EXTRACTION OF URSOLIC ACID FROM OCIMUM SANCTUM: MODULATORY EFFECT OF URSOLIC ACID ON HYDROGEN PEROXIDE INDUCED DNA DAMAGE IN HUMAN LYMPHOCYTES

EXTRACTION OF URSOLIC ACID FROM OCIMUM SANCTUM: MODULATORY EFFECT OF URSOLIC ACID ON HYDROGEN PEROXIDE INDUCED DNA DAMAGE IN HUMAN LYMPHOCYTES

Apsara Batra^*1 and V. Girija Sastry ²

Pharmaceutical Chemistry Division ¹, NCRD’S Sterling Institute of Pharmacy, Nerul, Navi-Mumbai, Maharashtra, India
Department of Pharmaceutical Chemistry ², University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India

ABSTRACT: The objective of the present study was to i) Extract ursolic acid , a pentacyclic triterpenoid from Ocimum sanctum; ii) develop a HPTLC based method for its quantification; and iii) investigate ursolic acid dependent adaptive response to cytotoxic effect of hydrogen peroxide. To elucidate the possible underlying mechanism(s), lymphocytes were co-incubated with hydrogen peroxide (0, 10, 25 and 100 mM) and ursolic acid (20 and 40 mg/ml incubation medium). After 2 h of incubation we investigated the profile of DNA strand breaks, intracellular reactive oxygen species (ROS), apurinic/apyrimidinic sites (AP sites) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) residues in DNA. Results showed that ursolic acid inhibited hydrogen peroxide induced DNA strand breaks. Ursolic also showed significant free radical scavenging as indicated by reduced ROS and decreased AP sites. The formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lymphocyte DNA was also inhibited. These findings clearly showed mechanistic basis for protective effect of ursolic acid against intracellular ROS and consequent generation of AP sites in genomic DNA.  In conclusion, our studies clearly suggest that ursolic acid might be a possible chemo-preventive phytochemical against oxidative stress.

DNA, AP sites, apoptosis, reactive oxygen species

INTRODUCTION: Exposure to environmental xenobiotics is often unavoidable and creates a great risk to human health ¹. Intake of sufficient amounts of antimutagens is believed to confer a preventive effect against environmental mutagens ^{2, 3}. Over the last two decades, an expanding body of evidence has demonstrated that edible plants as a whole, or their identified phytochemical ingredients, have substantial protective effects on onset of diseases such as carcinogenesis ⁴.

Studies have demonstrated that phytochemicals taken in isolation show adequate protective effects against mutagenesis ⁵. Phytochemicals are secondary metabolic products produced by plants in response to environmental stresses ⁶. Many protective effects of phytochemicals have been ascribed to their potential to destroy reactive oxidative species (ROS) that initiate carcinogenesis through oxidative damage of DNA ⁷.

In this context, plant triterpenoids are also well recognized for their antioxidant activities ⁸. These compounds scavenge free radicals and disrupt the free-radical chain reaction of lipid peroxidation, protect cell constituents against oxidative damage and therefore, limit the risk of various diseases associated with the oxidative stress ⁹. Ursolic acid (3ß hydroxy urs-12 en – 28 oic acid) is one such a triterpenoid found in plants of Ocimum sanctum. It has been reported to possess a wide range of pharmacological properties. Many protective effects of O. sanctum are determined by strong antioxidative properties of its constituent ursolic acid ¹⁰. Since so far it is not known whether antioxidant effect of ursolic acid could protects the genomic DNA of cells, the present study was planned to unravel the effect of ursolic acid on hydrogen peroxide (H₂O₂) induced DNA damage in lymphocytes. In particular, we focused on its effects upon purine/pyrimidine free sites (AP sites) on DNA and oxidative damage to guanine base as biomarker.

Lymphocytes are known to be highly sensitive to oxidative stress ¹¹. The protective effect of ursolic acid on mitigation of intracellular ROS level and reduction of AP sites might be determined to ascertain potential protective mechanisms. It has been well established that biological effects of H₂O₂ induce formation of ROS, which are known to cause oxidative stress to DNA, cause 8-hydroxydeoxyguanosine (8-OH-dG) formation and DNA strand breaks ¹². H₂O₂ induced free radicals react with DNA and inflict damage to purine and pyrimidine bases ¹³.

