that TS administration suppressed microglial activation, but enhanced HO-1 expression in LPS-injected mice. These results suggest that TS has anti-neuroinflammatory effects via inhibition of NF-κB signaling through the activation of HO-1, and that TS may be a therapeutical candidate for NDDs treatment.

Lipid-Lowering Activities of Cucurbitacins Isolated from Trichosanthes cucumeroides and Their Synthetic Derivatives.

In the ongoing efforts to discover natural cholesterol-lowering compounds, dihydrocucurbitacin B, isolated from Trichosanthes cucumeroides roots, was found to promote LDL uptake by upregulating LDLR protein in a PCSK9-dependent process. In this study, an in-depth investigation of T. cucumeroides roots afforded 27 cucurbitacins (1-27), including seven new cucurbitacins (1-7), and their structures were elucidated by spectroscopic data analyses. In order to gain insight into their structure-activity relationship, cucurbitacin derivatives (B1-11 and DB1-11) were synthesized. Evaluation of lipid-lowering activities of these cucurbitacins by an LDL uptake assay in HepG2 cells revealed that most of the compounds improved the LDL uptake rate, among which hexanorisocucurbitacin D (6) and isocucurbitacin D (21) exhibited the highest activities (rates of 2.53 and 2.47, respectively), which were comparable to that of the positive control, nagilactone B (rate of 2.07). According to a mechanistic study by Western blot analysis, compounds 6 and 21 dose-dependently increased LDLR protein levels and reduced PCSK9 protein levels, representing promising new lipid-lowering drug candidates.

Microwave-assisted extraction, characterization and immunomodulatory activity on RAW264.7 cells of polysaccharides from Trichosanthes kirilowii Maxim seeds.

Microwave-assisted extraction of polysaccharides from Trichosanthes kirilowii Maxim seeds (TKMSP) was optimized using Response surface methodology (RSM) base on Central composite design (CCD). The optimum extraction conditions are detailed as follows: liquid-solid ratio 42 mL/g, extraction temperature 80 °C, microwave power 570 W, extraction time 26 min. Under this conditions, the mean value of TKMSP yield 2.43 ± 0.45% (n = 3), which was consistent closely with the predicted value (2.44%). The five polysaccharides (TKMSP-1, TKMSP-2, TKMSP-3, TKMSP-4 and TKMSP-5) were isolated from TKMSP by DEAE-52. TKMSP-1, TKMSP-2 and TKMSP-4 were common in containing Man, Rib, Rha, GluA, GalA, Glu, Gal, Xyl, Arab and Fuc. However, there was no Fuc in TKMSP-3, while TKMSP-5 lacked GluA, GalA and Fuc. UV-vis and FT-IR analysis combined with molecular weight determination further indicated that the five fractions were polydisperse polysaccharides. A significant difference was achieved in the structural characterization of these five fractions. TKMSP exhibited immunosuppressive activity on RAW264.7 cells. It can be applied as a potential immunosuppressant agent in medicine.

CPF impedes cell cycle re-entry of quiescent lung cancer cells through transcriptional suppression of FACT and c-MYC.

Blockade of cell cycle re-entry in quiescent cancer cells is a strategy to prevent cancer progression and recurrence. We investigated the action and mode of action of CPF mixture (Coptis chinensis, Pinellia ternata and Fructus trichosanthis) in impeding a proliferative switch in quiescent lung cancer cells. The results indicated that CPF impeded cell cycle re-entry in quiescent lung cancer cells by reduction of FACT and c-MYC mRNA and protein levels, with concomitant decrease in H3K4 tri-methylation and RNA polymerase II occupancy at FACT and c-MYC promoter regions. Animals implanted with quiescent cancer cells that had been exposed to CPF had reduced tumour volume/weight. Thus, CPF suppresses proliferative switching through transcriptional suppression of FACT and the c-MYC, providing a new insight into therapeutic target and intervention method in impeding cancer recurrence.

Assessment of the 4-week repeated-dose oral toxicity and genotoxicity of GHX02.

