Antioxidant and Photo protective property of Chloroxylon swietenia leaf extract in human skin keratinocytes.

Sundararaju D; Ajit kumar Marisetti;********; C.V. Rao; Sengupta K*. Laila Impex R&D Centre Unit-1 Phase-III Jawahar Autonagar Vijayawada-520007, Andhra Pradesh. *Corresponding author: Ph. # (0866) 2541182 Fax# (0866) 2543484. Email: krishanu@lailanutra.com Abstract: Ultraviolet (UV) irradiation has been demonstrated to generate reactive oxygen species (ROS) causing oxidative stress and skin photoaging in human skin keratinocytes. In the present study, we examined the photoprotective effects of aq. Methanol extract of CSE. Pre-treatment with CSE significantly inhibited the production of ROS and cell viability in human skin keratinocytes (HaCaT) when compared to the UV-irradiated cells. Enzymatic, non-enzymatic antioxidants and lipid peroxide functions confirmed the decreased antioxidant activities, assessed by superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH) and increased endogenous lipid peroxidation. Based on presen results, we suggest that CSE might play an important role in the cellular defense mechanism against oxidative stress and induced by photoprotective effects of CSE were conformed with inhibitory effect of collagen degradation and measured hydroxyl proline levels in CSE pretreated HaCaT cellsexposed to UV radiation( photoaging). Key words: INTRODUCTION Oxidative stress initiated by reactive oxygen species (ROS) is a major contributor to the aging process (Ross et al). Skin is a major organ and target of oxidative stress, and during the skin aging process, ROS levels are increased and antioxidant defenses system lowers than the normal level (Laskin et al). The balance between ROS production and antioxidant defenses determines the degree of oxidative stress. Consequences of this stress include modification to cellular proteins, lipids DNA and skin aging (velino et al). The antioxidant system is an important defense mechanism against oxidative cell damage (vertuani et al).The antioxidant defense system that includes the enzymatic scavengers

Superoxide dismutase (SOD), catalase and glutathione. SOD rapidly converts superoxide to hydrogen peroxide, whereas catalase and glutathione converts hydrogen peroxide to water (Chae, H. et al 1999). Many studies suggest that age-related decrease in antioxidant enzyme activity is consistent with increased free radical damage that contributes to aging (Habdous et al). The UV induced generation of ROS can result in the structural and functional alteration of cutaneous proteins such as collagen which may contribute to photoging (Carbonare et al, 1992). Collagen is the one of the main building block of human skin, which gives more strength to the skin. Collagen is primarily composed of glycine, proline and hydroxyproline. It is one of the strongest proteins in nature and gives skin its strength and durability (http://training.seer.cancer.gov). As we age, it is believed that collagen begins to deteriorate and causes the skin to become thinner. ROS directly destroys tissue collagen by inhibiting the tissue inhibitor of matrix metalloproteinase (MMP). The use of antioxidants was reported to repair skin damage, loss of elasticity, wrinkling, and premature ageing. Antioxidants can inhibit many UV induced transduction pathways (Fguyer et al 2003). Daily intake of antioxidants may helpful to prevent oxidative stress, skin aging and several infectious diseases. Chloroxylon Swietenia is rich in antioxidant property and skin care properties (Sivakumar et al, 2008; Palani et al, 2010, Rao et al 2009). Therefore, the present study was to evaluate the potential applicability of C. swietenia extract for the prevention of oxidative stress and skin aging due to UV irradiation. As a part of experiment to evaluate the antioxidant potential activity and skin aging activity of C.swietenia extract. Chloroxylon swietenia DC belongs to family Rutaceae and is commonly known as East India satinwood in English. The leaves of C. swietenia are used by some tribal communities for anti-inflammatory activity, insecticide, mosquito repellent, wound healing and analgesic properties and anti-lice (The wealth of India 1992; Ravikiran et al, 2007, Kirthikar and Basu,1993; Senthil and Ramkumar, 2003). This plant extract is also used in antifertility activity (Sharma et al 1989), Hepatoprotective and antioxidant activity (Palani et al, 2010), tyrosinae inhibitory activity (Rao et al 2009). C. swietenia has been extensively investigated and a number of chemical constituents from the leaves, bark and roots of the plant have previously reported in a number of instances which

includes alkaloids, (Ravi kiran et al 2006; Talapatra 1968) cumarin (Mujumdar et al 1975;Govindachari et al, 1967) furoquinolines (Vorkoc et al 1977) and lignans (Lee, 1969).

