Phytosome Loading the Combined Extract of Mulberry Fruit and Ginger Protects against Cerebral Ischemia in Metabolic Syndrome Rats

The prevalence of ischemic stroke in metabolic syndrome (MetS) is continually increasing and produces a great impact on both qualities of life and annual healthcare budget. Due to the efficiency limitation of the current therapeutic strategy, the poor availability of polyphenol substances induced by the first pass effect and the beneficial effects of mulberry fruit and ginger on brain and MetS-related diseases together with the synergistic concept, the neuroprotective effect against ischemic stroke in MetS condition of phytosome containing the combined extract of mulberry fruit and ginger (PMG) has been considered. To explore the neuroprotective effect and possible underlying mechanism of PMG on brain damage in cerebral ischemic rat with MetS, male Wistar rats were induced MetS by high-carbohydrate high-fat diet (HCHF) for 16 weeks and subjected to the cerebral ischemia/reperfusion injury (CIRI) at the right middle cerebral artery (Rt. MCAO). PMG at doses of 50, 100, and 200 mg/kg were orally fed with for 21 days, and they were assessed brain damage, neurological deficit score, and the changes of oxidative stress markers, inflammatory markers, PPARγ expression, and epigenetic modification via DNMT-1 were performed. All doses of PMG significantly improved brain infarction, brain edema, and neurological deficit score. In addition, the reduction in DNMT-1, MDA level, NF-κB, TNFα, and C-reactive protein together with the increase in SOD, CAT, and GPH-Px activities, and PPARγ expression in the lesion brain were also observed. The current data clearly revealed the neuroprotective effect against cerebral ischemia with MetS condition. The possible underlying mechanism might occur partly via the suppression of DNMT-1 giving rise to the improvement of signal transduction via PPARγ resulting in the decreasing of inflammation and oxidative stress. In conclusion, PMG is the potential neuroprotectant candidate against ischemic stroke in the MetS condition. However, the clinical trial is still essential.


Introduction
Currently, stroke and metabolic syndrome (MetS) have been recognized as important health problems worldwide due to the increasing prevalence and the great impact on healthcare budget [1][2][3]. It has been demonstrated that MetS increases the risk of stroke [4][5][6] via the increase in intracranial atherosclerosis [7]. In addition, it can also increase oxidative stress and inflammation [8], the important factors playing the pivotal roles on the pathophysiology of ischemic stroke, and may increase severity of stroke [9]. Despite the increasing prevalence and importance of stroke with MetS, the therapeutic efficacy is not in the satisfaction level.
Recently, it has been demonstrated that the substance possessing antioxidant and anti-inflammation such as the combined extract of black stick rice and dill significantly improves brain damage and deficits in an animal model of ischemic stroke in MetS condition [10,11]. Our previous study also clearly demonstrates that the phytosome containing the combined extract of ginger rhizome and mulberry fruit (PMG) can also decrease oxidative stress and inflammation [12]. This raises the possibility that PMG which also possesses the aforementioned activities [13][14][15][16][17] can also protect against ischemic stroke in MetS condition. Currently, no data concerning the aforementioned effect are available. Therefore, this study was set up to determine the neuroprotective effect against ischemic stroke in the rat model of MetS induced by a high carbohydrate high fat (HCHF) diet. In addition, the possible underlying mechanism especially the roles of oxidative stress, inflammation, PPARγ signal transduction, and epigenetic mechanism modification were also determined.

Preparation of PMG.
The authentication of ginger rhizomes (Zingiber officinale Roscoe) which were harvested from Khon Kaen province, Thailand, was performed by the expert in pharmacognosy of the National Museum of THAI Traditional Medicine, Thailand and the voucher specimen No. 0002402 was deposited at the National Museum of THAI Traditional Medicine. Ripen mulberry fruit (Morus alba Linn. var. Chiangmai) used in this study was identified and kindly provided by the Queen Sirikit Department of Sericulture Center (Udon Thani province), Ministry of Agriculture and Cooperatives, Thailand, and the voucher specimen 61001 was deposited at Research Institute of Human High Performance and Health Promotion, Khon Kaen University, Khon Kaen, Thailand. The phytosome loading with the combined extract of ginger rhizome extract and ripen mulberry fruit was prepared as prepared according to the method of Palachai and coworkers [12]. In brief, 50% and 95% hydroalcoholic extract of mulberry fruit and ginger were prepared by using the maceration method. Then, the combined extract at a ratio of 1 : 1 was mixed and encapsulated with phosphatidylcholine matrix in order to produce phytosome containing the combined extract of mulberry fruit and ginger (PMG). The concentrations of active ingredients including total phenolic compounds, flavonoids, gingerol, cyanidin-3-Oglucoside, quercetin-3-rutinoside, ferulic acid, and gallic acid in PMG were as same as those mentioned in our previous study [12].

