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), Bouin’s fixative solution, Coomassie brilliant

blue, DCF (2’, 7’ dichloro fluorescein), Dulbecco’s 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 37°C with 95% and 5% (v/v) mixture of air and CO2.

Cell viability assay. Viability of cells in culture was measured by 3-[4,5-

dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HaCaT

cells were plated at 5×104 cells per well in 200 µL 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 37ºC 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 hank’s balanced salt solution (HBSS) and supplemented

with new medium. Cells were incubated with DCF in HBSS solution for 30 min at 37ºC

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 60–80% 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 25µl 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 4°C. To 50 l of

cell supernatant or lysate 150l saturated sodium chloride solution is added, allowed it to

stand for 10 min at 25°C, 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 μl)

was mixed with 10% TCA and incubated for 15 min at 4°C and then centrifuged at 2.2g

for 15 min at 4°C. To 300l of protein-free supernatant, 300l of fresh TBA reagent was

added, mixed thoroughly and incubated at 60°C 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 60-

80% 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 (5µg-20µg) 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 CO 2 incubator. The

same procedure was continued for 3 consecutive days and the collagen levels were

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 µl 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 µl

of standard or sample was added to each well. This was followed by addition of 100 µl of

Ehrlich’s 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 Students‘t’ 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 µg/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 (1–20 µg/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 Antioxidant activity on HaCaT

cells IC50 (µg/mL)CSE-Hexane >50

CSE-EtOAc 19.47

CSE-Methanol 9.52

CSE-Aq. Methanol 3.47

CSE-Water 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 µg/ mL). UV (50 mJ/cm)2 irradiation resulted in a dose-

dependent 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

µg/ 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 µg/ 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

µg/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 μg). 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 µg/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 (5-20µg/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 µg/ 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 µg/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.

Referencie:

1. Ross, C.; Alston, M.; Bickenbach, J.R.; Aykin-Burns, N. Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner. Exp. Dermatol. 2011, 20, 58–63. 2. Laskin, J.D.; Black, A.T.; Jan, Y.H.; Sinko, P.J.; Heindel, N.D.; Sunil, V.; Heck, D.E.; Laskin, D.L. Oxidants and antioxidants in sulfur mustard-induced injury. Ann. NY Acad. Sci. 2010, 1203, 92–100. 3. Vileno, B.; Jeney, S.; Sienkiewicz, A.; Marcoux, P.R.; Miller, L.M.; Forró, L. Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols. Biophys. Chem. 2010, 152, 164–169. 4. Dreger, H.; Westphal, K.; Weller, A.; Baumann, G.; Stangl, V.; Meiners, S.; Stangl, K. Nrf2-dependent upregulation of antioxidative enzymes: A novel pathway for proteasome inhibitor-mediated cardioprotection. Cardiovasc. Res. 2009, 83, 354–361. 5. Vertuani, S.; Angusti, A.; Manfredini, S. The antioxidants and pro-antioxidants network: An overview. Curr. Pharm. Des. 2004, 10, 1677–1694. 6. Chae, H. Z., Kang, S. W. & Rhee, S. G. Isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin. Methods Enzymol. 300, 219–226 (1999)7. Carbonare MD, Pathak MA: Skin photosensitizing agents and the role of reactive

oxygen species in photoaging. J Photochem Photobiol B: Biol 14: 105-124, 1992.

8. Introduction to Skin Cancer, U.S. National Cancer Institute SEER Training Website, http://training.seer.cancer.gov

9. Ravi Kiran, S. Sita Devi, E P. Janardhan Reddy,E K .Evaluation of in vitro antimicrobial activity of leaf and stem essential oils of Chloroxylon swietenia DCWorld J Microbiol Biotechnol (2008) 24:1909–1914.

10. Sivakumar T. Kanagasabai R. Sampathkumar R. Perumal P. Sivakumar, Gupta P.M. Mazumder U. K. (2008) 11th NAPRECA Symposium Book of Proceedings, Antananarivo, Madagascar p.201-213

11 Vorkoc J ., sedmera, p., evaluation of anti-inflammatory activity and toxicity related studies of chloroxylon sweitenia phytochem, 11,2647,(1977).12 Sharma AK, Chandan BK, Anand KK and Pushpangadan P (1989). Antifertility activity of Chloroxylon swietenia DC. In Rats. Zoologica Orietalis. 4(2): 50-55.

