**6. Green tea for treatment of acute ischemic stroke**

*Camellia sinensis* with the active ingredient EGCG inhibits neuronal cell death through apoptosis (increased expression of BCL-2 and decreased expression of Caspase-3) and necroptosis (decreased expression of TNFR1 and RIP 3) in the Rattus norvegicus model of Middle Cerebral Artery Occlusion (MCAO) [31].

A study conducted with R. norvegicus model Middle Cerebral Artery Occlusion (MCAO) found the effect of Camelia sinensis with the active ingredient EGCG on HO-1 expression. Inhibition of HO-1 expression started with doses of 1, 20, and 30 mg/kg BW. So that indicates that the antioxidant properties of green tea can inhibit HO-1 expression starting from the lowest dose in this study. The group with 30 mg/KgBW green tea extract intervention also showed a significant difference compared to the control group. Thus, green tea extract and its active ingredient, namely EGCG, showed an inhibitory effect on HO-1 expression. HO-1 protein is an active protein due to oxidative stress through the Nrf-2 pathway; this decrease in HO-1 expression indicates that the administration of green tea extract and its active ingredient (EGCG) can reduce oxidative stress in stroke models [30, 46].

The same study found no difference between the levels of HMGB-1 in control compared to all interventions, and this indicates that neither green tea nor the active ingredient EGCG affects HMGB-1 levels. The results that showed no difference indicated that HMGB1 in this study was secreted passively by stressed cells so that it could not be inhibited by EGCG or green tea extract [30, 46].

*C. sinensis* with the active ingredient EGCG also influences the expression of TNFR1 in the MCA model. Significant differences in TNFR1 expression in the intervention group of EGCG 20 mg/kgBW, EGCG 30 mg/kgBW, and green tea extract 30 mg/kgBW compared to the control group. The effect of EGCG and green tea extract on decreasing TNFR1 expression started at a dose of EGCG 20 mg/kgBW, and this shows that both green tea extract and its active ingredient, EGCG, can reduce inflammation that occurs in brain tissue affected by stroke. Inflammation is one of the pathways of cell damage caused by ischemia. TNFR1 protein is the primary receptor of TNF- which will cause the active process of necroptosis and inflammation [30].

Furthermore, a decrease in RIP3 expression was found after green tea intervention, either using the active ingredient in the form of EGCG or green tea extract. A significant difference was also found in the RIP3 expression of the intervention group EGCG 20 mg/kgBW, EGCG 30 mg/kgBW, and green tea extract 30 mg/kgBW compared to the control group. RIP3 protein is an executor protein in the apoptotic process, so the decrease in RIP3 expression indicates that EGCG and green tea extract can prevent necroptosis in the MCAO model. The inhibition of RIP3 expression was

initiated at a dose of EGCG 20 mg/kgBW. Green tea extract 30 mg/kgBW also has a similar effect to EGCG 30 mg/kgBW [30].

Bcl-2 is a protein that is a major regulator of mitochondrial permeability and the release of pro-apoptotic molecules. BCL-2, together with Bcl-xL, are anti-apoptotic proteins located in mitochondria and endoplasmic reticulum. In mitochondria, BCL-2 maintains mitochondrial integrity and prevents apoptogenic molecules' release [75–78]. *C. sinensis* with the active ingredient EGCG affects the expression of BCL-2. Based on reserch results, there were significant differences in the expression of BCL-2 in all intervention groups compared to the control group. In the intervention group, EGCG 20 mg/kgBW, EGCG 30 mg/kgBW and green tea extract 30 mg/kgBW found a very significant difference when compared to the control. These results indicate that both EGCG and green tea extract can increase anti-apoptotic protein so that it will prevent apoptosis [31].

A significant difference was found in the expression of Caspase-3. Caspase-3 expression was lower in the EGCG 30 mg/kgBW group and the green tea extract group. These results indicate that EGCG and green tea extract can prevent apoptosis by inhibiting the apoptotic execution pathway. The inhibition of this execution pathway is very important because there are three apoptotic pathways: the intrinsic or mitochondrial pathway, the extrinsic pathway, and the granzyme pathway. If one of them is inhibited, other pathways will activate apoptosis, but if the executor caspase is inhibited, the apoptotic pathways that are active can be inhibited. Inhibit the apoptotic pathway by increasing the anti-apoptotic protein or down-regulating the pro-apoptotic protein caspase-3 [30, 63, 66].

Our study also shows that green tea can increase anti-inflammatory mediators that will increase recovery after stroke. We also use animal model and immunohistochemistry to measure CD 206, a marker for M2-type mitochondria. According to our research, there is an increase in CD 206 expression in both Green tea and EGCG group [46, 79].

The study on the benefit of green tea should be conducted in the clinical setting to know about the benefit of green tea in acute ischemic stroke. Its potential benefit can be as an adjunct treatment for acute ischemic stroke besides standard treatment.