Experimental
Protocol. The experimental animals in this study were male Wistar rats, weighting 180-220 g (8 weeks old), from National Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand. The animals were kept under standard laboratory conditions at 23 ± 2°C and 12 : 12 hours light-dark cycle. They were provided food and water ad libitum and housed in standard metal cages (6 per cage) and allowed to accustom to the laboratory condition for one week. All procedures and experimental protocols were approved by the Institutional Animal Ethics Committee of Khon Kaen University (record no. IACUC-KKU 95/60).
After the acclimatization, all animals were randomly divided into 8 groups.
(a) Group I:Normal diet (ND) + vehicle: the animals in this group were provided a normal diet and treated with vehicle The normal diet used in this study contained 4.5% fat, 42% carbohydrate, and 24% protein whereas the HCHF diet contained 35% fat, 45% carbohydrate, and 20% protein. After the exposure to a 16-week-feeding period, rats which showed the full criteria of MetS as mentioned in the previous study [12] were recruited for further study. The recruited animals were randomly assigned to various treatments as mentioned earlier. Food, water intake, and body weight changes were recorded every week. Then, 21 days after treatment with the assigned substances, the MetS rats were subjected to the occlusion of the right middle cerebral artery and continually treated with the assigned substances for 14 days after Rt. MCAO induction. The neurological deficit scores were assessed at 24 hours after Rt. MCAO and every 7 days throughout the study period while brain infarction volume, brain edema, the determinations C-reactive protein in serum were determined at 24 hours after Rt. MCAO. The determination of oxidative stress markers including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and the expressions of DNA-methyltransferase 1 (DNMT-1), peroxisome proliferator-activated receptorgamma (PPARγ), nuclear factor kappa B (NF-κB), and tumor necrosis factor-alpha (TNFα) in the lesioned brain were performed at the end of the study. The experimental protocol was summarized in Figure 1 Oxidative Medicine and Cellular Longevity (I/R) induction technique. After the anesthetization with pentobarbital sodium at a dose of 60 mg/kg BW, the nylon monofilament coated with silicone was inserted from the right common carotid artery into the internal carotid artery and put advanced forward until felt the faint resistance, toward the junction of anterior and middle cerebral arteries. After the exposure to a 90-minute Rt. MCAO, the filament was withdrawn to allow reperfusion and wound was sutured [18].
2.4. Neurological Score Assessment. To assess the functional consequences of cerebral ischemia, an 18-scale-modified neurologic severity score (mNSS) was used as a tool for monitoring neurological deficit [19]. According to this assess-ment, various components including motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), reflex, and balance tests were evaluated. One point was given for the inability to perform each test, and an overall composite score was given to determine the neurological impairment. The details of grading scores assessed via an 18-scale-modified neurologic severity score (mNSS) test were provided in Table 1