13. Ravi kiran S. ; shiva reddy A. ; sita devi P. ; janardhan reddy K. ; insecticidal, antifeedant and oviposition deterrent effects of the essential oil and individual compounds from leaves of Chloroxylon swietenia DC . Pest management science. 2006:62- 1116-1121.14 Talapatra S.K., Bhattacharya M., Talapatra B.,Das, B.C.,J.Indian Chem. Soc.,45,861 (1968).15. Mujumdar R.B., Rama Rao A.V., Rathi, S.S.Venkataraman,K. Tetrahedron Lett., 11 867(1975).

16. Govindachari T.R.,Sathe, S.S. & Viswanathas N.,Tetrahedron Lett., 42 ,4183 (1967).

17. Lee K., Soine T.O., J. Pharm. Sci., 58 , 681, (1969).

18. Ponti V., Dianzani M. U., Cheeseman K., Slater T. F., Chemico-Biol. Interact., 23, 281—291 (1978).19. Colin C, Boussouira B, Bernard D, Moyal D, N guyen QL. Non invasive methods of

oxidative stress induced by lokw dose of ultraviolet in humans. Proc 18th Congr Int Fed

Soc Cosm Chem(IFSCC), Venezia, Oct 3-6, 1994, 1-23.

20. Wheeler, C.R., Salzman, J.A., Elsayed, N.M., et al. Automated assays for superoxide

dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal

Biochem 184 193-199 (1990).

21. Ohkawa H, Onishi N, Yagi K. Assay for lipidperoxidation in animal tissue by

thiobarbituric acid reaction. Anal Biochem 1979; 95:351-358.

22. Nguyen Xuan CUONG, Tran Anh TUAN, Phan Van KIEM, Chau Van MINH, Eun

Mi CHOI, and Young Ho KIM. New Cembranoid Diterpenes from the Vietnamese Soft

Coral Sarcophyton mililatensis Stimulate Osteoblastic Differentiation in MC3T3-E1

Cells. Chem. Pharm. Bull. 56(7) 988—992 (2008)

23. Brown, S., Worsfold, M., and Sharp, C. (2001) BioTechniques 30, 38–40

6. Habdous, M.; Herbeth, B.; Vincent-Viry, M.; Lamont, J.V.; Fitzgerald, P.S.; Visvikis, S.; Siest, G. Serum total antioxidant status, erythrocyte superoxide dismutase and whole-blood glutathione peroxidase activities in the Stanislas cohort: Influencing factors and reference intervals. Clin. Chem. Lab. Med. 2003, 41, 209–215. 18. Emerit, I.; Filipe, P.; Freitas, J.; Vassy, J. Protective effect of superoxide dismutase against hair graying in a mouse model. Photochem. Photobiol. 2004, 80, 579–582. 19. Alscher, R.G.; Erturk, N.; Heath, L.S. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 2002, 53, 1331–1341.

20. Bose Girigoswami, K.; Bhaumik, G.; Ghosh, R. Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes. Cell Biol. Int. 2005, 29, 761–767. 21. Rajeshkumar, N.V.; Ramadasan, K. Modulation of carcinogenic response and antioxidant enzymes of rats administered with 1, 2-dimethyl hydrazine by Picroliv. Cancer Lett. 2003, 191, 137–143. 22. Thu, V.T.; Kim, H.K.; Ha, S.H.; Yoo, J.Y.; Park, W.S.; Kim, N.; Oh, G.T.; Han, J. Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts. Pflugers. Arch. 2010, 460, 55–68.

11) Payá M., Ferrándiz M. L., Miralles F., Montesinos C., Ubeda A., AlcarazM. J., Arzneim.-Forsch./Drug Res., 43, 655—658 (1993).

