Treatment of Pimpinella Alpina Molk Improve Oxidative stress and Inhibit Liver Cellular Apoptosis in Rats Following UVB Irradiation : Is there Any Correlation ?

Objective: The effect of Pimpinella alpina Molk (PaM) in improvement of oxidative stress has been established, but did not for cellular apoptosis. This investigation was conducted to evaluate the effect of PaM on GPx and XO activities, hepatocytes apoptosis and their correlation following UVB radiation. Methods: Forty male rats were assigned into 8 groups: Nor-G, Neg7, Neg-15, PaM50-7, PaM100-7, PaM150-7, PaM100-15, and PaM150-15. UVB irradiation was given 10 minutes to each rat daily, blood and liver organ samples were taken in day 8 and day 16 to evaluate all variables. Results: Post Hoc statistical analysis showed that GPx activity in PaM150-15 was not significant lower compared to that of Nor-G, p > 0.05. Conversely, the activity of XO in PaM150-7 was not significant higher compared to that of Nor-G, p > 0.05. Expression of Bax and Caspase3 mRNA in PaM100-15 and PaM150 were comparable to that of Nor-G, p > 0.05. The strong negative correlation (-795, p < 0.01) was occur between GPx and XO. Similarly, there were strong negative correlations (-697 and -715, p < 0.001) between GPx and the expression of Bax and Caspase3 mRNA. Conclusion: PaM treatment with 100 150 mg daily dose for 15 days capable of improving oxidative stress marked by increase in GPx activity and decrease in XO activity and inhibit apoptosis characterized by decrease in the expression of Bax and caspase3 mRNA. There were strong negative correlations between antioxidant activity and apoptosis on Sprague male rats after UVB irradiation. Keyword: Pimpinella Alpina Molk; Apoptosis; Oxidative stress; UVB; Oxidative stress; apoptosis Correspondence to: Taufiqurrachman Nasihun, Department of Biochemistry, Medical Faculty of Sultan Agung Islamic University, Semarang, Central Java, Indonesia, E-mail: taufiq_rn@yahoo.com 1. Eni Widayati, Department of Chemistry, Medical Faculty of Sultan Agung Islamic University,Semarang, Central Java, Indonesia 2. Taufiqurrachman Nasihun, Department of Biochemistry, Medical Faculty of Sultan Agung Islamic University, Semarang, Central Java, Indonesia Bangladesh Journal of Medical Science Vol. 17 No. 01 January’18. Page : 118-128 Introduction Pimpinella alpina Molk (PaM), known as Purwoceng, is a medicinal plant growing in Dieng plateau Central Java Indonesia. Male population particularly who aged 40 years or above have traditionally consumed PaM to rejuvenate and increase their vitality1. According to the compression-of-morbidity hypothesis, reducing the cumulative lifetime morbidity is possible, as long as primary prevention measures is able to postpone the onset of chronic illness2,3. Most evidence indicates that daily intake of flavonoids from plants could prevent degenerative diseases and chronic illness.4-6 Considering, degenerative diseases and chronic illness are tightly associated with oxidative stress, therefore, daily intakes of flavonoids in midlife and late adulthood are important predictors of subsequent degenerative diseases and disability.3 The effect of PaM in improvement of oxidative stress following UVB and UVC irradiation has been established,7 but did not for cellular apoptosis. Considering, severe oxidative stress is prerequisite for apoptosis triggered by UVB and posses an important role in aging process and frailty,8,9 therefore, further research is needed to investigate the effect of PaM on antioxidant activity and hepatocyte cells apoptosis, and their relationship following UVB irradiation. Forty five percent of the total sunlight spectrum is UV light, consisting of UVA ranging from 315 400 nm, UVB ranging from 280 315 nm), and UVC ranging from 100 280 nm. The earth’s ozone DOI: http://dx.doi.org/10.3329/bjms.v17i1.35292


Introduction
Pimpinella alpina Molk (PaM), known as Purwoceng, is a medicinal plant growing in Dieng plateau Central Java Indonesia.Male population particularly who aged 40 years or above have traditionally consumed PaM to rejuvenate and increase their vitality 1 .According to the compression-of-morbidity hypothesis, reducing the cumulative lifetime morbidity is possible, as long as primary prevention measures is able to postpone the onset of chronic illness 2,3 .][6] Considering, degenerative diseases and chronic illness are tightly associated with oxidative stress, therefore, daily intakes of flavonoids in midlife and late adulthood are important predictors of subsequent degenerative diseases and disability. 