Various H₂O₂ induced biochemical changes occur in cells long before the loss of integrity ¹⁴. In this study, we focused on 8-hydroxy-2′-deoxyguanosine (8-oxo-dG), a DNA metabolite, as a marker for oxidative sensitivity. Guanine is the highly susceptible target for oxidative reactions because of its low redox potential ^15-17. Moreover, 8-oxo-dG is potentially mutagenic because of its ability to form base pairs with both cytosine and adenine ¹⁸. Thus, cellular 8-oxo-dG levels are one of the most mutagenic lesions, and the most abundant source of AP site in genomic DNA.

Delayed repair of AP sites can also result in replication induced double strand breaks (DSB). Therefore reducing AP sites in DNA may be an approach to decrease the adverse effects of oxidative stressors such as H₂O₂.

One of the objectives of present work was to isolate and quantify ursolic acid from O. sanctum. As most plant extracts are composed of complex phytochemical constituents, proper method is particularly desired for extraction of desired bioactive constituent from the extract of the plant. Our other objective was to evaluate anti-genotoxic effect of ursolic acid using human lymphocytes as model system. Existing evidence suggests that antioxidants of plant origin may reduce oxidative damage to DNA. We therefore asked whether ursolic acid has the potential to mitigate H₂O₂ induced cellular ROS, AP sites and 8-oxo-dG residues in DNA. Development of new therapeutic strategies using ursolic acid as antioxidant agent rests heavily upon the elucidation of its effect on DNA repair reactions.

MATERIAL AND METHODS:

Materials and Reagents: O. Sanctum whole plant powder was purchased from “Total Herb Solutions Pvt. Ltd., Mumbai”. The OS powder size used was 0.50–1.00 mm. Dulbacco’s modified Eagle’s medium (DMEM) for culturing human lymphocytes, dimethyl sulfoxide (DMSO), β-nicotinamide adenine di-nucleotide phosphate and hydrogen peroxide were purchased from Sigma (St. Louis, MO, USA). Penicillin, streptomycin, and phytohemagglutinin M were purchased from GIBCO, Invitrogen Corporation (UK). Heat-inactivated fetal bovine serum was purchased from Life Technology (UK). All solvents were purchased from Hi Media Ltd., Mumbai, India. Standard UA was purchased from Sigma Aldrich Chemical Company, USA. All the other reagents were purchased from reputed manufacturers and were of reagent grade.

Extraction and quantification of UA: The methanolic crude extract obtained was suspended in CHCl₃: MeOH (9:1) and subjected to gradient column chromatography by hexane, ethyl acetate and methanol in increasing polarity. The fractions were collected and monitored on TLC and tested for the presence of UA. And those fractions, which consisted of UA, were combined together and the solvent was removed under vacuum. Evaporation of the combined fractions containing UA led to a white solid. The isolated UA was well characterized by melting point, U.V. and I.R. spectra as well as by comparing with an authentic sample in TLC and HPTLC. Amount of UA in the extract was calculated by TLC using optical densitometry, with known concentrations of the samples and of commercial UA.  Different parameters affecting the extraction such as extraction time, solute to solvent ratio, extraction temperature were optimized and the final extraction experiment at the optimized conditions. The UA was quantified using Camag HPTLC apparatus with spotting device-linomat V Automatic Sample Spotter (Camag, Muttenz, Switzerland); Syringe-100mL (Hamilton), HPTLC chamber.-Glass twin trough chamber for 20´ 10cm plates (Camag); Densitometer.-TLC Scanner  with wincats software (Camag); HPTLC plates-20 ´ 10cm, pre-coated with silica gel 60, 0.2mm layer thickness (Merck, Darmstadt, Germany).