Chae, H. Z., Kang, S. W. & Rhee, S. G. Isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin. Methods Enzymol. 300, 219–226 (1999)

Scharffetter-Kochanek K, Brenneisen P, Wenk J, Herrmann G, Ma W, Kuhr L, Meewes C, Wlaschek M: Photoaging of the skin from phenotype to mechanisms. Exp Gerontol 35:307–316, 2000.

Payá M., Ferrándiz M. L., Miralles F., Montesinos C., Ubeda A., Alcaraz M. J., Arzneim.-Forsch./Drug Res., 43, 655—658 (1993).

Ponti V., Dianzani M. U., Cheeseman K., Slater T. F., Chemico-Biol. Interact., 23, 281—291 (1978).

Ohkawa, H., N. Ohishi and K. Yagi, 1979. Assay for lipid peroxide in animal tissues by

thiobarbituric acid reaction. Anal. Biochem., 95: 351-358.

Katiyar, S. K., F. Afaq, A. Perez and H. Mukhtar (2001) Green tea polyphenol (–)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress. Carcinogenesis 22, 287–294.

Scharffetter-Kochanek, K., M. Wlaschek, P. Brenneisen, M. Schauen, R. Blaudschun and J. Wenk (1997) UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol. Chem. 378, 1247–1257.

Nicholls, P., Fita, I., and Loewen, P.C. (2001) Enzymology and structure of catalases.

Adv Inorg Chem 51: 51–106.

I.N. Zelko, T.J. Mariani, R.J. Folz, Superoxide dismutase multigene family: a comparison

of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures,

evolution, and expression, Free Radic. Biol. Med. 33 (2002) 337–349.

J.M. Mates, C. Perez-Gomez, I. Nunez de Castro, Antioxidant enzymes and human

diseases, Clin. Biochem. 32 (1999) 595–603.

Nguyen Xuan CUONG, Tran Anh TUAN, Phan Van KIEM, Chau Van MINH, Eun Mi

CHOI, and Young Ho KIM. New Cembranoid Diterpenes from the Vietnamese Soft

Coral Sarcophyton mililatensis Stimulate Osteoblastic Differentiation in MC3T3-E1

Cells. Chem. Pharm. Bull. 56(7) 988—992 (2008)

Benaiges A, Marcet P, Armengol R, Betes C, Girones E: Study of the refirming effect of a plant complex. Int J Cosmet Sci 1998, 20:223-233.

Jenkins G: Molecular mechanisms of skin ageing. Mech Ageing Dev 2002, 123:801-810.

Y. Shindo, E. Witt, and L. Packer, “Antioxidant defense mechanisms in murine epidermis

and dermis and their responses to ultraviolet light,” Journal of Investigative

Dermatology, vol. 100, no. 3, pp. 260–265, 1993.

F’guyer, S., F. Afaq and H. Mukhtar (2003) Photochemoprevention of skin cancer by

botanical agents. Photodermatol. Photoimmunol. Photomed. 19, 56–72

Rittie L, Fisher GJ. UV-light-induced signal cascades and skin aging. Ageing Res Rev

2002; 1:705- 20.

Carbonare MD, Pathak MA: Skin photosensitizing agents and the role of reactive oxygen

species in photoaging. J Photochem Photobiol B: Biol 14: 105-124, 1992.

Introduction to Skin Cancer, U.S. National Cancer Institute SEER Training Website, http://training.seer.cancer.gov

Afaq, F., V. M. Adhami and H. Mukhtar (2005) Photochemoprevention of ultraviolet B

signaling and photocarcinogenesis. Mutat. Res. 57, 1153–1173.

Afaq, F. and H. Mukhtar (2001) Effects of solar radiation on cutaneous detoxification

pathways. J. Photochem. Photobiol. B 63, 61–69.

Sander, C. S., H. Chang, F. Hamm, P. Elsner and J. J. Thiele (2004) Role of oxidative

stress and the antioxidant network in cutaneous carcinogenesis. Int. J. Dermatol. 43, 326–

335.

Bansal, M. and Ananthanarayanan, V. S. 1988. The role of hydroxyproline in collagen

folding: conformational energy calculations on oligopeptides containing proline and

hydroxyproline. Biopolymers 27: 299-312,

Winsor, 1970;

Sweeney.T.M, 1970