## 4 Discussion  
With the increasing governmental regulation on the use of in-feed antibiotics in many commercial agricultural food production, various antibiotic alternative strategies have been developed and are being used in animal agriculture [15]. Among novel antibiotic strategies, phytochemicals have shown many benefits to promote growth, enhance immunity, and reduce oxidative stress in animal production and in disease management in recent years [16; 25]. Studies show that phytogenic extracts supplemented into the diet of broiler chicken not only improved digestibility and absorption [26; 27] but also enhanced immunity, reduced oxidative stress caused by pathogens, modulated gut microbiota, and improved animal health [16; 28]. This study investigated selected phytogenic extracts such as GRE, GTEC, GTED, and OPEC for their ability to prevent bacteria proliferation, kill protozoa (_E. tenella_), and mitigate the effect of disease-causing toxins associated with _C. perfringens_ in vitro. Our MIC and CFU results showed that selected phytogenic extracts GRE, GTEC, GTED, and OPEC mitigated the growth of _C. perfringens_ but at a higher concentration compared to BMD. The inhibitory action of phytogenic extracts including GRE, GTEC, and GTED on bacteria growth has been associated with their inherent antimicrobial properties [29; 30]. Therefore, our results agree with the findings of [31] which state that phytogenic product-based extracts altered the abundance of microorganisms in caecum cultures. In a similar manner, BMD increased the growth performance of broiler chickens and mitigated bacteria growth by increasing the diversity or richness of the intestinal microbiota [32]. This suggests that selected phytogenic extracts used in this study and BMD may be preventing the proliferation of pathogenic bacteria while promoting the growth of beneficial bacteria [33]. Phytogenic extracts containing catechins have been shown to bind to the bacterial lipid bilayer cell membrane, causing damage to it [34]. Likewise, the exposure of bacteria to flavonoids like quercetin and kaempfer found on onion skin degrades or inhibits bacteria growth [35]. In addition, in vitro evaluation of green tea and ginger extracts in _E. tenella_ sporozoites killing assay showed that GRE, GTEC, and GTED exert antimicrobial properties against coccidia. In this study, we used cNK-2, the core \(\alpha\)-helical region of cNK-lysin, as positive control, since it has been established that cNK2 notably exhibits a high antimicrobial activity and can kill _Eimeria_ sporozoites through the disruption of their membrane [36]. Our results show that 5 to 10 mg/mL of GTEC decreased the concentration of viable sporozoites compared to 100 mg/mL of cNK2. However, GTED had similar killing action at the same dosage compared to cNK2. The differences in the killing action of GTEC and GTED may be attributed to caffeine's ability to inhibit a wide variety of pathogenic microorganisms by passing through the cell wall of bacteria, where it inhibits DNA synthesis [37; 38]. Similarly, GRE reduced the concentration of sporozoites at a dosage of 1 to 10 mg/mL, compared to 100 mg/mL of cNK2. This aligns with the findings of a previous study that demonstrated the therapeutic properties of green tea to include anticoccidial properties [22]. Moreover, [PERSON] et al. [39] reported that ginger containing phenols and other compounds derived, such as gingerdiol, shogaols, gingerol, and gingerdione, may be responsible for the coccidiostatic effect of ginger when fed to broiler chickens. Overall, these results show that these selected phytogenic extracts have antimicrobial property. To further understand the effect of these selected phytogenic extracts on toxin encoding genes associated with _C. perfringens_, we analyzed the effect of these extracts on the expression of NetB, tpeL and Alpha toxin genes, as well as NetB production, by C. perfringens bacteria in vitro.