Herbal medicines are widely utilized for disease prevention and health promotion. GHX02 consists of mixtures including Gwaruin (Trichosanthes kirilowii), Haengin (Prunus armeniaca), Hwangryeon (Coptis japonica) and Hwangkeum (Scutellaria baicalensis). It has been purported to have therapeutic effectiveness in cases of severe bronchitis. Non-clinical safety testing comprised a single-dose oral toxicity study and a 28-day repeated-dose oral toxicity study with a 14-day recovery period, and genotoxicity was assessed by a bacterial reverse mutation test, in vitro chromosomal aberration test, in vivo mouse bone marrow micronucleus test and single cell gel electrophoresis assay (comet assay). In the single-dose oral toxicity study, the approximate lethal dosage is estimated to be higher than 5000 mg/kg in rats. Thus, the dosage levels were set at 0, 1250, 2500 and 5000 mg/kg/day in the 28-day repeated-dose oral toxicity study, and 10 male rats and 10 female rats/dose were administered GHX02. No clinical signs of toxicological significance were recorded in any animal during the dosing and the observation period in the single-dose study. The no-observed-adverse-effect level of GHX02 was 5000 mg/kg/day when administered orally for 28 days to male and female Sprague-Dawley rats. Despite increases in the frequencies of cells with numerical chromosomal aberration in the in vitro test, the increases were not considered relevant to the in vivo genetic risk. Except for the increase of in vitro numerical chromosomal aberration, clear negative results were obtained from other genetic toxicity studies.

Synergistic anticancer effect of combined use of Trichosanthes kirilowii with cisplatin and pemetrexed enhances apoptosis of H1299 non-small-cell lung cancer cells via modulation of ErbB3.

BACKGROUND: Lung cancer is one of the most common malignancies worldwide. To treat lung cancer, various anticancer drugs were developed and tested, but they failed because of drug resistance. In the present study, we tested herbal medicines, such as TK and CuD, as anticancer drugs to decrease side effects and resistance. METHODS: Cell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by an annexin V-FITC/PI assay. We performed RTK kit analysis. Levels of p-ErbB3, p-STAT3, p-NF-κB, and caspases were measured by western blot analysis. Nuclear staining of ErbB3 was measured by immunocytochemistry. Transcriptional activity of STAT3 and NF-κB was detected by STAT3 and NF-κB luciferase reporter gene assays. RESULTS: We found a synergistic effect of TK with CDDP and PXD in primary culture of human NSCLC tumor cells. The combination of CDDP/PXD and TK or CuD inhibited the proliferation of H1299 cells. The combination of CDDP/PXD and TK or CuD induced sub-G1 and G2/M cell cycle arrest in H1299 cells. The combination of CDDP/PXD and TK or CuD induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in H1299 cells. We found that TK suppresses p-ErbB3 expression and signaling. The combination of CDDP/PXD and TK or CuD inhibited p-AKT, p-Erk, and p-JNK signaling and suppressed Stat3 and NF-κB transcriptional activity in H1299 cells. More importantly, the combination of CDDP/PXD and TK or CuD inhibited p-ErbB3 and downstream molecules in H1299 cells. The combination of CDDP/PXD and TK or CuD inhibited ErbB2/ErbB3 dimerization. Our results clearly demonstrate that the synergistic effect of CDDP/PXD and TK or CuD inhibits cell growth and induces apoptosis by inhibiting ErbB3 signaling. CONCLUSION: The combination of CDDP/PXD and TK or CuD decreases cell proliferation and induces apoptosis by inhibiting ErbB3 signaling in H1299 lung cancer cells. TK or CuD could be useful as a compound to treat lung cancer. Additionally, targeting ErbB3 may also be useful for treating lung cancer.

TKP, a serine protease extracted from Trichosanthes kirilowii, inhibits the migration and invasion of colorectal adenocarcinoma cells by targeting Wnt/ß-catenin and Hedgehog/Gli1 signalings.

Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/ß-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/ß-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9.

Pharmacokinetics of cucurbitacin B from Trichosanthes cucumerina L. in rats.

