ABSTRACT
Background: The consumption of the fruits of cucurbitaceae plants is widely popular among Indians dueto their various nutritional and medicinal purposes. Some of these plants are well reported in Ayurvedadue to their potential therapeutic importance. In particular, the plants of this family are wellcharacterized by the presence of its bitter principle, Cucurbitacin E which differs within the speciesdue to its genetic variations.Objectives: The objective of the study was to develop a validated RP-HPLC method for standardization insome widely consumed cucurbits with cucurbitacin E as a marker compound.Materials and methods: The RP-HPLC method was developed with a reverse phase C18 column, usingacetonitrile and water (1% glacial acetic acid) as mobile phase (70:30 v/v). The flow rate and lmax wereoptimized at 1 mL/min and 230 nm respectively. The HPLC method was validated in terms of accuracy,specificity, sensitivity, and repeatability as per ICH guideline.Results: The calibration curve was found linear in the concentration range of 1e100 mg/mL. The % RSD ofprecision and recovery was found to be <2%, which confirms high repeatability of the method. The results indicated that the content of cucurbitacin E was highest (0.0663% w/w) in Cucurbita pepo whereasLagenaria siceraria contains the lowest (0.0356% w/w).Conclusion: The study was able to explore the variation of cucurbitacin E content in some selected foodplants of Cucurbitaceae family. The applicability of the method can be established in nutraceutical industry for the effective quality control of cucurbits for safe human consumption.
ABSTRACT
Cucurbitacin E (CuE) is previously reported to exhibit antitumor effect by several means. In this study, CuE acted as a tyrosine kinase inhibitor interfering with the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signaling pathway and subsequently induced apoptosis and cell cycle arrest in non-small-cell lung cancer (NSCLC) cell line A549. The apoptosis regulators, cleaved Caspases-3 and Caspases-9, were observed to be increased with the treatment of CuE. The activated transcription factor STAT3 and the apoptosis inhibitor protein survivin were also observed to be reduced. The cell cycle regulators, CyclinA2, cylinB1, CyclinD1 and CyclinE, were also investigated and the results suggested that the cell cycle was arrested at G1/G0 phase. Treatment of CuE also altered the existence status of most of the participants in the EGFR/MAPK signaling. Phosphorylation of EGFR enhanced significantly, leading to the alteration of members downstream, either total amount or phosphorylation level, notably, MEK1/2 and ERK1/2. Moreover, the results of molecular simulation brought an insight on the interaction mechanism between CuE and EGFR. In summary, CuE exhibited anti-proliferative effect against A549 cells by targeting the EGFR/MAPK signaling pathway.
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Objective To explore the influence of cucurbitacin E (CuE) on autophagy in human bladder cancer cell line T24 and further study its impacts on cell proliferation. Methods MTT assay was used to determine the proliferation inhibition capacity of CuE on T24 and western blot to check the impacts of CuE treatment on the expression of classic autophagy markers LC3A/B and p62. LC3 turnover assay and GFP-RFP-LC3 fluorescent assay were performed to determine autophagy flux. Western-blot was used to check the autophagy inhibition ability of 3-MA on CuE treatment and MTT assay and cell counting assay were used to check the influence of CuE-induced autophagy on cell proliferation with/without autophagy inhibition. Results CuE inhibited the proliferation of T24 and the IC50 in 24 h was about 0.75 μmol/L. CuE treatment increased the expression of LC3A/B Ⅱ and LC3A/B Ⅱ/Ⅰ ratio (P < 0.05) , but decreased the expression of p62 (P < 0.05) , indicating the induction of autophagy. Autophagy flux was induced because of positive LC3 turnover assay and the increase of yellow and red dots in GFP-RFP-LC3 fluorescent assay (P < 0.05). CuE-induced autophagy was inhibited by 3-MA (P < 0.05). With autophagy inhibition, CuE's proliferation suppression ability on T24 was attenuated (P <0.05). Conclusion CuE induces autophagy in bladder cancer cell line T24 and the induced autophagy positively contributes to the inhibitation of cell proliferation.