A stock solution of ursolic acid (90% pure, 72mg/mL) was prepared by dissolving 2mg of accurately weighed ursolic acid in methanol and diluting to 25mL with methanol. Aliquots (1 to 8mL) of stock solution were transferred to 10mL volumetric flasks and diluted with methanol to obtain standard solutions containing 7.2, 14.4, 21.6, 28.8, 36, 43.2, 50.4, and 57.6mg/mL ursolic acid, respectively. 10mL each of the standard solutions of ursolic acid (72 to 576 ng/spot) were applied and HPTLC was performed. After development, the plates were dried at room temperature in air, derivatized with liebermann burchard reagent and scanned at 530nm in absorbance mode using the tungsten lamp

The peak areas were recorded. Calibration curve of ursolic acid was obtained by plotting peak areas vs applied concentrations of ursolic acid.  20mL of suitably diluted sample was applied in triplicate on an HPTLC plate. The plate was developed and scanned as described above. The peak areas and absorption spectra were recorded, and the amount of ursolic acid was calculated using its calibration curve.

Spectroscopic methods: ¹H-NMR spectra were obtained with a JEOL JNM-AL400 NMR spectrometer. Chemical shifts were referenced to the solvent peak as an internal standard.

Isolation of lymphocytes and experimental design of co-incubation experiments: Blood samples were obtained from five donors (Three male and two female, healthy, non-smokers, 20–30 years old). Around 0.5 ml of human venous blood was added to 3.5 ml of DMEM (human lymphocyte culture media) supplemented with 20% of fetal bovine serum to which phytohemagglutanin (PHA) (50μg/ml), antibiotics (Penicillin 100 IU/ml and streptomycin 50μg/Ml) and heparin sodium salt, 5000 IU (0.4 Ml/100 ml) were added and incubated at 37⁰ C for up to 72 h.

Lymphocytes yield was usually 10⁶ per ml of blood with viability above 96% (approx). After 24 hours of culture initiation, lymphocytes were treated with ursolic acid at different doses (20 and 40 µM) and hydrogen peroxide (10, 50 and 100 µM) to study their effects on DNA damage. For protective effect, studies were carried out as follows:

Group 1; Sham control          Lymphocytes incubated with 0.05 % DMSO

Group 2; UA + H₂O₂            Lymphocytes exposed to UA (20 μg/ml media) and 10 µM H₂O₂

Group 3; UA + H₂O₂            Lymphocytes exposed to UA (20 μg/ml media) and 25 µM H₂O₂

Group 4; UA + H₂O₂            Lymphocytes exposed to UA (20 μg/ml media) and 50 µM H₂O₂

Group 5; UA + H₂O₂            Lymphocytes exposed to UA (20 μg/ml media) and 100 µM H₂O₂

Group 6; UA + H₂O₂            Lymphocytes exposed to UA (40 μg/ml media) and 10 µM H₂O₂

Group 7; UA + H₂O₂            Lymphocytes exposed to UA (40 μg/ml media) and 25 µM H₂O₂

Group 8; UA + H₂O₂            Lymphocytes exposed to UA (40 μg/ml media) and 50 µM H₂O₂

Group 9; UA + H₂O₂            Lymphocytes exposed to UA (40 μg/ml media) and 100 µM H₂O₂

For all experiments lymphocytes were exposed for 2 h. Ursolic acid was dissolved in DMSO at a concentration of 1000mM with subsequent dilutions in culture medium. For sham control DMSO was diluted in medium to a concentration corresponding to the 0.05 % DMSO in H₂O₂-treated groups.

Reduced glutathione (GSH) Estimation: Level of reduced glutathione was estimated as total non-protein sulphydryl group in the cell homogenates after precipitating the proteins by 5% tricholoroacetic acid (TCA). The supernatant was mixed with 0.2 M phosphate buffer (pH 8) and 0.6 M 5, 5′-dithio-bis (2-nitrobenzoic acid) and allowed to stand for 8-10 min at room temperature. The absorbance was recorded at 412 nm using a spectrophotometer (Thermo Scientific, UV 10), and level was calculated as nMole of -SH content/mg protein from standard curve made with reduced glutathione (GSH) and finally expressed as percentage change of GSH level ¹⁹.

Protein Estimation:

The amounts of protein present in the sample were estimated using bovine serum albumin (BSA) as standard by using Folin reagent ²⁰.