Oxidative Medicine and Cellular Longevity
Louis, MO, USA) in the dark at room temperature for 15 minutes and turned over every 5 min. Then, all brain slices were captured using a digital camera (Sony HDR-SR11 Handycam Camcorder; Sony Co. Ltd., Japan), and the brain infarction volume in cortex, striatum, and hippocampus was calculated with the computerized image analysis system [10]. The areas of the cerebral cortex, the outer layer covering all infrastructures, and the main subcortical structures such as the hippocampus and striatum were measured and calculated for the brain infarcted volume.
2.6. Brain Edema Assessment. Brain water content was measured by the standard wet-dry method [19]. After 24 hours of reperfusion, the sacrifice and brain isolation were carried out. The left and right hemispheres were weighed (wet weight) and were dried at 100°C for 24 hours to determine the brain dry weight. The degree of brain edema was calculated by the following equation: 2.7. Assessment of Oxidative Stress Status. The ipsilateral cortex was isolated and homogenized with 0.1 M potassium phosphate buffer solution in a ratio of 10 mg: 50 μl. The brain tissue homogenate was used for determining oxidative stress status including malonaldehyde (MDA) level and the scavenging enzyme activities including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). The protein concentrations in brain tissue homogenate were assessed by using a Thermo Scientific NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA), and the optical density was measured at the wavelength of 280 nm. MDA level, a lipid peroxidation product, was monitored by using thiobarbituric acid (TBA) reaction [20]. The reaction mixture containing 50 μl of brain tissue homogenate, 50 μl of 8.1% sodium dodecyl sulphate (SDS) (Sigma-Aldrich, USA), 375 μl of 0.8% of thiobarbituric acid (TBA) (Sigma-Aldrich, USA), 375 μl of 20% acetic acid (Sigma-Aldrich, USA), and 150 μl of distilled water (DW) was boiled at 95°C in the water bath for 60 minutes. After incubation, it was cooled with tap water. Then, 250 μl of distilled water and 1,250 μl of the solution containing n-butanol and pyridine (Merck, Germany) at the ratio of 15 : 1 was added, mixed together, and centrifuged at 4,000 rpm for 10 minutes. The upper layer was separated and measured the absorbance at 532 nm. The standard curve was prepared by using TMP (1,1,3,3-tetramethoxy propane) at the concentrations of 0-15 μM as reference (Sigma-Aldrich, USA). MDA level was expressed as ng/mg protein.
SOD activity determination was performed according to the method of Sun et al. [21]. In brief, 20 μl of brain tissue homogenate was mixed with 200 μl of the reaction mixture containing 57 mM phosphate buffer solution (KH 2 PO 4 ) (Sigma-Aldrich, USA), 0.1 mM EDTA (Sigma-Aldrich, USA), 10 mM cytochrome C (Sigma-Aldrich, USA) solution, 50 μM of xanthine (Sigma-Aldrich, USA), and 20 μl of xanthine oxidase (0.90 mU/ml) (Sigma-Aldrich, USA). The absorbance at 415 nm was recorded. SOD enzyme (Sigma-Aldrich, USA) activities at the concentrations of 0-25 units/ml were served as standard, and the results were expressed as units/mg protein.
CAT activity was monitored based on the ability of the enzyme to break down H 2 O 2 . In brief, 10 μl of brain tissue homogenate was mixed with the reaction mixture containing 50 μl of 30 mM hydrogen peroxide (in 50 mM phosphate buffer, pH 7.0) (BDH Chemicals Ltd, UK), 25 μl of 4M H 2 SO 4 (Sigma-Aldrich, USA), and 150 μl of 5 mM KMnO 4 (Sigma-Aldrich, USA). Following this process, an absorbance at 490 nm was measured [22]. CAT enzyme (Sigma-Aldrich, USA) at the concentration range between 10-100 units/ml was served as standard, and the result was expressed as units/mg protein. Beam balance testes (normal = 0, maximum = 6) -Balance with steady posture 0 -Grasps side of beam 1 -Hugs beam and 1 limb falls down from beam 2 -Hugs beam and 2 limbs falls down from beam, or spins on beam (>60 s) 3 -Attempts to balance on beam but falls off (>40 s) 4 -Attempts to balance on beam but falls off (>20 s) 5 -Fall off, no attempt to balance or hang on the beam (<20 s) 6 Reflex absence and abnormal movements (normal = 0, maximum = 4) -Pinna reflex (head shake when auditory meatus is touched) 1 -Corneal reflex (eye blink when cornea is lightly touch with cotton) 1 -Startle reflex (motor response to a brief noise from clapping hands) 1 -Seizures, myoclonus, myodystony 1 Maximum points 18 4 Oxidative Medicine and Cellular Longevity GSH-Px activity was also determined. In brief, a mixture containing 20 μl of brain tissue homogenate was mixed thoroughly with the reaction mixture consisting of 10 μl of 1 mM dithiothreitol (DTT) (Sigma-Aldrich, USA) in 6.67 mM potassium phosphate buffer (pH 7.0), 100 μl of 1 mM sodium azide (Sigma-Aldrich, USA) in 6.67 mM potassium phosphate buffer (pH 7.0), 10 μl of 50 mM glutathione (Sigma-Aldrich, USA) solution, and 100 μl of 30% hydrogen peroxide (BDH Chemicals Ltd, UK), and incubated at 25°C for 5 minutes. At the end of the incubation period, 10 μl of 10mM DTNB (5,5-dithiobis-2-nitrobenzoic acid) (Sigma-Aldrich, USA) was added and an absorbance at 412 nm was recorded at 25°C over a period of 5 min [23]. Various concentrations of GSH-Px enzyme (Sigma-Aldrich, USA) between 1-5 units/ml were served as standard and the result was expressed as units/mg protein.
2.9. Statistical Analysis. All data are expressed as mean ± standard error of mean (SEM). Statistical significance was evaluated by using a one-way analysis of variance (ANOVA), followed by the post hoc (Tukey) test. Student's t test was used for comparing the means for two groups. Statistical significance was regarded at p value < 0.05. All statistical data analyses were performed using SPSS version 21.0 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows).  aaa p values < 0.001; compared between naïve control rats which received normal diet and vehicle, and metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle, bbb p values < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle, and metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle and * , * * * p values < 0.05 and 0.001, respectively; compared to metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: highcarbohydrate high-fat diet; MCAO: middle cerebral artery occlusion; vitamin C: the vitamin C at a dose of 250 mg.kg -1 BW; piracetam: the piracetam at a dose of 250 mg.kg -1 BW; PMG 50, PMG 100, and PMG 200: the phytosome containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg.kg -1 BW, respectively. 8 Oxidative Medicine and Cellular Longevity