BACKGROUND: Cucurbitacin B is the major bioactive constituent in Trichosanthes cucumerina L. fruits, which the pharmacological properties have been studied for decades particularly an anti-tumor activity. The pharmacokinetic profile of this compound is still limited and investigation is needed for further phytopharmaceutical product development. This study aimed to investigate the pharmacokinetic profile of cucurbitacin B after administering the compound at different doses and routes to rats. METHODS: Male Wistar rats (n = 6) were treated by cucurbitacin B extracted from Trichosanthes cucumerina L. The cucurbitacin B was administered at 0.1 mg/kg intravenously or by oral gavage at 2-4 mg/kg. Blood samples and internal organs were collected serially within 24 h after administration. Urine and feces were collected from time 0 to 48 h. The level of cucurbitacin B in biological samples was determined by liquid chromatography-tandem mass spectrometry. RESULTS: The absolute oral bioavailability of cucurbitacin B was approximately 10%. The maximum concentration in plasma after normalization by dose ranged from 4.85-7.81 µg/L and the time to reach maximum value was approximately within 30 min after oral dosing. The level of cucurbitacin B in plasma increased proportionally to the given dose. After intravenous administration, cucurbitacin B had a large volume of distribution of about 51.65 L/kg and exhibited a high tissue to plasma concentration ratio, approximately 60 to 280-fold in several organs. Negligible amount of unchanged cucurbitacin B could be detected in urine and feces and accounted less than 1% of administered dose. CONCLUSION: Cucurbitacin B had low oral bioavailability, but could be distributed extensively into internal organs with a high volume of distribution and tissue to plasma ratio. Only negligible amounts of unchanged cucurbitacin B were excreted via urine and feces suggesting that the compound might be biotransformed before undergoing an excretion. Further studies of the metabolic pathway and tissue uptake mechanism are required to strategize the future development of cucurbitacin B into clinical studies.

[Anti-platelet aggregation and anti-thrombotic mechanism of Trichosanthis Fructus combined with aspirin based on network pharmacology].

To explore the anti-platelet aggregation and anti-thrombotic mechanisms of Trichosanthis Fructus combined with aspirin based on network pharmacology and the validation of arteriovenous by pass model in rats. The databases of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Drug Repositioning and Adverse Drug Reaction Chemical-Protein Interactome(DRAR-CPI),Universal Protein Resource(Uniprot) and the Database for Annotation,Visualization,and Integrated Discovery(DAVID) were used to predict protein targets and analyze biological pathway and signal pathway in the combination of Trichosanthis Fructus with aspirin. The effects of pretreatment with Trichosanthis Fructus pellets,aspirin pellets and their combination on thromboxane B2(TXB2),6-keto prostaglandin F1α(6-keto-PGF1α) and cyclic adenosine monophosphate(c AMP) in rat thrombotic model were studied. Through the study of network pharmacology,12 components of aspirin and Trichosanthis Fructus,including hydroxygenkwanin,quercetin and adenosine,were found to show the anti-platelet aggregation and anti-thrombosis mechanisms through9 common protein targets,such as SRC,RAC1,MAPK14,MAPK1,AKT1,and 14 common signaling pathways,such as VEGF signaling pathway. After the intervention with Trichosanthis Fructus pellets combined with aspirin pellets,the vascular endothslia growth factor(VEGF) signaling pathway can be activated to inhibit platelet aggregation and improve vascular endothelial function,and show the anti-platelet aggregation and anti-thrombosis mechanisms,which verify the results of the network pharmacology,and explain the anti-platelet aggregation and anti-thrombotic mechanisms of the combination of Trichosanthis Fructus pellets with aspirin pellets.

Comparative Analysis of Carbohydrates, Nucleosides and Amino Acids in Different Parts of Trichosanthes kirilowii Maxim. by (Ultra) High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry and Evaporative Light Scattering Detector Methods.

