Mangosteen Pericarp Inhibits Nuclear Factor κ B ( NF-κ B ) Activation and Reduces Expression of IcAM-1 in High cholesterol Diet Rat

Background: Atherosclerosis is widely viewed as an inflammatory disease with hypercholesterolemia being a dominant underlying risk factor. This study aimed to determine the effect of mangosteen pericarp in inhibition of NF-κB activation and ICAM-1 expression in rat fed with high cholesterol. Methods and Results: Various doses of crude extract mangosteen pericarp were administered to the high fat diet wistar rats and the activity of NF-κB measured by immunohistochemistry to assess nuclear NF-κB expression and the ICAM-1 expression. The high fat diet resulted significant increased serum LDL levels. Increased nuclear NFκB activation and ICAM-1 expression were also observed in high fat diet rats in concurrence with increased serum LDL. The inhibitory effect on NFκB activity and ICAM-1 expression was observed when 400 mg of mangosteen pericarp crude extract was administered and even showed a higher inhibitory effect in 800 mg of mangosteen pericap treated rats. The 800 mg extract treatment resulted in decreased ICAM-1 expression similar to those of non high fat rats. Conclusion: The administration of 800 mg mangosteen pericarp crude extract significantly inhibited NF-κB activation and reversed the expression of ICAM -1 to the normal level in high cholesterol diet rats.

believed to be initiated by retention of LDL particles in the lesionprone areas, which is followed by monocyte recruitment and their differentiation into cholesterol laden macrophage foam cells.Excessive cholesterol accumulation in macrophages exaggerates innate immune response that is manifested by up regulated production and secretion of inflammatory cytokines and chemokines, thus dramatically amplifying initial signal originated from the injured artery. 4olecular pathogenesis researches are warranted to dissect the mechanism and explore the possible in hibitory ways by a new effective drug.One alternative a candidate anti inflammatory agent is mangosteen pericarp which contains many antioxidant substances including xanthone. 5his study therefore aimed to examine whether mangosteen pericarp inhibits inflammatory processes through the transcription factor NFκB.The expres sion of ICAM 1, an adhesion molecular target of NFκB, was also measured to see the consequence of NFκB inhibition.

Materials and Methods
Thirty 34 monthold healthy male Rattus novergicus wistar strain (150 300 gram) were randomly divided into 5 groups.4 groups fed with high cholesterol diet and 1 group fed with non high cholesterol (as a con trol negative group).The compound of diet consist of 67% comfeed PARS, 33% of the flour, and water sparingly (normal diet), and hipercholesterol diet consists of 50% comfeed PARS, 25% flour, 2% cholesterol, 0.2% cholic acid, 5% oil (lard), and water.Mangosteen pericarp crude extract were administered by gastric sonde route.Among 4 groups of high cholesterol rats, 3 groups were treated with 200 mg/kg body weight (kbw), 400 mg/kbw and 800 mg/kbw of mangosteen pericarp crude extract, respectively.1 group of high fat diet rats without treatment functioned as a posi tive control group.After 12 weeks treatment, NFκB activation and ICAM1 expression was checked by immunohistochemistry methods, respectively.
Mangosteen crude extract preparation: First, Drying Process wash started by washing the hull of mangosteen cleanly, cutting in small size and finally keeping in the 80 degrees oven or with the sun hot until dry (moisture free), second, The process of extraction: Once dry, puree with a blender until smooth, weigh as much as 100 grams of dried sample (mangosteen hull) into the erlenmeyer glass size 1 liter, then soak with volume 900 ml of ethanol up, shake until completely mixed (± 30 minutes), let sit for 1 night until it settles, and third, The process of evaporation: take the top layer of a mixture of ethanol with active substances that have been fetched, put in the evaporation tube 1 liter, attach evaporation tube in evaporator, fill water bath with water until it is full, all pairs of sets of tools including rotary evaporator, heater water bath (set up to 90 degrees), let the solution of ethanol with active substance splitting existing in the pumpkin, wait until the flow stops dripping on ethanol reservoir tube (± 1.5 to 2 hours for 1 tube), the results obtained roughly 1/3 of the dry natural materials, put extraction results in a plastic bottle, store in freezer.