Effect of PMG on Brain
Damage. The effect of PMG on the neurological deficit score was shown in Figure 2. The results showed that the MetS rats which received MCAO and vehicle showed the significant increase in neurological deficit score at 1, 7, and 14 days after MCAO (p values < 0.001 all; compared to the HCHF+sham operation+vehicle group), whereas sham operation failed to produce neurological deficit. However, the changes induced by MCAO were counteracted by all doses of PMG at 1, 7, and 14 days after MCAO (p values < 0.001 all; compared to the HCHF+MCAO+vehicle group). In addition, both piracetam and vitamin C also significantly improved neurological deficit score at 1, 7, and 14 days after MCAO (p values < 0.05 and 0.01, respectively; compared to the HCHF+MCAO+vehicle group). Figure 3 showed that the MetS rats which received sham operation failed to produce brain infarction, whereas MCAO and vehicle significantly increased brain infarction volume in cortex, striatum, and hippocampus (p values < 0.001 all; compared to the HCHF+sham operation+vehicle group). Piracetam treatment significantly improved brain infarction in cortex and striatum (p values < 0.05 and 0.01, respectively; compared to the HCHF+MCAO+vehicle group), whereas vitamin C significantly attenuated brain infarction only in striatum (p values < 0.05; compared to the HCHF+MCAO +vehicle group). Interestingly, all doses of PMG produced the significant reduction in brain infarction volume in the cortex, striatum, and hippocampus (p values < 0.001 all; p values < 0.001 all; p values < 0.01 all; compared to the HCHF+MCAO+vehicle group).
The effect of PMG on brain edema was shown in Figure 4. The results revealed that the contralateral hemisphere of MetS rats which subjected to I/R failed to produce the significant changes of brain water content. However, the significant change in brain water content was observed in the  Oxidative Medicine and Cellular Longevity ipsilateral hemisphere of MetS rats which received MCAO and vehicle (p values < 0.01; compared to the HCHF+sham operation+vehicle group). All doses of PMG, vitamin C, and Piracetam treated groups significantly decreased brain water content (p values < 0.05 all; compared to the HCHF+MCAO+vehicle group). Table 2 demonstrated the effect of PMG on oxidative stress status in the cerebral cortex including the level of MDA and the activities of the main scavenger enzymes including SOD, CAT, and GSH-Px. It was found that sham operation failed to show the significant change in oxidative stress status. MetS rats which subjected to MCAO and received vehicle significantly decreased SOD, CAT, and GSH-Px activities (p values < 0.001 all; compared to normal diet group and p values < 0.001 all; compared to the HCHF+sham operation+vehicle group). MetS rats which subjected to MCAO and received vitamin C significantly increased SOD and GSH-Px activities but decreased MDA level (p values < 0.01, 0.05, and 0.05, respectively; compared to the HCHF+MCAO+vehicle group), whereas piracetam failed to produce the significant changes on aforementioned parameters. PMG treatment at doses of 50 and 100 mg/kg also produced the significant decrease in MDA level (p values < 0.05 all; compared to the HCHF+MCAO+vehicle group) together with the significant increase in SOD (p values < 0.05 and 0.001, respectively; compared to the HCHF+MCAO+vehicle group), CAT (p values < 0.01 all; compared to the HCHF+MCAO+vehicle group), and GSH-Px activities (p values < 0.05 and 0.001, respectively; compared to the HCHF+MCAO+vehicle group) in the cerebral cortex of MetS rats with MCAO. In addition, PMG treatment at a dose of 200 mg/kg also significantly decreased MDA but increased SOD and GSH-Px (p values < 0.05, 0.01, and 0.05, respectively; compared to the HCHF+MCAO+vehicle group). However, no significant change of CAT was observed in MetS rats with cerebral ischemia and received the high dose of PMG.