Fluorimetric Analysis of DNA Unwinding (FADU) to quantify strand breaks: The modified procedure for FADU was followed with minor modifications. Human lymphocytes at the concentration of 5×10⁶ cells/ml were treated with 0.05 % DMSO control or different combinations of ursolic acid and hydrogen peroxide in a final volume of 1 ml for 2 hour. To study the ameliorative effect of UA on DNA strand breaks induced by H₂O₂, the lymphocytes were treated with H₂O₂ plus UA for 1 hour in 5% CO2 incubator at 37°C. The treatment was terminated by the addition of 5 ml ice-cold saline (0.9 % NaCl).

The treated and control cells were centrifuged at 1000 rpm for 10 minutes at 4°C, pellet was obtained and re-suspended in solution B and the volume was made up to 2.0 ml. The suspended lymphocytes were processed for FADU assay. In brief, measurement of DNA strand breaks by FADU is based on partial denaturation (unwinding) of double-stranded DNA under controlled alkaline and temperature conditions. DNA strand breaks are sites where unwinding of DNA can start. Briefly, after infliction of DNA damage, cell lysis was performed. Adding a neutralising solution terminated unwinding. To quantify the amount of DNA remaining double stranded, a commercially available fluorescence dye (SybrGreen) was used as a marker for double-stranded DNA.

Measurement of intracellular reactive oxygen species (ROS): Lymphocytes were loaded with 5 lM 20, 70-dichlorofluorescin diacetate (DCFH-DA) for 1 h. Following washing with DPBS, ROS levels were determined by measuring the fluorescent intensity at excitation wavelength 485 nm and emission wavelength 530 nm using Fluorescence microplate reader.

Quantification of purine/pyrimidine free sites (AP sites) in genomic DNA: Genomic DNA was isolated from lymphocytes using geneispin mammalian genomic DNA prep kit (Bangalore genei Pvt. Ltd. Peenya, Bangalore). AP sites in DNA were quantified using BioVision DNA damage quantification kit (Biovision Research Products, California, USA).

8-oxo-dG Quantification:

H₂O₂ induced oxidative stress was evaluated by competitive enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI, USA) that measured 8-oxo-dG, a marker of oxidative DNA damage, in the liver. Briefly, genomic DNA was extracted (Bangalore Genei Pvt. Ltd. Peenya, Bangalore, India) and suspended in 135 μl of 20 mM sodium acetate (pH 4.8). DNA was digested to nucleotide with nuclease P1 (40 U/ml) at 37 °C for 1 h. Then, 15 μl of 1 M Tris/HCl (pH 7.4) was added to the samples and they were then treated with alkaline phosphatase (25 U/ml) at 37 °C for 1 h.

The 8-oxo-dG assay in the digested DNA solution was based on the competition between 8-oxo-dG and 8-oxo-dG acetylcholinesterase (AChE) conjugate (8-oxo-dG tracer) for a limited amount of 8-oxo-dG monoclonal antibody. The 8-oxo-dG levels were expressed as 8-oxo dG (ng/ml)/DNA (mg/ml).

Statistical analysis:

All values were expressed as mean ± standard error of means of samples with n and p indicated in the Results section. Statistical analyses were performed using Student’s t-test (GraphPad QuickCalcs, San Diego, California, USA). Values with P < 0.05 were considered statistically significant.

RESULTS:

Extraction and quantification of ursolic acid: Preliminary experiments showed that ursolic acid was available in free form. Consequently, ursolic acid was quantified from the methanolic extract. The content of ursolic acid in methanolic extract of O. sanctum was quantified at 0.926 % w/w (Rf: 0.51). 20 and 40 mg per ml media concentrations of purified ursolic acid were used for their anti-oxidative effect against 10, 25, 50 and 100 mM concentrations of hydrogen peroxide in medium containing lymphocytes as shown in Figure 1.

The ¹H NMR spectrum (DMSO-d6) of ursolic acid contained the signals for seven skeletal methyl groups of which five were singlets at d 0.69 (3H, s, H-24), 0.73 (3H, s, H-26), 0.87 (3H, s, H-25), 0.89 (3H, s, H-23), 1.07 (3H, s, H-27) and two were doublets at d 0.78 (3H, d, J = 5.9 Hz, H-29) and 0.96  (3H, d, J = 6.8 Hz, H-30). The signal for H-5 and H-9 appeared as singlet at d 0.65 and 1.55 respectively.