3The effect of PaM in improvement of oxidative stress following UVB and UVC irradiation has been established, 7 but did not for cellular apoptosis.Considering, severe oxidative stress is prerequisite for apoptosis triggered by UVB and posses an important role in aging process and frailty, 8,9 therefore, further research is needed to investigate the effect of PaM on antioxidant activity and hepatocyte cells apoptosis, and their relationship following UVB irradiation.Forty five percent of the total sunlight spectrum is UV light, consisting of UVA ranging from 315 -400 nm, UVB ranging from 280 -315 nm), and UVC ranging from 100 -280 nm.The earth's ozone DOI: http://dx.doi.org/10.3329/bjms.v17i1.35292layer efficiently absorbs UV radiation up to about 310 nm.Accordingly, all UVC and most of UVB light are consumed by ozone before they reach the surface of the earth. 10,11However, current depletion of the ozone layer is significantly increasing the amount of UVB radiation that reaches the surface of earth.3][14] .These disorders were mostly attributable to photoproducts such as [cyclobutane pyrimidine dimers (CPD)] and (6-4) pyrimidine-pyrimidone [ (6-4)PPs], increase in production of reactive oxygen species (ROS) and alteration of antioxidant defensive system leading to cellular oxidative stress.Oxidative stress is defined as the imbalances in the equilibrium between pro-oxidants and antioxidants status in cellular systems, in favor of oxidants which results in damaging the cells. 15,16.The increase in ROS constitutes a major cause of DNA fragmentation and initiates cellular damages leading to apoptosis. 17here are growing evidences that ROS and ROSmodulated molecules participate in both intrinsic and extrinsic apoptotic pathways. 18Considering ROS, particularly OH• which is resulted from Fenton reaction are vastly distributed in systemic circulation following UVB radiation, thus the cellular damages, DNA fragmentation, and apoptosis occur not just in skin but also in other deeper cells 19 .There are various evidences that increase in ROS such as -and H 2 O 2 is associated with the arrest of cell proliferation.Similarly, generation of oxidative stress in response to various external stimuli has also been implicated in the activation of transcription factors and to the triggering of apoptosis. 20,21A study was reported by Higuchi and co workers indicated that increase in ROS inducing apoptosis mediated by xanthin oxidase (XO) and caspase 3 activity. 22On the other hand, study was reported by Wedi et al. pointed out that cellular apoptosis (eosinophil) can be blocked by glutathione and N-acetylcysteine. 23oreover, the study was reported by Nasihun and Widayati indicated that treatment of PaM capable of improving oxidative stress following UVC radiation in rats.The improvement of oxidative stress was marked by decreased in concentration of MDA and 8-OHdG, otherwise increased in concentration of total antioxidant capacity (TAC) and super oxide dismutase (SOD) activity 7 .Apoptosis is a word come from Greek and used to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. 24Moreover, in the biological system, apoptosis then used to describe the intrinsic cell suicide program, in contrast to necrosis.The morphological feature of apoptosis is characterized by global cell shrinkage, cell blabbing, and chromatin condensation accompanied by nuclear and DNA fragmentation into specific fragment sizes (180 -200 base paired) and so called DNA ladder pattern. 24.Apoptosis have a pivotal role in the aging process and age-associated carcinogenesis in mammals.The rate of apoptosis is prominent in most types of aging cell populations and organs, such as brain, cardio-vascular system, immune system, endocrine system, intestine, reproductive system and eye. 25,26The increase in apoptosis in these various organs regarded as protective mechanism of organism against the accumulation and spread defective cell.In contrary, when the dismantling system is predominance the age-associated decline and deterioration in tissue and organ structure and function are induced.Accordingly, apoptosis has a pivotal role as sentinel homeostasis pathway in organ and tissues.However, there are growing evidence that high rate of apoptosis can result in tissue's degeneration, 26,27 , while low rate of apoptosis allows either dysfunctional cells to accumulate or differentiated immune cells to persist. 