Screening Effective Sites of Momordicae Semen-Epimedii Folium and Anti-lung Cancer Mechanism of Its Prescription / 中国实验方剂学杂志

ObjectiveTo preliminarily confirm the effective anti-lung cancer sites of Momordicae Semen and Epimedii Folium and study their mechanism of action. MethodOn the basis of preliminary research， the extraction method of Momordicae Semen and Epimedii Folium was optimized and the effective parts were screened under the guidance of pharmacological effects. Different ethanol elution and water elution sites of Momordicae Semen and Epimedii Folium were obtained through adsorption and elution with D101 macroporous resin. The methylthiazolyldiphenyl-tetrazolium bromide （MTT） colorimetric assay was used to detect the effects of total drug extracts and different elution sites on the proliferation of various tumor cell lines， and to screen for the optimal elution site and tumor sensitive strains. Flow cytometry was used to detect the effect of the elution sites of Momordicae Semen and Epimedii Folium on intracellular reactive oxygen species （ROS） and apoptosis in A549 cells. Western blot was used to compare the expressions of tumor protein 53 （p53）， Bcl-2-associated X protein （Bax）， cysteinyl aspartate specific proteinase-3 and 9 （Caspase-3 and Caspase-9） proteins in A549 cells. ResultThe inhibitory effect of Momordicae Semen on the proliferation of A549 cells was better than the kernel of Momordicae Semen， with 50% inhibitory concentration （IC50） being （86.83±2.88） mg·L-1 and （95.10±18.13） mg·L-1， respectively. The effect of total extracts of Epimedii Folium on A549 anti proliferation IC50 value was （4.71±0.81） mg·L-1. The IC50 values of the 40%， 60%， and 80% ethanol and anhydrous ethanol eluted macroporous resins of the total extracts of Momordicae Semen and Epimedii Folium inhibiting A549 proliferation were （45.32±4.38）、 （14.95±0.73）、 （17.07±1.76）、 （14.46±2.35）、 （51.7±2.26）、 （12.37±0.67）、 （20.29±0.93）、 and （3.43±0.91） mg·L-1， respectively. Compared with the normal group， the 1∶1 combination of Momordicae Semen and Epimedii Folium inhibited A549 cell proliferation in a time-dependent and concentration-dependent manner. Compared with the normal group， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased intracellular ROS expression （P<0.01）. Compared with the normal group， 12.5， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of A549 cell apoptosis （P<0.01）. Compared with the normal group， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of p53 in A549 cells （P<0.01）. Compared with the normal group， 12.5， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of Bax （P<0.01）. Compared with the normal group， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly reduced the expressions of Caspase-3 and Caspase-9 （P<0.01）. ConclusionThe anti-tumor effect of Momordicae Semen is better than that of the kernel of Momordicae Semen. The anti-tumor substances of Momordicae Semen and Epimedii Folium mainly concentrate in the 60% ethanol to anhydrous ethanol elution site. A549 cells are sensitive to the 1∶1 combination of Momordicae Semen and Epimedii Folium， which can effectively inhibit the cell proliferation. The mechanism may be related to increasing the generation of ROS in A549 cells， promoting their apoptosis， increasing the expressions of apoptotic proteins such as p53 and Bax， and reducing the expressions of Caspase-3 and Caspase-9.

Chemical Constituents, Toxicology and Pharmacological Effect of Momordicae Semen / 中国实验方剂学杂志

Momordicae Semen a traditional toxic Chinese medicine, which was first recorded in Kaibao Bencao of the Northern Song Dynasty. It has the effects in reducing swelling, dispersing knot, and attacking sores. It is mainly distributed in South China, such as Guangxi and Guangdong. It is also distributed in Southeast Asian countries, such as Thailand and Vietnam. The present study showed abundant chemical components extracted from Momordicae Semen, including steroids, sterols, volatile oils and fatty acids. Among them, 30 terpenoids, 102 compounds in volatile oil, 6 sterols and 19 fatty acids have been identified. Aqueous extracts and alcohol extracts of Momordicae Semen have the toxicity, and the toxicity decreases with the increase of oil content. The main toxic components reported in the literatures are cochinchinin and saponins. Pharmacological studies have shown that in addition to its traditional anti-cancer, anti-inflammatory, antibacterial and other pharmacological effects, Momordicae Semen also exhibited many pharmacological effects, such as anti-ulcer, anti-oxidation and immune regulation. In recent years, there have been increasingly more research reports on Momordicae Semen. By studying relevant domestic and foreign literatures from 1964 to 2019 in China National Knowledge Infrastructure, Wanfang, PubMed and Web of Science, chemical constituents, pharmacological effects and toxicological research of Momordicae Semen were summarized, which will provide reference for further research and application of Momordicae Semen in the future.

