Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism.

(1) Background: Toad venom (Bufonis Venenum, known as 'Chansu' in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins.

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi-components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.

[Correlative study of bufogenin constituents in venom of Bufo bufo gargarizans].

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.

[Major Ionic Features and Possible Controls in the Groundwater in the Hamatong River Basin].

In order to study the major ion chemistry and controls of groundwater, 59 groundwater samples were collected and their major ions measured in the Hamatong River Basin. The hydrogeochemical characteristics of groundwater in this basin were analyzed by means of mathematical statistics, Piper triangular diagrams, Gibbs figures, and ionic relations, and the water chemical evolution and ion sources of the Hamatong River Basin were determined. The results showed that Ca2+ was the main cation in the groundwater, accounting for 22.1% to 72.4% of the total cations, with an average value of 48.7%. HCO3- was the main anion, accounting for 35.3% to 97.5% of the total anions, and with an average value of 80%. Total dissolved solids concentration ranged from 93.3 mg·L-1 to 521.1 mg·L-1 with a median value of 219.1 mg·L-1. The hydrochemical types of groundwater are HCO3-Ca, HCO3-Ca·Mg, and HCO3-Ca·Na. Chemical weathering rates of carbonates and silicates were estimated, and the chemical composition of groundwater samples located in the middle of Gibbs model indicated that the major chemical process of groundwater was controlled by rock weathering. Silicate weathering is believed to significantly contribute to dissolved solute compositions, and carbonate weathering played an important role as the source of dissolved ions.

[Hydrochemical Characteristics of Groundwater and the Origin in Alluvial-proluvial Fan of Qinhe River].

To study the hydrochemical characteristics of groundwater and the factors controlling the alluvial-proluvial fan of the Qinhe River, 60 samples were collected including 9 surface water samples and 51 groundwater samples. The distribution characteristics of the hydrochemistry and major controlling factors of hydrochemical evolution processes of surface water and groundwater in the study area were analyzed by using mathematical statistics, the Schakerev classification, Piper diagrams, Schoeller diagrams, Gibbs plots, and ion ratios. The results show that:① the major anions and cations of surface water and groundwater in the study area are Ca2+, Mg2+, SO42-, and HCO3-. The recharge sources of surface water and groundwater are closely related; ② the major hydrochemical types of the groundwater are HCO3-Ca, HCO3-Ca·Mg, and HCO3·SO4-Ca·Mg, while the major hydrochemical type of surface water is HCO3·SO4-Ca·Mg; and ③ the major ions of surface water and groundwater are affected by carbonate mineral weathering and dissolution, ion exchange, and evaporation. Sulfuric acid and carbonic acid were involved in carbonate mineral weathering and dissolution.

[The prevalence and influencing factors of abuse and negligence against elderly in rural areas of Anhui province].

OBJECTIVE: To investigate the prevalence and influencing factors related to abuse and negligence against the elderly in the rural areas. METHODS: 975 elderly over 60 years from 41 counties in Anhui province were included. All participants completed an anonymous questionnaire including items as: educational background, marital condition, income, child-discipline, rude action to parents, daily activities, physical functions, having chronic illness, abuse and negligence against the elderly, etc. RESULTS: In the last year, rates of common physical abuse, serious physical abuse, emotional abuse, financial exploitation, negligence, overall abuse and negligence against the elderly were 4.5%, 1.5%, 26.9%, 4.9%, 7.2%, 29.9% respectively. Among the 281 victims, 80.4% reported that they were suffered more than 3 times of abuse and neglect episodes, and 34.9% reported that they were suffered more than 2 forms of abuse and negligence. The primary sadism was carried out by the daughter-in-law or son-in-law (43.2%) of the elderly. Low activity on daily life and having chronic illness were the risk factors causing common physical abuse while better education was the protective factor to it. Low ability in managing daily activity of living was the risk factor causing serious physical abuse. Less active on daily life and having rude action to parents were the risk factors to emotional abuse, but being strict with their children was the protective factor to emotional abuse. Less active on daily life, often beating their children and having rude action to parents were the risk factors related to financial exploitation. Less active on daily life, having rude action to parents and having bad physical functions were the risk factors causing negligence. Less active on daily life and having rude manner to parents were the risk factors of overall elderly abuse and negligence, but being strict with their children was protective factor to the abuse and negligence against the elderly. CONCLUSION: High prevalence on abuse and negligence against the elderly was seen in the rural areas of China. Different forms on elderly abuse and negligence were affected by different factors that called for more attention to be paid to those elderly with lower ability in managing their daily life.