28Therefore, appropriate apoptosis is necessary in order to prevent age-related diseases and cancer.Initiation of apoptosis by the cell is governed by gene activities, leading to cellular self-destruction through the intrinsic or the extrinsic pathways.Apoptosis process is initiated by various inducers from both internal and external millieu, followed by the cell death commitment and execution.DNA damage due to UVB irradiation activates and stabilizes p53 in nucleus and cytoplasm.Moreover, p53 may drive intrinsic and extrinsic apoptotic pathway through amplifying the apoptotic signal consisting of apaf 1, cell death receptor Fas (CD95), PUMA, Bax, Bak, and caspases.These protein pro-apoptosis are evoked by activation of stress activated protein kinases (SAPK) following UVB irradiation. 29,30n intrinsic apoptosis pathway, the DNA damages induce mitochondrial permeability transition pore complex, which controls mitochondrial membrane potential to release cytochrome c.In concert with apaf-1, procaspase 9 , and ATP, cytochrome c forms apoptosome death complex (ADD), that proteolitically convert procaspase9 to caspase9, and then activate caspase7,6 and caspase3 resulting in DNA fragmentation and cell death.On the other hand, in extrinsic apoptosis UVB and ROS induces Fas and Fas ligand (FasL) or tumor necrosis factor (TNF) with TNF receptor 1 (TNFR1).Furtehrmore, Fas-FasL complex bound with Fas Associated Death Domain (FADD), subsequently activates caspase8 and other caspases executing apoptosis via Death Effectors Domain (DED). 31tudies about PaM have been documented particularly to increase expression of BcL 2 otherwise decreased expression of Caspase3 and inhibition of apoptosis in penile and prostate cells. 32,33Indeed those PaM effect were unlikely through antioxidant capacity instead of hormone stimulation effect in castrated male rats.It was possible, because PaM contained stigmasterol, is a natural weak androgen that believed as androgen precursor.Stigmasterol may be converted to testosterone in peripheral tissues catalyzed by enzyme of 3-βOHsteroid and 17-βOH steroid dehydrogenase. 34Testosterone is an androgen hormone capable of developing masculinity, increase sexual desire, vitality, and rate of fertility in male. 35,36n addition to stigmasterol, another significant constituent of PaM is flavonoids 37 , constituting a natural potent botanical antioxidant, radical scavenger, and metal ion chelator agent. 38Flavonoids have been documented capable of reducing ROS level, oxidative stress, and biological aging.Based on these previous data, this study was proposed to elucidate that PaM treatment capable of decreasing XO activity, followed by increase in activity of GPx, leading to pro-oxidant antioxidant balance after UVB irradiation.This pro-oxidant and antioxidant balance providing cellular damages due to oxidative stress an opportunity to undertake denovo formation for self repair, indeed prevent cellular apoptosis.

Matrial and Methods Animal Models and Research Procedure
In this experimental study, the post test only control group was adopted as design research.Forty Sprague Dawley (SD) male rats, 6 months old, and + 300 gram body weight (BW), were assigned into 8 groups randomly as follow: 1).Normal group (Nor-G), rats got no UVB irradiation and no PaM treatment.2).Negative control group for seven days (Neg-7) and 15 days (Neg-15), rats got UVB irradiation only.3).PaM treatment group, was divided into two arms: first arm comprise three groups: PaM50-7, PaM100-7, and PaM150-7, in these groups all rats were radiated with UVB and treated with PaM 50 mg, 100 mg, and 150 mg doses per day respectively during 7 days; second arm comprise 2 groups: PaM100-15, and PaM150-15, all rats in these groups were expose to UVB for 7 days and administered PaM with dose of 100 mg and 150 mg respectively during 15 days.The seven days longer was proposed to provide cell an opportunity to undertake denovo formation for repairing.All rats were kept in acclimatization for one week with environmental controlled temperature (20 0 C-24 0 C), constant humidity (55 -60%), and controlled photoperiod (12 h light and 12 h dark) properties before beginning of experiment.During the study all rats in all groups were given standard dietary intake (Ain 93) and tap water adlibitumly.At day 8 of study, blood samples and liver organs from rats in Nor-G, Neg-7, PaM50-7, PaM100-7, and PaM150-7 were taken and measured the activity of GPx and XO, and the expression of Bax and capase 3 mRNA, meanwhile, at day 16 with the similar method, blood samples from rats in Neg-15, PaM100-15, and PaM150-15 were taken for asses the same variables.All measurement of variables was undertaken at Inter University Program (PAU) of Gajah Mada University (UGM) Yogyakarta Indonesia.The study was ethically approved.

