## 4 Discussion  
The tendency to use traditional medicines in the treatment of many diseases has been increasing. Numerous studies have proven that plant-based medicines have not only demonstrated the ability to prevent and treat diseases effectively, but also limited side effects and adverse effects of modern medicines. Plants are rich in natural bioactive compounds, such as phenolics, flavonoids, and their derivatives. These compounds have obtained attention because of their bioactivities and physiological functions, including antioxidant, anti-allergic, anti-inflammatory, antimicrobial, and antidiabetic activities [42]. From methanol crude extract of _E. hirta_ (EH-Me extract) containing multiple different groups of phytoconstituents, liquid-liquid extraction technique can be employed to separate many different fractions. As a result, five extracts of _E. hirta_ were obtained based on their polarity by specific solvents including petroleum ether, chloroform, ethyl acetate and butanol extracts. Qualitative phytochemical analysis of five extracts of _E. hirta_ was carried out to evaluate the bioactive compounds in each extract. The concentration of bioactive compounds in the plant extracts depends on the solvent polarity that was used in the extraction preparation [33; 43]. The extraction method is the first step to separate phytochemicals from raw materials. According to the principle of extraction, solvent extraction is the most popularly used method. In this method, solvent polarity significantly affects the efficiency of extraction and constituents of phytochemicals. Depending on chemical nature, a variety of secondary metabolites are extracted in solvents of different polarity as no single solvent may reliably extract all the phytochemical and antioxidant compounds present in the plant material. The serial exhaustive extraction method involves the successive extraction with solvents of increasing polarity from non-polar (petroleum ether) to more polar solvent (water) to ensure the extraction of a wide range of compounds with different polarity. The ascending order of the polarization of solvents is petroleum ether \(<\) chloroform \(<\) ethyl acetate \(<\) butanol \(<\) methanol. Therefore, methanol was used to extract raw material. In relation to the solvent used, the highest concentrations of total phenolic and flavonoid contents were found in EH-EA extracts. EH-EA had the highest total phenol content  
Figure 3: Cytotoxicity of _E. hirta_ L. on NCI H460 and Hep G2 cells. (\(254.96\pm 10.05\) mg GAE/g extract) and the highest total flavonoid contents (\(27.66\pm 0.73\) mg quercetin/g extract). The results of the determination of total phenolic content and total flavonoid content of these extracts were almost compatible with previous studies [44]. From the above results, it can be predicted that EH-EA extract has better activity in biological activity trials. For the purpose of screening biological activities of these extracts, we investigated antibacterial, antioxidant, lipid peroxidation inhibitory and anticancer properties. Five _E. hirta_ extracts were investigated to evaluate their antibacterial activity against pathogenic bacteria including two strains of Gram-positive bacteria and three strains of Gram-negative bacteria using the agar disc diffusion method. The strains of bacteria selected for antibatesting cause diarrhea, acute diarrhea and pneumonia, etc., which are diseases with a high mortality rate. _E. coli_ are a large and diverse group of aerobic bacteria in the environment and food, and either live in the digestive tract of humans or animals. _E. coli_ have contributed to many important functions in human life such as stimulating immunity, improving the metabolization of sugars, and preventing the attack of other bacteria from entering the digestive system. However, they can cause diseases such as urinary tract infection, meningitis, etc. _S. aureus_ is a major bacterial human pathogen that causes a wide variety of clinical manifestations.