Abstract: UV radiation-induced damages may result in pre-cancerous and cancerous lesions and acceleration of skin aging. It involves an imbalance of the endogenous antioxidant system that leads to the increase of free radical levels. Antioxidant pretreatment might inhibit such imbalance. In the present study, the photoprotective effect of ursolic acid (UA; 3β-hydroxy-urs-12-en-28-oic acid), a dietary polyphenolic phytochemical, has been examined in the UVB-(280-320 nm) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentrations of ursolic acid (1, 5 and 10 μg mL-1) for 30 min, were irradiated and lipid peroxidation and antioxidant defense were examined. UVB-irradiated lymphocytes exhibited increased levels of lipid peroxidation and disturbances in antioxidant status. Ursolic acid pretreatment resulted in significant reduction in thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LPH) levels. Further, antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin-C (Vit-C) and vitamin-E (Vit-E) were normalised in ursolic acid pretreated plus UVB-treated lymphocytes. The maximum dose of ursolic acid (10 μg mL-1) normalized the UVB induced lipid peroxidation, indicating the photoprotective effect of ursolic acid in human peripheral lymphocytes under in vitro condition.
INTRODUCTION
Phototoxic effect induced by UVB (280-320 nm) radiation involve the generation of Reactive Oxygen Species (ROS) resulting in oxidative damage (Wu et al., 2006; Steenvoorden and Beijersbergen Van Henegouewwn, 1997). ROS generated due to UVB irradiation results in DNA damage and lipid peroxidation (Katiyar et al., 2007). Further, reactive oxygen species are shown to activate transcription factors such as AP-1 and NF-κB, which may contribute to cell proliferation and/or apoptotic cell death (Ichihashi et al., 2003). It has been demonstrated previously that oxidative stress induced by UVB-radiation can lead to alteration in antioxidant enzyme levels, apoptosis and cell death (Cejkava et al., 2000; Kimura et al., 2000).
Herbal medicine derived from plant extracts is being increasingly utilized
to treat a wide variety of clinical disease with relatively little knowledge
of their modes of action. Polyphenols are complex group of chemicals that
are widely distributed throughout the plant kingdom and thus form an integral
part of the human diet (Manach et al., 2004). It has been suggested
that dietary polyphenol protect against a variety of diseases including
cancer and cardiovascular disease and there has been an increased interest
in these compounds from both consumers and food manufactures (Geleijnse
et al., 2002).
| Fig.1: | Urosolic acid (3 β-hydroxy-urs-12-en-28-oic acid) |
Ursolic acid (UA; 3 β-hydroxy-urs-12-en-28-oic acid), a pentacyclic triterpenoid, exists widely in natural plants, which is present in many kinds of medicinal plants, (Fig. 1) such as Eriobotrya japonica, Rasmarinus officinalis and Glechoma hederaceae (You et al., 2001) in the form of free acid or as aglycones of triterpenoid saponins (Ovesna et al., 2006). It exhibits antiinflammatory (Wang et al., 2005), anticarcinogenic (Liu, 2005), antiulcer (Hsinshiha et al., 2004), antihyperlipidemic (Somova et al., 2003) and hepatoprotective (Sarananan et al., 2006) activities. Although protective effect of ursolic acid against UVA was evaluated in HaCaT human keratinocytes (Lee et al., 2003), no sufficient work has been carried out to study its protective effect against UVB-mediated oxidative stress in human lymphocytes. Lymphocytes have been used to develop non-invasive bioassays to screen human population for toxicant exposure and these cells have been used to determine exposure and susceptibility to the toxicants (Rajendra Prasad et al., 2005). Lymphocytes are most studied and contain variety of redox and free radical scavenging systems (Halliwell and Gutteridge, 1998). Hence, studies on lipid peroxidation and antioxidant enzymes in blood lymphocytes could be of immense significance in identifying intracellular oxidative damage in the individuals, who could be at risk to UVB induced oxidative damage. The purpose of the present study was to evaluate the impact of ursolic acid on UVB-mediated oxidative stress in human lymphocytes under in vitro condition
MATERIALS AND METHODS
Chemicals
Ursolic acid, heat inactivated fetal calf serum (FCS), thiobarbituric
acid (TBA), phenozine methosulphate (PMS) nitroblue tetrazolium (NBT),
5,5-dithiobis 2-nitrobenzoic acid (DTNB) and nicotinamide adenine dinucleotide
(NAD) were purchased from (Sigma chemical Co., St. Louis, USA). Other
chemicals for blood lymphocyte cultures (RPMI-1640, penicillin, streptomycin,
L-glutamine) and reduced glutathione (GSH) were purchased from (Himedia,
Mumbai). All other chemicals and solvents were of analytical grade and
obtained from (SD Fine Chemical, Mumbai and Fisher. Inorganic and Aromatic
Limited, Chennai).