PaM Extract
Pimpinella alpina Molk was obtained from market and extracted by soxhlet methods from the whole plant with ethanol as a solvent.UVB Irradiation UVB radiation was delivered using UV light sources fluorescent sun lamp FS72T12-UVB-H emitting a UVB weave length ranging from 280-320 nm, with a peak of 312.5 nm at the distance of 25 cm, was measured from cage floor.The average flux intensity at cage floor measured with digital UV light meter YK-35UV was 9.3 j/m 2 /sec.Hairless rats in all groups except in Nor-G were placed in plastic cage exposed to 1.6 kJ per M 2 or equivalent to minimal erythematic dose (MED) for 30 minutes per day during seven days.

GPx Measurement
A Glutathione peroxidase level was determined by the method of Paglia and Valentine, using a commercially available kit (Ransel; Randox Laboratories, UK). GPx catalyze the oxidation of glutathione by cumene hydroperoxide.In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to its reduced form with a concomitant oxidation of NADPH to NADP.The NADPH-consumption rate was monitored at 340 nm.The expression was calculated by using the relative standard curve method of quantification and reported as a fold change of gene expression.

Statistical Analysis
Anova and Post Hoc test were adopted as statistical analysis in 95% of confidence interval or P < 0.05 was considered statistically significant.All statistical analysis were done use computer program.This study has been approved by the ethic committee, medical faculty of Sultan Agung Islamic University Semarang Central Java Indonesia.Neg-7.One way anova statistical analysis pointed out that there were significant differences among groups, p < 0.001.

Activity of GPx and XO
Post Hoc statistical analysis showed that GPx activity in Neg-7 and Neg-15, were lower significantly compared with that of Nor-G, p < 0.001, whereas the GPx activity in Neg-7 were significant higher compared with that of Neg-15, p < 0.001.The GPx activity in PaM50-7, PaM100-7, and PaM150-7 was higher significantly compared to that of Neg-7 and Neg-15, p < 0.001.The GPx activity in PaM50-7, PaM100-7, and PaM150-7 was lowering significantly compared to that of Nor-G, p < 0.001.Interestingly, GPx activity in PaM150-15 was not significant lower compared to that of Nor-G, p > 0.05.(Figure 1) The activity of XO in Neg-7 and Neg-15 were higher significantly compared to that of Nor-G, p < 0.001, whereas the activity of XO in Neg-7 was lower significantly compared to that of Neg-15, p < 0.001.In contrary, the activity of XO in PaM150-15, PaM100-15, PaM150-7, PaM100-7, and PaM50-7 was lowering significantly compared to that of Neg-7 and Neg-15, p < 0.001 respectively.The XO activity in PaM50-7 was higher compared to that of Neg-15, p < 0.001.Special for the level of XO in PaM150-7 was higher compared to that of Nor-G; however that difference was not significantly, p > 0.05.(Figure 1) The Expression of Bax and Caspase 3 mRNA Post Hoc analysis indicated that the expression of Bax and caspase 3 mRNA in Neg-7 and Neg-15 was significant higher compared to that of Nor-G, p < 0.001.Whilst the expression of Bax mRNA in Neg-15 was significant lower compared to that of Neg-7, p < 0.05.Conversely, there was no significant difference expression of caspase3 mRNA in Neg-7 and Neg-15, p > 0.05.The expression of Bax mRNA in PaM50-7, PaM100-7, PaM150-7, PaM100-15, and PaM150-15 was significant lower compared to that of Neg-7, p < 0.001.Interestingly, the expression of Bax mRNA and caspase3 in all PaM groups when compared to that of Neg-15, the results were varies.