Effect of PMG on Biochemical
Changes. The effect of PMG on inflammatory markers such as NF-κB, TNFα in cortex was assessed and the results were shown in Figures 5  and 6. It was found that MetS rats which subjected to sham operation failed to produce the significant change on the expression of NF-κB in the cerebral cortex. MCAO significantly enhanced NF-κB expression in the aforementioned area (p values < 0.001; compared to normal diet fed rats which received vehicle; p values < 0.001; compared to compared to the HCHF+sham operation+vehicle group). The elevation of NF-κB expression was attenuated significantly by vitamin C and all doses of PMG (p values < 0.05, 0.05, 0.001, and 0.05, respectively; compared to the HCHF HCHF+MCAO+vehicle HCHF+MCAO+PMG100 HCHF+MCAO+vitamin C HCHF+MCAO+PMG200 Figure 7: Effect of various doses of PMG on C-reactive protein in serum. Data are presented as mean ± SEM (n = 6/group). aaa p values < 0.001; compared between naïve control which received normal diet and vehicle, and metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle, bbb p values < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle, and metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle and * , * * p values < 0.05 and 0.01, respectively; compared to metabolic syndrome+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MCAO: middle cerebral artery occlusion; vitamin C: the vitamin C at a dose of 250 mg.kg -1 BW; piracetam: the piracetam at a dose of 250 mg.kg -1 BW; PMG 50, PMG 100, and PMG 200: the phytosome containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg.kg -1 BW, respectively. 10 Oxidative Medicine and Cellular Longevity +MCAO+vehicle group). Piracetam failed to produce the significant change of this parameter. The effect of PMG on TNFα expression in the cerebral cortex was shown in Figure 6. The similar pattern of changes was also observed. MetS rats which exposed to sham operation failed to produce the significant change on TNFα expression. However, MCAO produced a significant increase in TNFα expression (p values < 0.001; compared to normal diet-fed rats which received vehicle; p values < 0.001; compared to compared to the HCHF+sham operation+vehicle group). This elevation was counteracted by vitamin C treatment (p values < 0.05; compared to the HCHF+MCAO +vehicle group). In addition, all doses of PMG also significantly decreased TNFα expression (p values < 0.001 all; compared to the HCHF+MCAO+vehicle group).
The effect of PMG on serum C-reactive protein level was also investigated and results were shown in Figure 7. MetS rats which subjected to sham operation failed to produce the significant modulation on this parameter whereas MCAO significantly enhanced C-reactive protein level in serum of MetS rats (p values < 0.001; compared to normal diet-fed rats which received vehicle; p values < 0.001; compared to compared to the HCHF+sham operation+vehicle group). These changes were attenuated by vitamin C and both medium and high doses of PMG (p values <0.05, 0.05, and 0.01, respectively; compared to the HCHF+MCAO+vehicle group).
The effect of PMG on PPARγ expression in the cerebral cortex was shown in Figure 8. It was found that MetS rats induced by HCHF diet which subjected to sham operation failed to produce the significant modulation on PPARγ expression, whereas MCAO significantly decreased PPARγ expression in the cerebral cortex of MetS rats (p values < 0.001; compared to normal diet-fed rats which received vehicle;  Figure 9 showed the modification effect of PMG on the epigenetic mechanism was assessed by using DNMT-1 as indicator. Sham operation failed to produce the significant modulation on DNMT-1 expression in the cerebral cortex of MetS rats induced by HCHF diet whereas MCAO significantly enhanced DNMT-1 expression in the cerebral cortex of MetS rats (p values < 0.001; compared to normal diet-fed rats which received vehicle; p values < 0.01; compared to compared to the HCHF+sham operation+vehicle group).