Blood Samples
Blood samples were aseptically collected in heparinized sterile tubes
from median cubital vein of non smoking healthy individuals (22-25 years).
Lymphocytes were isolated using Ficoll–Histopaque (Sigma, USA) and
cultured as described provisionally (Boyum, 1968). Blood was diluted 1:1
with Phosphate Buffered Saline (PBS) and layered onto histopaque/with
ratio of blood and PBS; Histopaque maintained at 4:3. The blood was centrifuged
at 1340 rpm for 35 min at room temperature. The lymphocyte layer was removed
and washed twice in PBS at 1200 rpm for 10 min each and then washed with
(RPM1-1640) media.
Study Design
Cultured lymphocytes were divided into six groups; in each group
six samples were processed.
| Group 1: | Normal lymphocytes without any treatment. |
| Group 2: | Normal lymphocytes with 10 μg mL-1 of ursolic acid. |
| Group 3: | UVB-irradiated lymphocytes for 30 min. |
| Group 4: | UVB-irradiated lymphocytes pretreated with 1 μg mL-1 of ursolic acid. |
| Group 5: | UVB-irradiated lymphocyte pretreated with 5 μg mL-1 of ursolic acid. |
| Group 6: | UVB-irradiated lymphocytes pretreated with 10 μg mL-1 of ursolic acid. |
Treatment of the Cells
Thirty minutes prior to irradiation three test-doses (1, 5 and 10 μg
mL-1) of ursolic acid were added to the grouped normal lymphocytes.
Preliminary studies were carried out to ensure that whether this concentration
had any toxic effect by trypan blue dye exclusion test. Before exposure
to UV light, the cell cultures were washed twice with PBS. Non-irradiated
lymphocytes showed decrease in viability over the 30 min period of incubation.
Irradiation Procedure
For UVB irradiation cells were irradiated in 35 mm Petri dishes containing
2 mL of PBS and covered with a UV permeable with a UV permeable membrane
to prevent contamination. A battery of TL 20W/20 fluorescent tubes (Heber
scientific) served as UVB source which had a wave length range set 280-320
nm peaked at 312 nm and an intensity of 2.2 mW cm-2 for 9 min.
The total UVB-irradiation was 19.8 mJ cm-2, corresponding to
an average value of 1.52x0-3 mJ cell-1. After irradiation
the lymphocytes were kept at room temperature for 30 min and then subjected
to biochemical assays.
Biochemical Estimation
Lymphocytes were suspended in 130 mM KCl plus 50 mM PBS containing
0.1 mL of 0.1 M dithiothreitol and centrifuged at 20,000 x g for 15 min
(4°C). The supernatant was taken for biochemical estimations. In each
group six samples (n = 6) were processed. The level of lipid peroxidation
was determined by analyzing TBA-reactive substance according to the protocol
of Niehaus and Samuelson (1968). The pink coloured chromogen formed by
the reaction of 2-TBA with breakdown products of lipid peroxidation was
measured. The lipid hydroperoxides (LPH) levels were determined by analyzing
BHT-reactive substance according to the protocol of Jiang et al.