MATERIALS AND METHODS Reagents and chemicals Aprotinin, Bovine serum albumin (BSA), Bouins fixative solution, Coomassie brilliant blue, DCF (2, 7 dichloro fluorescein), Dulbeccos modified eagles medium (DMEM), Dimethyl sulfoxide (DMSO), Formaldehyde, Methanol, Glucose, Glutamine, Glycine, Glutathione, Heat inactivated fetal bovine serum (FBS), Leupeptin, Malonaldehyde, Nicotinamide adenine dinucleotide phosphate (NADPH), Penicillin,

Phenylmethylsulfonyl fluoride (PMSF), Phenazine meta sulphate (PMS), Potassium periodate, Purpald, Sirius red dye, Superoxide dismutase (SOD), Tris, Tris HCl, Triton X-100 and Thiobarbituric acid (TBA) chemicals were purchased from Sigma-Aldrich (Bangalore, India). All other chemicals were purchased from SD Fine Chemicals Limited (Mumbai, India) Plant material Fresh leaves of Chloroxylon swietenia were collected from the reserved forests of Palvancha region, Andhra Pradesh, India. The material was taxonomically identified by Dr. K. Narasimha Reddy, Taxonnmist, Laila Impex R&D Centre, Vijayawada, India. A voucher specimens, (LIH *******) are deposited in the Laila Impex herbarium house of plant taxonomy division, Laila Impex R&D centre, Vijayawada. Extraction The shade dried leaves of C. swietenia DC were milled into a coarse powder (6.8 kg) and extracted using non-polar to polar solvents i.c with n-hexane (5 x 25 L), ethyl acetate (5 x 25 L), methanol (5 x 25 L), 60% aqueous methanol (3 x 20 L) and water (2 x 20 L) in a 60 liters reactor. The extracts of each solvent were concentrated under reduced pressure to yield 155 g thick paste, 341 g thick paste, 700 g thick paste, 230 g powder, 100 g powder from n-hexane, ethyl acetate, methanol, 60% aqueous methanol and water

respectively. The paste and powder form of extracts were used to screen for antioxidant and skin care activities. Cell culture The immortalized human skin keratinocytes (HaCaT) were cultured in DMEM containing 4.5 g/L of glucose and L- glutamine supplement with 10% FBS, 100 g/mL penicillin and 1000 U/mL streptomycin at 37C with 95% and 5% (v/v) mixture of air and CO2. Cell viability assay. Viability of cells in culture was measured by 3-[4,5dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HaCaT cells were plated at 5104 cells per well in 200 L of DMEM in a 96-well microtiter plate and cultured overnight followed by treatment with CSE, after treatment with CSE cells exposed to UV treatment at a dose of 50 mJ /cm2. After 24 h, of UV exposure, the cells were replaced with fresh media containing MTT for 2 h at 37C and the absorbance was measured at 570 nm using a microplate reader. Measurement of the reactive oxygen species (ROS) generation The DCF-DA method was used to detect the intracellular ROS levels (Rosenkranz et al). HaCaT cells were seeded into 96 well plates (5500cell/well). Next day the cells were washed two times with hanks balanced salt solution (HBSS) and supplemented with new medium. Cells were incubated with DCF in HBSS solution for 30 min at 37C after incubation discarded solution. Now the cells were treated with different concentrations of the C. swietenia (LI/PD/148/4) in HBSS. Levels of relative fluorescence units (RFU, produced by ROS catalyzed oxidation) were measured by turner biosystems with excitation wave length at 460 nm and emission at 580 nm. Treatment of cells: Superoxide Dismutase (SOD), Catalase (CAT), Glutathione, lipid peroxidation and Hydroxyproline HaCaT cells were maintained in DMEM culture media up to a of 6080% confluence and then pretreated with different concentrations of aq. methanol (LI/PD/148/4) extract of CSE in 0.1% (vol vol) DMSO. After 24 hour cells were washed with HBSS and were irradiated with UV-50 mJ/ cm2/4 hour for 4 days (using a custom designed Research Irradiation Unit (Daavlin, Bryan, OH). Cells were harvested for SOD, catalase, Glutathione (GSH), lipid peroxidation and hydroxyproline.