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Objective To establish HPLC method for simultaneous determination of five active ingredients of cucurbitacin B, cucurbitacin E, praeruptorin A, praeruptorin B, and praeruptorin E in Infantile Qingfei Pills (IQP), and study the contents changes of the five effective components in IQP before and after 60Co-γ ray irradiation. Methods The Agilent-C18 column (250 mm × 4.6 mm, 5 μm) was adopted and the detection wavelength was 230 nm and 321 nm with the flow rate of 1.0 mL/min. The mobile phase consisted of A (methanol: acetonitrile) and B (0.5% glacial acetic acid solution) for gradient elution, and column temperature was 25 ℃. Irradiation does of 2, 4, 6, 8 kGy were selected to irradiate IQP respectively. The contents of five active components in IQP were compared before and after irradiation, and the significant condition was observed by t-test. Results The linear range of cucurbitacin B, cucurbitacin E, praeruptorin A, praeruptorin B, and praeruptorin E were 0.049—1.247, 0.079—1.973, 0.056—1.406, 0.028—0.705, and 0.028—0.693 μg, The average recovery were 101.2%, 99.7%, 99.9%, 98.9%, and 100.5%, and RSD were 0.6%, 0.5%, 1.2%, 1.1%, and 1.2%, respectively. After irradiation of 2, 4, 6, and 8 kGy, the effective components contents of cucurbitacin B, cucurbitacin E, praeruptorin A, praeruptorin B, and praeruptorin E were changed. After t-test in groups, the content change of cucurbitacin B was significantly after irradiation over 6 kGy (P < 0.05). Conclusion The established method has a high recovery rate, good repeatability, which is simple and practical and can be used for quality control of IQP. The changes of each component are not significant with the radiation no more than 6 kGy, which can provide a reference for the sterilization of IQP.
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Objective: To establish an HPLC method for the content determination of cucurbitacin B and E in cucurbitaceae plants, and evaluate the quality of cucurbits by grey relational analysis.Methods: A Diamonsil-C18 column (250 mm×4.6 mm, 5 μm) was adopted and the UV detection wavelength was 234 nm at the flow rate of 1.0 ml·min-1.The mobile phase consisted of 0.1% acetic acid and acetonitrile with gradient elution, and the column temperature was 30℃.In addition,grey relational analysis was carried out for the comprehensive evaluation.Results: The linear range of cucurbitacin B and E was 0.894-44.715 μg·ml-1(r=0.999 6) and 0.257-12.825 μg·ml-1(r=0.999 7),respectively.The average recovery was 98.8%-100.3% and 98.0%-100.1%(RSD＜0.88%,n=6), respectively.According to the grey correlation analysis on the components, Cucumis melo L had the best quality, and Momordica cochinchinensis (Lour.) Spreng showed the worst quality.Conclusion: The established method is rapid and convenient, which can be used for the content determination of cucurbitacin B and E in cucurbits, and the comprehensive quality evaluation of cucurbits.
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Aim To study the mechanism of cucurb-itacin E ( CuE )-induced autophagy in HeLa cells. Methods Improved MTT assay was adopted to meas-ure the effect of CuE on cell proliferation. Western blot was used to determine the phosphorylation levels of downstream signaling proteins of mTORC1 and the ex-pression of autophagy associated proteins. ResultsCuE inhibited the proliferation of HeLa cells in a dose-dependent manner, and the 24-h IC50 of CuE was 4. 01μmol· L-1 . CuE significantly inhibited the phospho-rylation of p70 S6 K in a time-and dose-dependent man-ner as evidenced by decreased phosphorylation levels of the mTORC1 substrate. Meanwhile, the expression of LC3-II, a marker for autophagosome formation, was elevated by CuE treatment, and was further increased in the presence of chloroquine. Furthermore, CuE re-duced the levels of p62/SQSTM1 . These results indi-cated that CuE induced autophagy in HeLa cells. The decreased levels of phosphorylated ULK1 S757 were posi-tively correlated with autophagy induction in HeLa cells. Conclusion CuE is likely to induce autophagy through inhibiting mTORC1 activity.