(1992). Superoxide dismutase (SOD) activity was assayed by the method
of Kakkar et al. (1984), based on the inhibition of the formation
of (NADH-PMS-NBT) complex. Catalase (CAT) activity was assayed by the
procedure of Sinha (1972) quantifying the hydrogen peroxide after reacting
with dichromate in acetic acid. The activity of glutathione peroxidase
(GPX) was assayed by the method of Rotruck et al. (1973) a known
amount of enzyme preparation was allowed to react with hydrogen peroxide
(H2O2) and GSH for a specified time period. Then
the GSH content remaining after the reaction was measured. The total GSH
context was measured by the method of Elliman (1959). This method was
based on the development of a yellow colour when 5,5-dithiobis 2-nitrobenzoic
acid was added to compounds containing sulphydryl groups. The ascorbic
acid was estimated by the methods of Roe and Kuether (1969) the red coloured
compound when treated with sulphuric acid and then adding 2,4 - dinitrophenyl
hydrazine in the presence of thiourea solution. α-tocopheral was
estimated by the method described by Baker et al. (1980).
Statistical Analysis
Statistical analysis was performed by one-way (ANOVA) followed by DMRT
taking p<0.05 to test the significant difference between groups.
RESULTS
In this study, the concentration of TBARS and LPH increased significantly
in UVB irradiated lymphocytes (Table 1). Ursolic acid
pretreated lymphocytes showed progressively decreased concentrations of
TBARS and LPH when compared with UVB-irradiated cells and even 1 μg
mL-1 of ursolic acid pretreatment significantly decreased the
levels of lipid peroxidation indices in UVB-irradiated lymphocytes. UVB-exposure
significantly decrease the SOD, CAT activities in this study and pretreatment
with ursolic acid results in significant increase in the SOD, CAT activities
as ursolic acid concentration increases (Table 2). Present
study also shows (Table 3) that UVB-irradiation caused
a significant decrease in the GPx activities and GSH levels when compared
with the normal lymphocytes. Ursolic acid pretreatment significantly restored
the GPx activities and GSH levels to normal when compared with UVB-exposed
groups. UVB-irradiated group decrease vit-C, vit-E levels and pretreatment
with ursolic acid result in significantly increases in the vit-C, vit-E
levels as ursolic acid concentration increases (Table 4).
| Table 1: | Effect of ursolic acid on the levels of TBARS and LPH in normal, UVB-irradiated and ursolic acid pretreated lymphocytes |
| Values are given as means±SD of six experiments in each group; Values not sharing a common superscript different significantly at p<0.05 (DMRT) | |
| Table 2: | Effect of ursolic acid on the activities of SOD and CAT in normal, UVB-irradiated and ursolic acid pretreated lymphocytes |
| Values are given as means±SD of six experiments in each group; Values not sharing a common superscript different significantly at p<0.05 (DMRT); *: Enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in 1 min; **: μmol of hydrogen peroxide consumed per min | |
| Table 3: | Effect of ursolic acid on the GPx activities and GSH levels in normal, UVB-irradiated and ursolic acid pretreated lymphocytes |
| Values are given as means±SD of six experiments in each group; Values not sharing a common superscript different significantly at p<0.05 (DMRT); ***: μg of glutathione consumed per min | |
| Table 4: | Effect of ursolic acid on the levels of Vit-C and Vit-E in normal, UVB-irradiated and ursolic acid pretreated lymphocytes |
| Values are given as mean±SD of six experiments in each group. Values not sharing a common superscript different significantly at p<0.05 (DMRT) | |
DISCUSSION
The studies on development of novel agents with anti-photoaging capabilities particularly from natural resources including various plants have been intensively performed. The UVB radiation is the most described physical attack it causes cellular damage resulting in both pre-cancerous and cancerous lesions and acceleration of aging (Casagrande et al., 2006). Probably the genesis of pathologies due to UVB exposure is a consequence of the generation of free radicals. The resulting imbalance between oxidants and antioxidants shifts the redox-sensitive signal transduction pathways and gene expression. These molecular changes may be involved in the pathogenesis of photo damages (Fuchs, 1998).