Superoxide Dismutase (SOD): The NADH/PMS/ NBT system was used to determine the superoxide anion scavenging activities of the compounds (Paya 1993; Ponti 1978). Briefly, generation of superoxide anion was measured in a reaction mixture containing cell lysate or SOD (25l/well) were added in 96 well, add 200l reaction mixture which contains 9.8mM NADH, 6.2mM NBT and 100mM EDTA in 50mM PBS at pH 7.4, and add 25l PMS. After thoroughly mixing, the samples were incubated 5 min at room temperature. The absorbance was read at 550nm using a microplate reader. Superoxide dismutase was expressed as U /mg protein. Protein estimation was carried out by Barford method. Catalase activity: The activity of catalase was determined by a photometric method (Colin et al 1994; Wheeler, et al 1990). Briefly, the cell lysate or formaldehyde (50l/well) were added in 96 well, then add 25l of 250 mM phosphate buffer, 25l of 12 M methanol, 5l of H2O2. The total reaction was incubated 20 min at room temperature. After incubation add 75l of purpald, incubated 20 min at room temperature, and then add 25l potassium periodate, mix well in the plate by shaking on orbital shaker. The absorbance was read at 550 nm using a microplate reader. Catalase was expressed as mol /mg protein. Glutathione Estimation of glutathione was done by the method of Grunert and Phillips (1951). Briefly the metaphosphoric acid, extract of cell lysate was saturated with sodium chloride and allowed to stand for 15-30 min and centrifuged at 14000 g for 20 min at 4C. To 50 l of cell supernatant or lysate 150l saturated sodium chloride solution is added, allowed it to stand for 10 min at 25C, and then add 25l of Sodium nitroprusside (0.0067M) and 25l of Na2CO3(1.5 M) with NaCN (0.067M). The colored complex developed is measured immediately at 550 nm. Glutathione is expressed in terms of g/ mg of protein cell lysate. Malondialdehyde (MDA): Lipid peroxidation was determined by measuring the amounts of malondialdehyde (MDA) according to the method of Ohkawa et al. (1979). Briefly, the cell lysate (400 l) was mixed with 10% TCA and incubated for 15 min at 4C and then centrifuged at 2.2g

for 15 min at 4C. To 300l of protein-free supernatant, 300l of fresh TBA reagent was added, mixed thoroughly and incubated at 60C for 1 hour in water bath. Then OD was measured at 532 nm for the assay of MDA. Lipid peroxide is expressed in terms of nM of MDA mg of protein cell lysate. Treatment of collagen recovery: HaCaT cells were maintained in DMEM culture media up to a confluence of 6080% and splitted into 24 well plates (30000 cell/well). Next day media was removed and cells were washed with HBSS and the cells were pretreated with different concentrations of (5g-20g) aq. methanol (LI/PD/148/4) extract of CSE in 0.1% (vol vol) DMSO and cells exposed to UV-50 mJ/cm2 for 4 hour. After exposure cells were washed with HBSS and replaced with given treatment in regular media and kept back in CO2 incubator. The same procedure was continued measured on day 4. Collagen recovery assay: The collagen content was quantified by a Sirius-red based colorimetric assay (Nguyen et al 2008). Briefly, the cells were washed with PBS for three times followed by fixation with bouins fluid for 1 hour at room temperature. After fixation, the fixation fluid was removed and culture dishes were washed by immersion in running tap water for 15 min. The plates were air dried and stained with Sirius-red dye reagent for 1 hour under mild shaking on shaker. Thereafter, the solution was removed and the culture was washed with 0.1 N HCl to remove any non-bound dye. The stained material was dissolved in 0.1 N NaOH, and incubates for 30 min under continuous shaking. The dye containing solution was transferred to micro plate and absorbance was measured at 550nm. Hydroxyproline: Collagen deposition was estimated by determining the hydroxyproline content of the cell lysate (Brown, et al). The cell lysates were hydrolyzed in 1 ml of 5 N HCl for overnight at 110 C. The next day reaction was neutralized with 2.5 N NaOH. The hydroxyproline assay was done in a 96-well plate by adding 100 l of oxidizing solution to wells. The oxidizing solution was prepared by adding 6 mg of chloramine T/ml of oxidation buffer (3 ml of isopropyl alcohol, 1.65 ml of H2O, 1.95 ml of citrate-acetate buffer, pH 6). 50 l of standard or sample was added to each well. This was followed by addition of 100 l of for 3 consecutive days and the collagen levels were