Discussion
The current study has clearly demonstrated that the phytosome containing the combined extract of mulberry fruit and ginger significantly improves neurological deficit and brain damage in an animal model of stroke in MetS condition induced by the I/R injury at the middle cerebral artery in HCHF diet-fed rats. The oxidative stress status, inflammation, and PPARγ together with DNMT-1 are also improved.

Oxidative Medicine and Cellular Longevity
Our data have demonstrated that MetS rats with cerebral ischemia show the elevation of oxidative stress and inflammatory markers in the brain. These findings are corresponding with our previous study [11]. These changes give rise to brain damage which in turn induces the failure of ATPase pumps which resensitive to free radical reactions and lipid peroxidation. The impairment of ATPase pumps especially Na + -K + ATPase triggers brain edema [25] which in turn significantly influences the degree of neurological deficit [26]. Based on these pieces of information, we suggest that the improvement of brain edema, brain damage, and neurological score deficit in this study may occur partly via the improvement of oxidative stress and inflammation. It has been revealed that both oxidative stress and inflammation are under the influence of PPARγ. PPARγ agonists suppress both processes and give rise to the neuroprotection against cerebral ischemia [27,28]. Recent findings also reveal that the expression of PPARγ is under the influence of epigenetic modification, the modification of the mediator between genetic and environment. DNA hypermethylation suppresses the expression of PPARγ which in turn suppresses the anti-inflammatory cytokine production via the activation of M2 macrophages [29] which in turn increases inflammatory reaction, cell proliferation, and differentiation of foam cells leading to atherosclerosis and ischemic stroke [30,31]. Therefore, the increase PPARγ expression in this study may partly relate to the decrease of DNA methylation.
These findings are corresponding with our data. It has been shown that PMG significantly decreases methylation of DNA via the reduction of DNMT-1 expression which in turn increases PPARγ expression. Taken all data together, we suggest that PMG significantly suppresses the expression of DNMT-1 giving rise to the increase in PPARγ expression leading to the decrease in inflammatory markers such as TNFα, NF-κB, and C-reactive protein leading to the decrease of brain infarction and brain edema resulting in the improvement of neurological deficit. The increase in PPARγ expression may also improve both parameters just mentioned either via the decrease in oxidative stress status [35] which in turn directly decreases both brain infarction and brain edema [36] or indirectly via the reduction of inflammation and results in the improvement of both brain infarction and brain edema as mentioned earlier. Since PMG also possesses both antioxidant and anti-inflammation [12], it may be possible to improve brain infarction and brain edema via direct its effect.
It has been revealed that both ginger extract and mulberry fruit extract can improve oxidative stress status by enhancing antioxidative enzymes giving rise to the reduction of brain damage and dysfunction [13,14,37]. Based on this information and the antioxidant and anti-inflammation of PMG mentioned earlier, we suggest that the neuroprotective effect of PMG observed in this study may be associated with the combined extract of mulberry fruit and ginger of PMG.

Conclusion
The present study has demonstrated that PMG is the potential candidate to protect against brain damage and dysfunction induced by the occlusion of the middle cerebral artery

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Oxidative Medicine and Cellular Longevity in MetS condition. PMG can produce the direct suppression effect on oxidative stress and inflammation. In addition, it can increase antioxidant enzymes which in turn decrease oxidative stress status. Moreover, the decrease of both inflammation and oxidative stress status can also occur as the result of the suppression of DNMT-1 giving rise to the increase of PPARγ expression leading to the decrease in oxidative stress and inflammation and finally result in the improvement of brain infarction and brain dysfunction. The improvement of brain edema appears to occur as the result of the improvement of inflammation either via the increased PPARγ expression induced by the suppression of DNMT-1 mentioned earlier or via the reduction of oxidative stress as shown in Figure 10. However, further research in clinical trial study is very much essential to confirm this neuroprotective effect.

Data Availability
I confirm that data are available and will be provided on request because during this period, all data are in the process of petty patent registration.

Conflicts of Interest
The authors declare that they have no conflicts of interest.