Immunohistochemistry using the monoclonal anti-ICAM-1
Cells are washed with HEPES buffer for 30 minutes and fixation with methanol for 5 minutes, dry and wash with PBS pH 7.4, application of 3% H 2 O 2 for 10 minutes and wash with PBS pH 7.4.Bloking using serum 5% FBS containing 0.25% Triton X100 and incubation for 1 hour at room temperature.Wash with PBS pH 7.4 give drops with antiGoat IgG (secondary antibodies) biotin labeled and incubation for 1 hour, wash with PBS pH 7.4 and give drops with SAHRP for 40 minutes, then wash with PBS pH 7.4 and apply for HRP substrate, i.e.DAB (Diamono Benzidine).Counterstain with Mayer hematoxilin for 10 minutes, rinse with tap water and washing with dH 2 O. Drain and shut the cover glass.
Observe under the microscope with magnification up to 200 x until 100 endothelial cells is achieved by three different laboratory personnel who are experi enced.Expression of ICAM1 in endothelial cells are seen in the brown color on the cell membrane endothe lial cell.Note the number of endothelial cells that are brown in between the endothelial cells of 100.

Observation on the expression of NF-κB activation Aortic Tissue by Immunohistochemistry
Slide washed with PBS pH 7.4 one time for 5 minutes, endogenous bloking using 3% peroxide H2O2 for 20 minutes, wash using PBS, pH 7.4, three times for 5 minute, bloking using unspesifik protein 5% FBS containing 0.25% Triton X100, wash using PBS, pH 7.4, three times for 5 minutes.Incubation using monoclonal antibodies of NFκB activation (p65) (LabVision), during the 60 minutes, wash using PBS, pH 7.4, three times for 5 minutes, incubation using anti mouse HRP conjugated for 40 minutes.Wash us ing PBS, pH 7.4, three times for 5 minutes.Give drops with a DAB (Diamino Benzidine) and incubation for 10 minutes.Wash using PBS, pH 7.4, three times for 5 minutes, wash using dH20, for 5 minutes.The counterstaining using Mayer Hematoxilen incubated for 10 minutes and wash using tap water, rinse using dH2O and dried.Mounting using entelan and cover with cover glass, and observe the light microscope.
Serum LDL cholesterol: total cholesterol levels in the blood of every mg/dl, is called hypercholesterolemia if taken more than 2 times the levels of control.
Translocation of NF-κB activation: endothelial cell number which is express p65 (NFκB activation) in the cytoplasm and nucleus of the endothelial cells that are painted with immunohistochemistry method on one cell well disc.It said if the number of p65 transloca tions in the cell nucleus more than in the cytoplasm.Endothelial cells that express p65 NFκB activation marked with a brown color in the cytoplasm and the nucleus.Measured using a semiquantitative method (endothelial cell number that brown among 100 en dothelial cells) and viewed with a light microscope (nikon) at 200 x magnification.

Expression of ICAM-1:
The number of endothelial cells that express ICAM1 protein in the cytoplasm of endothelial cells that were painted by immunohis tochemistry methods on one well disc.Endothelial cells expressing ICAM1 is characterized by brown color on the cell membrane.Measurement of endothelial cells expressing ICAM1 using a semiquantitative method (the number of endothelial cells brown among the 100 endothelial cells) and viewed with a light microscope (Nikon) at 200X magnification.

Statistical Analysis
Data analysis, which were performed on One way ANOVA, and continued with Post Hoc Analyses, to examine the effect of mangosteen pericarp with variety doses about NFκB activation and ICAM1 expression on high cholesterol diet treatment group, significance level 0.05.Analyses were performed with SPSS version 17.0 for Windows (SPSS, Inc, Chicago, Illinois).

Figure 1. In the negative control group (A) occurs the expression of NFκB activation (arrow), the positive control group who received highcholesterol diet (B) increased expression of NFκB activation (arrows).
On treatment with mangosteen peel extract dose of 200 mg/kg (C) is still there appeared to be increased expression of NFκB activation.But at a dose of 400 mg / kg (D) and the dose of 800 mg / kg (E) began to appear decreased expression of NFκB activation.
Figure 2. In the negative control group (A) occurs the expression of ICAM1 (arrow), the positive control group who received highcholesterol diet (B) increased expression of ICAM1 (arrows).On treatment with mangosteen peel extract dose of 200 mg/kg (C) is still there appeared to be increased expression of ICAM 1.But at a dose of 400 mg/kg (D) and the dose of 800 mg / kg (E) began to appear decreased expression of ICAM 1.
Table 2.In the negative control group occurred expression of NFκB activation with the lowest value     highcholesterol diet increased the mean expression of ICAM 1, with the highest yields (51.33).On treatment with mangosteen peel extract dose of 200 mg/kg was still an increase in the mean expression of ICAM1 (45.67), and with increasing doses starting to look a decrease in the mean expression of ICAM 1, at doses of 400 mg/kg (37.50) and at doses of 800 mg/kg (26.67).