Ehrlichs reagent to each well. After incubating the samples at 60 C for 45 min, the samples were cooled to room temperature. The amount of hydroxylproline present in samples was determined by the absorbance measured at 562 nm. Statistical analyses: Statistical analyses were carried out using sigma plot. Regression analysis was used for calculation of IC50 values for all in vitro assays. Differences among the tested antioxidants were analyzed by using one-way ANOVA followed by Studentst test. Results Cell Viability Assay Fig-1, showed the effect of various concentrations of CSE (daily 4 hours exposure) on HaCaT cells vialbility for 72 hours against UV irradiation. The cell viability against UV irradiation (50 mJ/cm2) was found to be 70.44% or 85.84% from 1 to 20 g/mL when compared to control. These results displayed that CSE significantly reduced UV-induced cell death in HaCaT cells. Fig-1: Effect of CSE on the viability of UV irradiated HaCaT cells(Fig: Legend) Fig-1. CSE pretreatment protects HaCaT cells from UV-mediated decrease in cell viability. HaCaT cells were treated with different doses of CSE (120 g/mL) for 2 hours after which the cells were exposed to UV (50 mJ/cm2) radiation. After UV exposure, HBSS was removed and fresh media was added. At 72 hours after UV irradiation, percent of cell viability was evaluated by MTT assay. Generation of ROS Table-1 displayed the CSE extract showed potent anti-oxidant activity by inhibiting Reactive Oxygen Species (ROS) generation in UV exposed HaCaT cells. HaCaT cells were pretreated with CSE and then irradiated with UV at 50 mJ/cm2 for 2 hours. Intracellular ROS generation was evaluated by 2,7-dichlorofluorescein (DCF) fluorescence. Table-1: effect of CSE-hexane, CSE-EtOAc, CSE-Methanol, CSE-Aq. methanol, and CSE-Water extract.

Test compound CSE-Hexane CSE-EtOAc CSE-Methanol CSE-Aq. Methanol CSE-Water

Antioxidant activity on HaCaT cells IC50 (g/mL) >50 19.47 9.52 3.47 13.57

Anti-oxidant effects of CSE in HaCaT cells: CSE inhibits UV mediated decrease SOD: We assessed the SOD levels in HaCaT cells and found that treatment of HaCaT cells with CSE resulted increase in SOD levels in a dose dependent manner (5-20 g/ mL). UV (50 mJ/cm)2 irradiation resulted in a dosedependent decrease in SOD levels (Fig. 2a). CSE inhibits UV mediated decrease in catalase levels: Similar way treatment of cells with CSE resulted in increased of catalase enzyme. UV (50 mJ/cm2) irradiation resulted in a dose-dependent decreased in catalase levels. Pretreatment of cells with CSE (5-20 g/ mL) prior to UV-mediated decrease in catalase content (Fig. 2b). CSE inhibits UV mediated decrease in glutathione levels: Further we assessed the GSH levels in HaCaT cells, and found the UV (50 mJ/cm)2 irradiation exhibited in a dose-dependent decrease in GSH levels (Fig. 2c). But the treatment of HaCaT cells with CSE resulted in increased levels of GSH in a dose dependent manner (5-20 g/ mL). Fig: 2a, 2b and 2c: Effect of CSE on the superoxide dismutase, catalase and glutathione of UV irradiated HaCaT cells:(Legend) Inhibitory effects of CSE on UV mediated increase in SOD, catalase and glutathione levels in HaCaT cells. HaCaT cells were pretreated with different doses of CSE (5-20 g/mL). After which the cells were exposed to UV (50 mJ/cm2) for 72 hours (the cells were exposed to 4 hours per day), the media were removed and cells were washed once with HBSS and then fresh HBSS was added. 72 hours post-UV, the cells lysates were prepared for SOD, catalase and GSH estimation. *P < 0.01 versus control and UV. CSE inhibits UV mediated increased in LPO in HaCaT cells:

As shown in Fig-3. UV irradiation of HaCaT cells resulted in a significant induction of LPO measured in terms of malondialdehyde equivalent when compared with untreated cells. UV-mediated increase in LPO was found to significantly inhibit in HaCaT cells which were pretreated with CSE (5-20 g). Thus, inhibition of LPO by CSE resulted in the reduction of risk factors associated with UV radiation. Fig-3: Effect of CSE on the lipid peroxidation of UV irradiated HaCaT cells. (Legend) Inhibitory effects of CSE on UV mediated increase in LPO in HaCaT cells. HaCaT cells were pretreated with different doses of CSE (5-20 g/mL). After which the cells were exposed to UV (50 mJ/cm2) for 72 hours (the cells were exposed to UV for 4 hours per day), the media were removed and cells were washed once with HBSS and then fresh HBSS was added. 72 hours post-UV, the cells were harvested and cell lysates were prepared for LPO estimation. Photoprotective effect of CSE HaCaT cells: Collagen content: Further to evaluate the photoprotective effect of CSE, we measured the collagen and hydroxyproline content in CSE treatment in UV irradiated HaCaT cells. Collagen levels in UV (50 mJ/cm)2 irradiated HaCaT cells were to increase in CSE treatment group (5-20g/mL) in a dose dependent manner when compared to control and UV alone (Fig-4a). Hydroxy proline content: to conform the inhibitory effect of CSE on glycogen degradation we assessed the hydroxy proline (HP) levels in HaCaT cells, and found that treatment of HaCaT cells with CSE resulted in increased level of hydroxy proline in a dose dependent manner (5-20 g/ mL). where as in control cells UV (50 mJ/cm)2 irradiation resulted in a dose-dependent decrease in hydroxyl proline levels (Fig-4b). Fig-4a and 4b: Effect of CSE on the collagen and hydroxyl proline content of UV irradiated HaCaT cells: (Fig; Legend) Inhibitory effects of CSE on UV mediated increase in collagen content and hydroxyl proline levels in HaCaT cells. HaCaT cells were pretreated with different doses of CSE (5-20 g/mL). After which the cells were exposed to UV (50 mJ/cm2) for 72 hours (the cells were exposed to 4 h/day). After 72 hr media were removed and the cells were

washed once with HBSS and then fresh HBSS was added. 72 hours of post-UV treatment cell lysates were prepared for estimation of collagen and hydrohyproline content. DISCUSSION There is a two new finding of this study, to identify the inhibition of antioxidant and anti-skin ageing activity. Several studies have produced clear evidence that there is a relation between the oxidative damage lades to skin ageing. Researchers pay attention on to the relation of oxidative stress and skin ageing. Accumulation of ROS in the human body to causes several pathophysiological diseases. Enzymatic antioxidant defense of the organism includes: SOD, CAT, and GSH. Superoxide dismutase protects a cell from toxic effect of superoxide radicals as it catalyzes the dismutation reaction of the radicals (Katiyar, 2001; Scharffetter,1997) ROS are causes skin damage by UV exposure, scavenging of these reactive species could prevent the oxidative reactions and subsequently protect skin from the damaging effects of UV exposure. Therefore, the researchers to identify the useful of botanical antioxidants to reduce harmful effect of UV radiation by scavenging ROS is a novel approach to delay the process of photoaging. One such natural product is Chloroxylon swieteniea, which is commonly available plant in India. The crude extract and fractions of C. swieteniea prove tyrosinae inhibitory activity (ref). The authors have believed that Chloroxylon swieteniea is naturally occurring antioxidant-rich botanicals and effective in reducing the harmful effect of UV radition mediated oxidative damage, skin damage. Our results showed that the percentage of viable cells was markedly reduced after UV-irradiation as compared to control group. Cellular antioxidant mechanisms may be overwhelmed by excessive free radical generation altering the redox status of cell and affects cell viability. CSE protects the cells from UV-mediated cell death (Fig-1) by its strong antioxidant activity (Table-1), and therefore CSE plays a significant role in preventing photo-biological damage. Antioxidant enzymes, such as SOD and Catalase are the play an important role in antioxidant defense mechanism. SOD plays a vital role in scavenging superoxide anion which are formed during the early stages of oxidative stress, and in preventing skin aging (Emerit, et al 2004). SOD catalyzes the conversion of superoxide to hydrogen peroxide