Screening combination ratio and exploring mechanism of Momordicae Semen and Epimedii Folium / 中国中药杂志

The aim of this paper was to obtain low toxicity and high efficiency anti-tumor Chinese medicine through screening the combination ratios of Momordicae Semen and Epimedii Folium, and to explore the anti-tumor mechanism of the combination of two drugs by observing their effect on apoptosis-related proteins in cancer cells. Methyl thiazolyl tetrazolium(MTT) assay was used to observe the effect of drug combination on the proliferation of tumor cells from different tissue sources. The effects of the combination of the two drugs on tumor cells were analyzed by Compusyn software. Plate cloning assay was used to observe the effect of combination of these two drugs on the proliferation of A549 cells in vitro. The expression of reactive oxygen species(ROS) and apoptotic proteins p53, Bcl-2 and Bax were compared by using ROS kit and Western blot. Lewis lung cancer model was used to observe the anti-tumor effect of drugs in vivo. The results showed that the anti-tumor effect of their ethanol extract was more significant than that of water extract, and the anti-proliferation effect was strongest when the ratio was 1∶1(P<0.05). Compusyn analysis showed that the combination of the two drugs had synergistic effect. Further studies showed that after combined use, the number of clonogen formation in A549 cells was significantly reduced(P<0.01); ROS production was increased; the expression of apoptosis-related protein p53 was up-regulated, and the ratio of Bcl-2/Bax was decreased. In vivo animal study showed that the tumor inhibition rate was 53.06%(P<0.05) in the high dose group. As compared with the single use of the two drugs, the combination of the two drugs had more significant anti-proliferative effect on tumors, and the optimum ratio was 1∶1. The combination of the two drugs at a ratio of 1∶1 inhibited the proliferation of various tumor cells, and had no significant effect on normal liver cells LO2 when compared with other ratios. Therefore, it can be preliminarily inferred that the combination of the two drugs may have the effect of synergism and detoxification. Further studies showed that the combination of the two drugs can significantly inhibit the proliferation of A549 cells, and its mechanism may be related to the activation of endogenous apoptotic pathway. In vivo experiments also showed that the tumor inhibition rate increased with the increase of drug concentration.

Optimization of processing technology of Momordicae Semen cream by Box- Behnken design-response surface methodology / 中草药

Objective To optimize the optimum processing technology of Momordicae Semen cream using Box-Behnken design-response surface method (BBD-RSM). Methods The overall desirability (OD) of indexes of the content of 3-O-β-D- glucuronic acid methyl ester, β-sitosterol, and oleanolic acid and the content of fatty oil in gypsogenin was comprehensively evaluated, and BBD-RSM with three factors and three levels was used to study the effect of baking temperature and time, and pressing time on the processing technology of Momordicae Semen cream. Results According to the experiment, the optimum processing conditions were optimized: the baking time 1.68 h, the baking temperature 80 ℃, the pressing time 30 min, and the OD value was 0.777. Considering the actual situation, the optimum processing technology of Momordicae Semen was obtained by fine-tuning the baking time (A). That was, the baking time was 1.7 h, the baking temperature was 80 ℃, and the pressing time was 30 min. Also, the calculated OD values were 0.779, 0.783, 0.766, and its RSD was 1.15% through the obtained conditions in parallel with three batches of samples. Conclusion The processing technology optimized by Box-Behnken design-response surface method has the advantages of stable, good model prediction effect and a new processing technology for Momordicae Semen cream production.