Effect of high dietary cholesterol on the activation of NF-κB in the positive control than the negative control
After the test conducted by One way ANOVA and Post Hoc analysis, as found in Figure 3 obtained the result that there are significant differences in the expression of NFκB activation between the mice fed a cholesterol     diet (positive control group) with rat not given a diet high in cholesterol (negative control group), (positive control: 71.83; negative control : 29.67; p = 0.000).
In Figure 4 obtained the result that there are sig nificant differences in ICAM1 expression between rat fed a cholesterol diet (positive control group) with mice not given a high cholesterol diet (negative control), (positive control : 51.33; negative control : 20.50; p = 0.000).

Effect of various doses of mangosteen peel extracts on NF-κB activation and ICAM -1 expression in aortic endothelial cells of mice that were given high-cholesterol diet
Based on the ANOVA test shows that there is a sig nificant difference in effect between the five treatment groups on the expression of NFκB activation, and expression of ICAM1 (pvalue = 0.000).
Based on the results of Post Hoc test seen that the addition of mangosteen peel extracts on the mice fed highcholesterol diet lowers the expression of NF κB activation and ICAM1.Dose of 400 mg/kg of mangosteen peel extract has the effect of decreased expression of NFκB (pvalue = 0.000) and ICAM1 (pvalue = 0.000) when compared with positive control group.Dose of 800 mg/kg of mangosteen peel extract has the effect of decreased expression of NFκB (p value = 0.000) and ICAM 1 (pvalue = 0.000) when compared with positive control group.
Thus concluded that the addition of mangosteen peel extract of 800 mg/kg in mice fed a high cholesterol diet is the best dose to reduce the expression of NFκB activation and expression of ICAM1, although the inhibitory effect of ICAM 1 expression and activation NFκB has been significant with a dose of 400 mg/kg with significant results.

Discussion
Dyslipidemia is marked by elevated levels of plasma cholesterol and (or) triglycerides (TG) or low levels of highdensity lipoprotein (HDL) that contribute to the development of atherosclerosis. 6herosclerosis is a chronic disease of the arte rial wall where both innate and adaptive immuno inflammatory mechanisms are involved.Inflammation is central at all stages of atherosclerosis.It is implicated in the formation of early fatty streaks, when the en dothelium is activated and expresses chemokines and adhesion molecules leading to monocyte/lymphocyte recruitment and infiltration into the subendothelium.It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading to rup ture and thrombus formation.Cells involved in the atherosclerotic process secrete and are activated by soluble factors, known as cytokines. 7DL oxidation has a key role in the activation and facilitation of atherogenesis.The signals in the form of oxidized lipoproteins and reactive oxygen species (ROS) lead to the activation of transcription factors such as nuclear factorκB (NFκB) that are involved in the regulation of expression of ICAM1. 8any studies have shown that free radicals cause oxidative damage to fats, and nucleic acids.Antioxidants seem very important in the prevention of degenerative diseases (including heart disease and blood vessels), because it can inhibit the for mation of the substrate chain reaction that can be oxidized. 9nvitro studies had explained that activation of oxLDLmediated endothelial LOX1linked signaling pathway causing proinflammatory respons.LOX1 activation can stimulate ROS production and activate nuclear factor κB (NFκB).Activation of NFκB and translocation into cell nucleus, result in increase of proinflammatory and adhesion molecules coding genes, i.e. tumor necrosis factorα, ICAM1, and VCAM1. 10xygen radicals react readily with cellular phos pholipids and proteins, causing lipid peroxidation and oxidation of thiol groups with subsequent alteration of membrane ultrastructure and dysfunction of various cellular proteins.The antioxidants are known to inter fere with the free radical formation, and antioxidant reserve and enzyme capacity are significantly reduced following ischemia and reperfusion. 5Several studies have shown that antioxidant nu trients and (or) natural medicine positively modulate the susceptibility of LDL to oxidation and enhance the antiatherogenic properties of HDL, thus playing an important role in the prevention of cardiovascular diseases. 11t has been reported that some free radical scaven gers and antioxidants prevent arrhythmias and cardiac injury induced by myocardial ischemiareperfusion.Xanthones have been described as strong scavengers of free radicals and antioxidants.They exhibit concentra tiondependent scavenging activity toward superoxide anions, hydroxyl and peroxyl radicals. 5he fruit hull of mangosteen, G. mangostana, has been widely used as an antiinflammatory medicine in Southeast Asia for many years. 11angostin contained in mangosteen peel, that is an effective inhibitor of LDL peroxidation in vitro as indicated by lagtime to oxidation and malondialdehyde production.Derivatives of the parent compound have been produced and may offer further benefits and therapeutic potential. 12lavonoids and quinones have received the most attention as phenolic antioxidant derivatives and much is known about the structural requirements for antioxidant activity.Therefore, so many other phenolic compounds such as xanthone have been shown to act as scavengers of various oxidizing species; superoxide anion (O 2 -), hydroxy and peroxyradicals. 8Evidence that antioxidant micronutrients poten tially reduce the risk of CHD comes from four major sources.First, studies of antioxidant supplementation in animal models of atherosclerosis have generally shown a reduction in disease.Second, many studies have now shown that antioxidant supplementation in healthy subjects or patients with CHD can reduce lev els of free radical damage products and protect LDL against oxidation.Third, large scale epidemiological studies generally show that low intakes of antioxi dants are associated with increased cardiovascular risk after correcting for other risk factors.Thus, there is a plausible case supported by experimental studies, animal experiments, and epidemiology linking oxida tive stress and atherosclerosis.The key test of such a hypothesis is whether increased antioxidant intake can be shown to prevent the clinical manifestations of atherosclerosis in humans.Several published ran domised studies have now considered this issue, and others are currently ongoing.Early results have not been encouraging. 14ere is substantial evidence to suggest that xan thones and xanthone derivatives may be potentially useful as pharmacological agents in the treatment or prevention of cardiovascular diseases, including ischemic heart disease, atherosclerosis and hyperten sion.The protective effects of xanthones in the car diovascular system may be due to their antioxidant, antiinflammatory, platelet aggregation inhibitory, antithrombotic and or vasorelaxant activities.In par ticular, the antagonism of endogenous NOS inhibitors by xanthones may represent the basis for improved endothelial function and for reduction of events as sociated with atherosclerosis.However, the precise effects of xanthones need to be further elucidated in animal experiments in vivo and in humans.Moreover, pharmacokinetics, toxicity and structural optimization of xanthones should also be explored. 5