In this study the levels of lipid peroxidation has been significantly increased in UVB irradiated cells (Table 1). The increase in the levels of TBARS and LPH indicates the activation of lipid peroxidation in UVB-irradiated lymphocytes. Lipid peroxidation induced by UVB-radiation is known to be due to the attack of free radicals on the fatty acid component of membrane lipids. Present results shows that ursolic acid renders protection against UVB-radiation induced oxidative stress. This may be due to its antioxidative property. The antioxidant effects of ursolic acid on lipid peroxidation in liver microsomes, leukemic cells and myocardial cell were already documented (Sarananan et al., 2006; Ovesna et al., 2006; Senthil et al., 2007).
The free radical scavenging and antioxidant property of ursolic acid have been recently proved by Dufour et al. (2007). It was thought that this antioxidant property is due to the polyphenolic methyl group present in ursolic acid (Zhang et al., 2001). In this study reduced SOD, CAT, GPx activities and GSH levels were observed in UVB-irradiated lymphocytes (Table 2 and 3). Similar results were obtained by Cajkova et al. (2000) in corneal epithelium cells and by Isoherranen et al. (1997) in He La cells, when these cells were exposed to UVB-irradiation. SOD protects the cells against superoxide radical, which can damage the membrane (Michaelson, 1977). CAT primarily causes decomposition of hydrogen peroxide (H2O2) to H2O at a much faster rate GPx also plays an important role in the removal of lipid hydroperoxides. Therefore a reduction in the activity of these enzymes during UVB-exposure can result in a number of deleterious effects due to the accumulation of superoxide radicals and H2O2. Pretreatment with ursolic acid increased the activities of SOD, CAT in UVB-irradiated lymphocytes and thus ursolic acid could exert a beneficial action against pathological alterations caused by the UVB-radiation. Further the increased activity of SOD, CAT, GPx and GSH in UVB-irradiated lymphocytes is mainly because of the antioxidant sparing action of ursolic acid. Since ursolic acid prevents the formation of ROS the syntheses of these enzymes are not affected (Mortin-Aragon et al., 2001).
Further present study shows (Table 4) UVB-irradiation caused a significant decrease in the levels of Vit-C and Vit-E in irradiated groups when compared with the normal lymphocytes. The observed decrease in the levels Vit-E and Vit-C may be due to their increased utilization for scavenging hydroxy and/or oxygen derived radicals. Vitamin-C and Vit-E may play a role in preventing lipid peroxidation under experimental and clinical conditions. Lymphocytes with ursolic acid (1, 5 and 10 μg mL-1) prior to irradiation protected Vit-C and Vit-E depletion resulting from the radiation effect. In this study 10 μg mL-1 of ursolic acid pretreatment protects Vit-C and Vit-E levels in UVB-irradiated lymphocytes. The results shows that ursolic acid renders protection against UVB-radiation induced oxidative stress. Previously, ursolic acid and other triterpenes have been reported to show photoprotective activity by inhibiting UV-modulated signal transduction pathways in various experimental models (Both et al., 2002; Yarosh et al., 2000). Studies shows ursolic acid has significantly suppressed the UVA-induced reactive oxygen species production, lipid peroxidation and p53 accumulation in HaCaT human keratinocytes (Lee et al., 2003).
CONCLUSION
It is evident from the present study that ursolic acid offers a remarkable protection against UVB-induced oxidative stress. According to our data and those previously reported in the literature, the photoprotective activity in terms of inhibition of lipid peroxidation and sustaining antioxidant status could explain the beneficial action of ursolic acid against pathological alterations caused by the presence of free radicals which occur during UVB exposure.