In PaM50-7, PaM1007, and PaM150-7, there was no significant difference expression of Bax mRNA compared to that of Neg-15, p > 0.05.On the other hand, in PaM100-15 and PaM150-15, the expression of Bax mRNA was significant lower compared to that of Neg-15, p < 0.05.Moreover, the expression of Bax mRNA in PaM150-7, PaM100-15, and PaM150-15 was no difference compared to that of Nor-G, p > 0.005.There was no significant difference expression of caspase3 mRNA in PaM50-7 and PaM100-7 when compared to that of Neg-7, p > 0.05.Meanwhile, the expression of caspase3 mRNA in PaM150-7, PaM100-15, and PaM150-15 was significant lower compared to that of Neg-7, p < 0.001.On the other hand, there was no significant difference expression of caspase3 mRNA in PaM50-7, PaM100-7, and PaM150-7 compared to that of Neg-15, p > 0.05.Whereas, the expression of caspase3 in PaM100-15 and PaM150-15 was significant lower compared to that of Neg-15, p < 0.05.The expression number of caspase3 in PaM100-15 and PaM150-15 was lower compared to that of Nor-G, however the difference was not significant, p > 0.05.(Figure 1)

Correlation among Variables
Based on the Pearson correlation analysis there were strong correlation among variables.The strong negative correlation (-795, p < 0.01) was occur between GPx and XO.Similarly, there were strong negative correlations (-697 and -715, p < 0.001) between GPx in one hand and the expression of Bax and caspase3 mRNA on other hand.In contrary, there was positive correlation (+610, p < 0.01) between the expression of Bax and caspase3 mRNA.(Figure 2) Discussion At present, it is well known that UVB irradiation capable of inducing DNA damage directly by absorption of energized UVB photons or indirectly through photosintizer molecule, thus creating an excited singlet state.DNA damages by an excited singlet may occur either through direct interaction with DNA molecule, thus induce free radical formation, or via energy transfer to molecular oxygen or named as photodynamic action, leading to ROS production. 11,39he production of ROS by photodynamic action give rise several type of DNA damage including strand breakage, base modifications, and DNA-protein cross-linkage. 39,40There is growing evidence that the ROS which is generated by UVB radiation comprise OH•, O-, and H2O2, 39 were able to induce oxidative stress characterized by increase in MDA and 8-oxo-2'-deoxyguanosine (8OHdG), otherwise decrease in total antioxidant capacity (TAC) and GPx activity, 7 and subsequently apoptosis.
The result of the present study was in line with those previous studies.In this result indicated that following UVB irradiation GPx activity in Neg-7 and Neg-15 was significant lower compared to that of normal.
In contrary, XO activity in Neg-7 and Neg-15 was significant higher compared to that of normal.This result suggested that decrease in GPx activity and followed by increase in XO activity demonstrated that cells were suffering from oxidative stress.It was confirmed by the study of Higuchi and co workers indicated that increase in ROS capable of inducing oxidative stress and apoptosis mediated by XO and caspase 3 activity. 22On the other hand, in vitro study was reported by Li and co workers showed that 6-CySeCD, a novel GPx also proven capable of improving DNA damage induced by UVB. 41It was plausible since GPx serve as an antioxidant protector of lipid membrane and other cellular components. 42n addition, in the present study also demonstrated that UVB irradiation during 7 days and seven days aftermath (day 15) was blank without UVB radiation, however the UVB radiation effect remain occur.It was demonstrated by the increased in XO activity and decreased in GPx activity significantly in Neg-15 compared to those of Neg-7.This result suggested that, although the UVB irradiation was halted, however the destruction effect of ROS triggered by UVB remain work continuously as well as oxidative stress.It was possibly caused by oxidant or oxidative stress memory of cells, however further research is needed.The similar result was reported by numerous studies, demonstrating that activities of SOD, Catalase, and Glutathione reductase were reduced after a single UVB irradiation.However, when exposure to UVB was prolonged for more than 12 weeks, the activity of SOD was also raised. 43.