Conclusion
There is an increased activation of NFkB and increased expression of ICAM1 significantly (p < 0.05) in rat fed high cholesterol diet (positive control) compared to negative control.There are inhibition to activation of NF κB and decreased expression of ICAM1 in rat fed high cholesterol diet with mangosteen peel extract in various doses.The 400 mg and 800 mg dose of mangosteen peel can inhibit NF κB and ICAM1 significantly.

Figure 1 .
Figure 1.A, B, C, D, E Activation of NFκB Expression in aortic endothelial cells.

Figure 3 .
Figure 3. Graph of the effect of high dietary cholesterol on the activation of NFκB in aortic endothelial cells.Data expressed as average number, n = 6, significant if the value of p <0.05.

Figure 4 .
Figure 4. Graph of the effect of high dietary cholesterol on the activation of ICAM1 in aortic endothelial cells.Data expressed as average number, n = 6, significant if the value of p <0.05.

Figure 5 .Figure 6 .
Figure 5. Graph Effect of mangosteen peel extract on NFκB activation in endothelial cells aorta rats fed highcholesterol diet.Data expressed in mean number of endothelial cells expressing NFκB activation, n = 6, significant when the pvalue <0.05.* Normal diet vs High cholesterol diet p = 0.000 a Normal diet vs (High cholesterol diet with 800 mg extract) p = 0.005 b High cholesterol diet vs (High cholesterol diet with 200 mg extract) p = 0.0788 c High cholesterol diet vs (High cholesterol diet with 400 mg extract) p = 0.006 d High cholesterol diet vs (High cholesterol diet with 800 mg extract) p = 0.000 e (High cholesterol diet with 200 mg extract) vs (High cholesterol diet with 400 mg extract) p = 0.000 f (High cholesterol diet with 400 mg extract) vs (High cholesterol diet with 800 mg extract) p = 0.000

Table 1 .
Mean of Cholesterol, Triglycerides, LDL and HDL level

Table 3 .
In the negative control group occurred expression of ICAM 1 with the lowest value (20.50), whereas the positive control group who received

Table 2 .
Mean NFκB activation in aortic endothelial cells.

Table 3 .
Mean ICAM1 expression in aortic endothelial cells.