Trichosanthes kirilowii Maxim. is one of the original plants for traditional Chinese medicines Trichosanthis Fructus, Trichosanthis Semen, Trichosanthis Pericarpium and Trichosanthis Radix. Amino acids, nucleosides and carbohydrates are usually considered to have nutritional value and health-care efficacy. In this study, methods involving high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD), UV-visible spectrophotometry and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) were established for quantifying carbohydrates (fructose, glucose, stachyose, raffinose and polysaccharide), fourteen nucleosides and twenty one amino acids. Moreover, sixty-three samples from nine different parts, including pericarp, seed, fruit pulp, stem, leaf, main root, main root bark, lateral root and lateral root bark of T. kirilowii from different cultivated varieties were examined. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. The results showed that the average content of total amino acids in roots (15.39 mg/g) and root barks (16.38 mg/g) were relatively higher than for others. Contents of nucleosides in all parts of T. kirilowii were below 1.5 mg/g. For carbohydrates, fruit pulp has a higher content than others for glucose (22.91%), fructose (20.63%) and polysaccharides (27.29%). By using partial least-squared discriminate analysis (PLS-DA), Variable importance in the projection (VIP) plots and analysis of variance (ANOVA) analysis, the characteristic components of the different organs (fruit, stems and leaves, roots) were found. This analysis suggested there were potential medicinal and nutritive health care values in various parts of the T. kirilowii, which provided valuable information for the development and utilization of T. kirilowii.

Discrimination of Trichosanthis Fructus from Different Geographical Origins Using Near Infrared Spectroscopy Coupled with Chemometric Techniques.

Near infrared (NIR) spectroscopy with chemometric techniques was applied to discriminate the geographical origins of crude drugs (i.e., dried ripe fruits of Trichosanthes kirilowii) and prepared slices of Trichosanthis Fructus in this work. The crude drug samples (120 batches) from four growing regions (i.e., Shandong, Shanxi, Hebei, and Henan Provinces) were collected, dried, and used and the prepared slice samples (30 batches) were purchased from different drug stores. The raw NIR spectra were acquired and preprocessed with multiplicative scatter correction (MSC). Principal component analysis (PCA) was used to extract relevant information from the spectral data and gave visible cluster trends. Four different classification models, namely K-nearest neighbor (KNN), soft independent modeling of class analogy (SIMCA), partial least squares-discriminant analysis (PLS-DA), and support vector machine-discriminant analysis (SVM-DA), were constructed and their performances were compared. The corresponding classification model parameters were optimized by cross-validation (CV). Among the four classification models, SVM-DA model was superior over the other models with a classification accuracy up to 100% for both the calibration set and the prediction set. The optimal SVM-DA model was achieved when C =100, γ = 0.00316, and the number of principal components (PCs) = 6. While PLS-DA model had the classification accuracy of 95% for the calibration set and 98% for the prediction set. The KNN model had a classification accuracy of 92% for the calibration set and 94% for prediction set. The non-linear classification method was superior to the linear ones. Generally, the results demonstrated that the crude drugs from different geographical origins and the crude drugs and prepared slices of Trichosanthis Fructus could be distinguished by NIR spectroscopy coupled with SVM-DA model rapidly, nondestructively, and reliably.

Hierarchical extraction and simultaneous determination of flavones and triterpenes in different parts of Trichosanthes kirilowii Maxim. by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry.

Trichosanthin (root of Trichosanthes kirilowii Maxim.) has received much attention, but T. kirilowii has received little systematic analysis. A comprehensive evaluation of flavones and triterpenes in nine different parts of T. kirilowii would, thus, be of value to fully understand the potential medicinal properties of T. kirilowii. A hierarchical extraction-simultaneous determination method was established, which based on different types of components had different response on the MS detector. The hierarchical extraction method extracted 3, 29-dibenzoyl rarounitriol solely, which response lowly on the MS detector in the mixture of one step extract, in order to obtain accurate results in the next UHPLC-MS/MS assay. By this method, ten flavones, three tetracyclic triterpenoids, and one pentacyclic triterpenoid in the extracts of T. kirilowii were determined. The results showed the level of flavonoids in leaves was higher than those in the other samples, tetracyclic triterpenoids mainly existed in root and root bark, and pentacyclic triterpenoid, especially 3, 29-dibenzoyl rarounitriol was concentrated in the seed kernel. According to principal component analysis, the F values of the leaves of all varieties were generally greater than those in other parts. The leaves might provide a supplementary source of flavonoids, and the root and root bark could be a good choice for tetracyclic triterpenoids. The analysis results revealed the distribution of flavones and triterpenes in different parts of T. kirilowii and provided reference for the research and rational utilization of T. kirilowii.