The result of the present study also implied that UVB irradiation with 1.6 kJ per M 2 or equivalent to minimal erythematic dose (MED) for 30 minutes per day during seven days worked appropriately.Study was reported by Nasihun as to UVC irradiation on rats have similarity result to this finding.Because of UVC irradiation between 190 and 280 nm (peak 254 nm) at a distance of 20 cm for 30 minutes daily was able to increase level of MDA and 8OHdG, conversely decrease GPx activity and TAC concentration as markers of oxidative stress. 70][41] It was plausible, since the increase in lipid peroxidation, MDA, and 8OHdG level, followed by decrease in SOD, Catalase, and GPx activities are prerequisite for cellular damages insulted by UVB. 11s mentioned above, H 2 O 2 , OH • , and O -, were ROS resulted from UVB irradiation, by which several cellular components such as cellular membrane, mitochondrial and nuclear DNA suffers from damages. 30,42Considering, apoptosis is resulted from enormous and unrepaired DNA damages and DNA damages is attributable to excessive oxidative stress, hence modify the degree of oxidative stress with antioxidant flavonoids in particular may inhibit apoptosis. 7,41lutathion peroxydase is an enzyme dependent on the micronutrient selenium (Se), plays a critical role in the reduction of lipid and hydrogen peroxides. 44If GPx activity is decreased, more hydrogen peroxide is present, which leads to direct tissue damage and activation of nuclear factor-кB-related inflammatory pathways.On the other hand, XO is an enzyme catalyzes the reaction of hypoxanthine to xanthine and xanthine to uric acid.In both steps, molecular oxygen is reduced, forming the superoxide anion followed by the generation of hydrogen peroxide. 45n addition, various studies have also proven that intake exogenic antioxidant particularly from plants were able to counteract oxidative stress.It were attributable to phenolic compounds contained in plants. 46n the present study following treatment of PaM the GPx activity in PaM50-7, PaM100-7, and PaM150-7 was higher significantly compared to that of Neg-7 and Neg-15.In contrary, the activity of XO in PaM150-15, PaM100-15, PaM150-7, PaM100-7, and PaM50-7 was lowering significantly compared to that of Neg-7 and Neg-15.This result suggested that administration of PaM with 50 -150 mg during 7 -15 days were able to elevate antioxidant activity and lowering pro-oxidant activity, therefore ameliorate the oxidative stress in rats after UVB radiation.Interestingly, GPx activity in PaM150-15 and XO activity in PaM150-7 was not significant lower and higher respectively compared to those of Nor-G.This result suggested that dosing of PaM with 150 mg for 7 -15 days was capable of increasing GPx activity and decreasing XO activity comparable to those of normal condition.This result corroborated by the study was reported by Nasihun et al. that supplementation of PaM was capable of increasing GPx activity and total antioxidant capacity, otherwise decrease MDA and 8OHdG levels in rats following UVC radiation. 7Albeit UVB and UVC posses difference wavelength, however they have a common target on DNA to damage.
The DNA damaged was induced by UVB and UVC maid occur both directly by cyclobutane pyrimidine dimers (CPD) and indirectly by ROS production. 7,11ast evidences pointed out that both CPD and ROS were able to trigger cellular apoptosis. 11iver apoptosis may be induced by viral infection, excessive alcohol consumption, exposure any type of chemical carsinogenic, and DNA damage caused by high concentration of intra cellular ROS.In addition, the liver resident cells such as hepatocytes, cholangiocytes, activated stellate cells, and Kupffer cells express high levels of cell-deathassociated receptors.Fas/FasL signalling has been largely implicated in liver pathophysiology but the mitochondrial intrinsic pathways are also involved in liver homeostasis. 47n the present study UVB irradiation have been proven induced liver cells (hepatocytes) apoptosis in rats characterized by increase in Bax and caspase3 mRNA expression in Neg-7 and Neg-15 compared to those of normal and PaM groups.Cellular apoptosis occurring after UVB irradiation may through both intrinsic and extrinsic pathways.The intrinsic apoptosis pathway can be triggered by both excessive ROS and DNA damages, by which mitochondrial DNA release Cytochrome c and Growth arrest DNA damage-inducible gene 45β (GADD45beta) regulates apoptotic cell death in response to DNA damage is upregulated.Moreover, Cytochrome c holoform along with apaf-1, procaspase 9 , and ATP form apoptosome death complex (ADD), which proteolitically activate and convert procaspase 9 to caspase 9 and then activate caspase7 , 6, and caspase3, acting as apoptosis executor. 