Study on mechanism of differentiation of esophageal carcinoma Kyse30 cells induced by p-hydroxylcinnamaldehyde / 中草药

Objective To investigate the effects of p-hydroxylcinnamaldehyde (CMSP) on cell proliferation, migration, cell cycle and the expression level of malignant biomarkers, and to investigate the underlying mechanism of differentiation of esophageal carcinoma Kyse30 cells (ESCC cells). Methods The effect of different concentration of CMSP at 0, 10, 20, and 40 μg/mL on viabilities of ESCC cell lines (Kyse30, Eca109, and Kyse180) for 24, 48, and 72 h was determined by MTS assay. Optical microscope and scanning electronic microscopy (SEM) were used to observe the morphologic changes of Kyse30 cells. The effect of CMSP at different concentration on cell cycle distribution and apoptosis of Kyse30 cells was assessed by flow cytometry analysis. ELISA was used to detect the effect of CMSP on expression of tumor related antigens (CEA and SCC) and malignant biomarkers (IL-6 and MIC-1) in Kyse30 cells at protein secretion level. Influence of different concentration of CMSP on migration and invasiveness of Kyse30 cells were determined by colony-formation, wound healing and Transwell assays. Western blotting was used to evaluate the effect of CMSP on expression of protein biomarkers C-myc and N-myc of Kyse30 cells and the related proteins in RhoA-MAPK pathway. Results The proliferation of esophageal cancer cell lines (Kyse30, Eca109, and Kyse180) was significantly inhibited by CMSP in a dose- and time-dependent manner. The cell cycle Kyse30 was blocked in G0/G1 phase. After the treatment with CMSP, Kyse30 cells showed typical dendrite-like cellular protrusions, and the percentage of such elongated cells was significantly and progressively increased with the increase in CMSP concentration (P 0.05). CMSP could decrease the expression of CEA, SCC, IL-6, and MIC-1 both in protein secretion levels significantly in a dose- and time-dependent manner (P < 0.05, 0.01). Western blotting analysis showed that C-myc and N-myc proteins were all decreased significantly in Kyse30 cells after treatment with CMSP (P < 0.05). CMSP significantly inhibited the proliferation and migration ability of Kyse30 cells (P < 0.05) and induced cell differentiation; The protein levels of p-P38 was significantly increased (P < 0.01), while protein levels of ERK1/2, SAPK/JNK, and GTP-RhoA were obviously decreased in Kyse30 cells after treatment with CMSP (P < 0.01). Conclusion CMSP suppressed the proliferation and induced the differentiation of Kyse30 cells through regulating the RhoA-MAPK signal pathway, which might provide new potential strategies for ESCC treatment.

Inhibitory effect of p-hydroxylcinnamaldehyde from Momordicae Semen on growth of mouse melanoma xenograft in vivo andstudy on its mechanism / 中草药

Objective: To investigate the inhibitory effect of p-hydroxylcinnamaldehyde (PHD) from Momordicae Semen seeds on growth of mouse melanoma B16 cells in vivo. Methods: The inhibitory effect of PHD on growth of mouse melanoma B16 cells was measured by MTS method. Morphological changes of B16 cells were observed by phase contrast microscope. The xenograft tumor models of B16 cells in mice were established and divided into two groups: The mice in treatment group were treated with PHD (2 mg/kg) and in control group (treated with equal volume of normal saline). The growth of xenograft tumors in mice was observed and their sizes and weights were measured. The expression of Tyr, MMP-9, S-100B, p-P38, and p-ERK in tumor tissues was detected by immunohistochemical method. Morphological changes of lung and liver tissues in mice were observed by HE staining. Results: PHD had obvious inhibitory effect on the growth of B16 cells in vitro (P < 0.01). The morphological changes of B16 cells were typically differentiated after treated with 20 μmol/L PHD for 48 h. The average volume and weight of tumor tissues in mice of PHD treatment group were significantly decreased as compared with those of the control group (P < 0.01). Compared to the control group, the expression levels of Tyr and p-P38 in PHD treatment group were increased (P < 0.05), while the expression levels of MMP-9, S-100B, and p-ERK were decreased (P < 0.05). No obvious morphological changes were found in liver and lung tissues of mice in PHD treatment group and the control group. However, lung tumor metastasis was found in control group mice. Conclusion: PHD has inhibitory effect on the growth of xenograft tumor of mouse melanoma cells in mice.