A preliminary study of the antitumor activity of Agkistrodon tissue extracts and to investigate their cytotoxic effect on glial cells. This is a preliminary study of the anti-tumor activity of Agkistrodon tissue extracts.
1 Materials and Methods
1.1 Instruments and Reagents
- Electrophoresis microplate reader (USA) 86-3.
- Ultrasonic extraction instrument (Shanghai Ultrasonic Instrument Factory).
- CO2 incubator, 96-well plates (imported).
- Reagents: RPMI1640 as medium, DMSO, phosphate buffer saline (PBS), standard protein (purchased from the Ministry of Health).
- Agkistrodon: purchased from a pharmacy with genuine herbs.
- Cell lines: glioblastoma (not included).
1.2 Methods
1.2.1 Extraction of Agkistrodon Composition
The purchased Agkistrodon was crushed into powder, precisely weighed at 0.5g, and placed in a 25mL conical flask. 20mL of 30% ethanol was added accurately, soaked for 3 hours, ultrasonically extracted for 10 minutes, rested, and then ultrasonically extracted again. The extract was centrifuged at 4000rpm/min for 15 minutes, and the supernatant (15mL) was precisely drawn out, dried in a water bath at 60°C, resulting in the Agkistrodon extract.
1.2.2 Electrophoresis Analysis of Acutobin Extract
Approximately 0.5g of the extract was taken, 2mL of 0.2M phosphate buffer saline (PBS) was added, mixed, and reserved. Control solution: standard protein at 0.1mg was reserved. 10μL of the above samples and 5μL of control fluid were respectively spotted on the same plastic sheet for electrophoresis mobility (PAGE).
1.2.3 Cytotoxicity of Agkistrodon Extract on Glial Cells by MTT Assay
Glial cells in the logarithmic growth phase were added to 96-well culture plates at a concentration of 1X10^5 cells/well, cultured in a CO2 incubator for 24 hours. Different concentrations of Agkistrodon extract were added to the experimental wells (four control wells were done simultaneously). After culturing for 48 hours, MTT (100μL, 1mg/mL) was added to each well and incubated for four hours. DMSO (20μL) was added, and the cells were fully dissolved (30 minutes). The absorbance value A was measured at 570nm using a microplate reader. The inhibitory rate was calculated by the following formula:
Inhibition rate (%) = (A value of control well - A value of experimental well) / A value of control well × 100%.
2 Results
2.1 Electrophoresis Analysis Results
Electrophoresis was performed on the plastic sheet staining (silver staining) with the standard protein. The results showed that the molecular weight of Agkistrodon tissue extracts was 1.8Kd, and no other components were identified in this study.
2.2 Cytotoxicity Results of Agkistrodon Extracts on Glial Cells
The results showed that the ingredients in Agkistrodon tissue extracts have a certain inhibitory effect on glial cells.
Table 1. Inhibition of glial cells by extracts at different concentrations:
| Concentration (μg/mL) | Inhibition Rate (%) |
|-----------------------|--------------------|
| 200 | 51.2 ± 1.98 |
| 150 | 46.8 ± 2.11 |
| 75 | 38.5 ± 1.67 |
| 37.5 | 31.3 ± 1.32 |
| 18.75 | 27.6 ± 0.99 |
3 Discussion
Although modern medicine has widely used Agkistrodon in the clinical treatment of malignant tumors, literature reports are more common in oral clinical treatments. However, its chemical composition analysis and anti-tumor mechanism of action are less reported. Literature reports indicate that Agkistrodon contains hemolytic protein, cholesterol, amino acids, and other substances. To further explore the anti-tumor effects of Agkistrodon, an ultrasonic extraction experiment was conducted on Agkistrodon tissue, followed by a preliminary electrophoretic analysis and cytotoxicity experiments on the extract material for active ingredient testing. Experimental results show that the extract of Agkistrodon possesses antitumor activity. Our group will conduct further analysis and purification of Agkistrodon tissue extracts, studying the inhibitory mechanism of the active ingredients of Agkistrodon's antitumor effect, aiming to play a larger role in the clinical application of Agkistrodon in anti-tumor therapy.