plus dioxygen. Hydrogen peroxide (H2O2) is a harmful by-product of many normal metabolic processes. However, CAT is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide (Bose Girigoswami,et al 2005). CAT catalyzes the formation of water and oxygen from hydrogen peroxide and prevents oxidative damage (Rajeshkumar,et al 2003). The free radical scavenging and antioxidant property of CSE have been recently proved by senthil et al (ref). In this study SOD, CAT levels were observed in UV irradiated HaCaT cells (Fig-2a&2b). Therefore a reduction in the activity of these enzymes activity during UV exposure can result in a number of deleterious effects due to the accumulation of superoxide radicals and H2O2. Pretreatment with CSE increased in the SOD and CAT in UV irradiated HaCaT cells and thus CSE could exert beneficial action pathological alterations caused by the UV radiation. Further increased activity of SOD, CAT in UV irradiated HaCaT cells are mainly because of the antioxidant sparing action of CSE. Glutathione is an important intracellular nonprotein sulfhydryl peptide with multiple functions ranging from antioxidant defense to modulation of cell proliferation.GSH plays a central role in the maintenance of cellular homeostasis, regulating signaling pathways modulated by oxidative stress and protects skin cell against oxidative injury (ref). UV-mediated loss of cell viability is associated with a marked decrease in GSH content, indicating an impairment of the antioxidant pool, causing an increase in ROS, and may predispose the cell to a lower defense against condition of oxidative stress. GSH depletion was significantly and dose dependently inhibited by pretreatment of cells with CSE (Fig-2c). CSE due to its antioxidant activity protects cells from UV-induced oxidative stress by increasing their antioxidative status. Oxidative damage to lipids and proteins, an immediate consequence of UV radiation to skin, occurs most readily in the superficial layers (32). Sustained oxidative insult as a result of UV exposure causes LPO, which leads to the accumulation of MDA, a stable end product of LPO, indirectly suggesting the generation of ROS. Increased LPO caused by UV irradiation may evoke immune and inflammatory responses and activate gene expression resulting from membrane- dependent oxidative damage (REF). CSE significantly reduced peroxide accumulation, suggesting that CSE due to its antioxidant activity could scavenge ROS and inhibit the reaction of LPO (Fig-3).

The collagen composed of amino acid (hydroxyproline) is the major component of extra cellular tissue, which gives strength and support. Breakdown of collagen liberates free hydroxyproline and its peptides; measurement of the hydroxyproline could be used as an index for collagen turnover. CSE treated HaCaT cells showed significant increased collagen content and hydroxyproline content when compared with the control group. Conclusion: Interestingly, CSE treatment dose-dependently increased SOD, Catalse and GSH levels in UV-exposed human keratinocytes. Whereas, CSE potentially reduced lipid peroxidation, as evidenced by dose-dependent decrease in MDA level in UV exposed cells. Taken together, these data demonstrate that the aqueous-methanol extract of C. swietenea leaves have strong anti-oxidant potential. Therefore, we conclude that this herbal extract might be useful for relief from UV induced oxidative damage of skin.

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