48The activities of these caspases result in DNA fragmentation and cells death.Regulation of apoptosis was controlled by various pro-apoptosis protein such as Bax and Bak and anti apoptosis protein such as Bcl2 and Bcl-xL.Accordingly, in this context, occurrence of apoptosis is fully determined by the ratio between these pro and anti apoptosis protein which generated by severe oxidative stress triggerred by XO.A study was reported by Higuchi et al. showed that apoptosis can be induced by xanthin and XO.In the low level, XO may induce apoptosis mediated by protein like caspase3.In the high level, XO may induce necrosis also mediated by protein like caspase3.Conclusion was taken by the author that albeit apoptosis and necrosis could be induced by ROS, however, enzymes were engaged in this process is protein like caspse3 instead of caspase3. 22The result of the present study is in disagreement with the Higuchi study, since in this study expression of Bax and caspase3 mRNA increased following UVB irradiation.Considering, both Bax and caspase3 mRNA constitute pro-apoptosis protein, hence can be implied that increase in ROS after UVB radiation result in apoptosis mediated by Bax and caspase3.The available literatures have also confirmed that apoptosis triggered by UVB was mediated by caspase3 in intrinsically and extrinsically fashion.In the extrinsic apoptosis, UVB trigger and activate FAS/CD95 death-receptor pathway and in turn recruits caspase-8 to form the CD95 death inducing signaling complex (DISC).Oligomerization of caspase-8 upon DISC formation drives its activation through self-cleavage.Caspase-8 then eventually activates downstream effector caspases such as caspase3.Similarly, in intrinsic apoptosis, ROS which is produced by UVB is able to damage membrane mitochondrial and activate its protein effectors downstream and activation of caspase3 later on. 11,49,50he result of the present study also indicated that activities of GPx and XO were increased and decreased respectively following PaM treatment with 100 and 150 mg doses for 15 days.This result demonstrated that PaM containing flavonoid have been proven capable of increasing GPx activity and decrease XO activity.This circumstance bring about cells may able to circumvent from oxidative stress and prevent cell undergo apoptosis and death.Administration of PaM with 100 and 150 mg dose daily during 15 days was able to decreased expression of Bax mRNA and caspase3.There are mounting evidences that Bax/Bak and caspase3 are protein pro-apoptosis.2][53] Protein pro-apoptotic Bax/Bak are the target of JNK induced apoptotic signalling pathway.On the other hand, protein anti apoptotic such as Bcl2/BclxL are phosphorilated and inhibited by JNK. 54Therefore decrease in expression Bax and caspase3 mRNA going to prevent cellular apoptosis.Analysis correlation of Pearson also indicated that there is a negative correlation between GPx and XO activities.In addition, there was a positive correlation between XO and mRNA and Caspase3 (figure 2).Taken together the results of the present study imply that increase in oxidant invariably followed by decrease in antioxidant and apoptosis after UVB irradiation.

Conclusion
In conclusion, treatment of PaM with 100 and 150 mg dose per day during 15 days was capable of improving cellular oxidative stress and inhibits apoptosis.

Acknowledgment
The author greatly appreciated to Yulianto and Didik as a laboratory staff and pathological experties respectively who assisted in carrying out of this study.The research was supported and funding by Indonesian ministry of high education and research PaM on GPx, XO, Bax, and Caspase3 mRNA Expression After treatment of PaM for 7 and 15 days, all variables including activities of GPx, XO in serum, and the expression number of Bax and Caspase 3 of liver organs were measured at day 8 and 16 and the results shown in table 2. This result indicated that the highest level of GPx was occur in Nor-G, followed by PaM150-15, PaM100-15, PaM150-7, PaM100-7, PaM50-7, NeG-7, and the lowest was in Neg-15.In contrary the lowest level of XO, was found in Nor-G, followed by PaM150-7, PaM150-15 and PaM100-7, PaM100-15, PaM50-7, Neg-7, and the highest was in Neg-15 (table 2).The lowest expression of Bax mRNA was in PaM150-15, followed by Nor-G, PaM100-15, PaM150-7, PaM50-7, PaM100-7, Neg-15, and the highest was in Neg-7.Moreover, for the expression of caspase 3 mRNA, the lowest was occur in PaM150-15, followed by Nor-G, PaM100-15, PaM100-15, PaM150-7, Neg-15, PaM100-7, PaM50-7, and the highest was in Xanthine oxidase was determined by instruction method manufactured by BioVisison.The principal measurement of XO in this assay is XO oxidizes xanthine to to hydrogen peroxide (H 2 O 2 ) which react stoichiometrically with OxiRed TM probe to generate color (at ʎ = 570 nm) and fluorecence (at Ex/Em = 535/587 nm).Considering the color or fluorecence intensity is proportional to XO content, the XO activity can be accurately measured.This kit detects 1 -100 mU XO in 100 µl reaction volume.