FIG.1:  EXISTENCE OF URSOLIC ACID IN O. SANCTUM SPECIES AS INDICATED BY HIGH PERFORMANCE-TLC (HPTLC) ANALYSIS OF METHANOLIC EXTRACT. 1A) DENSITOGRAM OF URSOLIC ACID IN METHANOLIC EXTRACT WHEREIN URSOLIC ACID FROM METHANOLIC EXTRACT SHOWED RF VALUE OF 0.51; 1B) URSOLIC ACID IN METHANOLIC EXTRACT ALONG WITH PURE UA STANDARD.  THE CONTENT OF URSOLIC ACID IN METHANOLIC EXTRACT OF O. SANCTUM WAS QUANTIFIED AT 0.926 % W/W.

The signal for olefin proton and proton at position 3 appeared as doublet of doublets at d 5.11 (1H, dd, J = 13.7; 3.5 Hz; H-12) and 2.98 (1H, dd, J = 5.2; 9.5 Hz, H-3). Multiplet signals were observed for each two protons at d 1.55 (2H, m, H-16), 1.57 (2H, m, H-1), 1.56 (2H, m, H-22), 1.45 (2H, m, H-2), 1.25 (2H, m, H-21), 1.23 (2H, m, H-7) and 1.02 (2H, m, H-15). Doublet of doublets was also observed for two protons at position 11 with different coupling constants at d 2.09 (2H, dd, J = 13.7; 3.5 Hz, H-11). Further signals at d 2.48 (1H, d; J = 11.1 Hz, H-18), 1.51 (1H, m, H-20), 1.49 (1H, m, H-6a), 1.28 (1H, m, H-19), 1.26 (1H, m, H-6b) were observed in the spectrum.

Ursolic acid mitigated oxidative strand breaks in DNA: Fig. 2 shows the protective effect of ursolic acid against oxidative stress induced by H₂O₂ in lymphocytes. When the cells were treated with ursolic acid at the doses of 20μg/ml, they showed approximately 17, 43, 61 and 79 % of DNA strand breaks after exposure to 10, 25, 50 and 100μM of H₂O_2, respectively as shown in Fig. 2A. These were very significant when compared to the control experiments (14%).  However, exposure to 40 μg/ml of ursolic acid reduced strand breaks to 15, 17, 36 and 46 % after exposure to 10, 25, 50 and 100 μM of H₂O₂ as shown in Fig.2B_. The maximum protection by ursolic acid was observed at 40 μg/ml showing 17 % strand breaks after exposure to 25 μM of   H₂O₂ as shown in Fig.2B.

FIG. 2:  URSOLIC ACID INHIBITED HYDROGEN PEROXIDE INDUCED STRAND BREAKS IN DNA OF HUMAN LYMPHOCYTES 2A) EFFECT OF URSOLIC ACID (20mG/ML) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE; 2B) EFFECT OF URSOLIC ACID (40mG/ML) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE.

All values represent mean ± standard error of samples (in triplicate) from 3 experiments in each group.  (*P < 0.05, **P < 0.01: indicates significance of difference between control and ursolic acid treatments).

Ursolic acid alleviated H₂O₂ induced intracellular ROS level and mitigated decrease in glutathione levels: To evaluate the activated oxygen metabolism of lymphocytes, the intracellular production of ROS in lymphocytes was analyzed as shown in Fig.3 after oxidative stress either with ursolic acid or with DMSO as control. Dose (H₂O₂) dependent increase in intracellular ROS was observed.

Compared to control levels, ROS levels rose up (P < 0.01) more than 2-fold (approx. 277%) after co-incubation with 100 mM of H₂O₂ and 20 μg/ml of ursolic acid as shown in Fig. 3A. However, compared to 40μg/ml ursolic acid treatment, ROS levels were observed to decline when 40 μg/ml of ursolic acid was used against 100 mM of H₂O₂ as shown in Fig.3B.

FIG.3: EFFECT OF URSOLIC ACID TREATMENT ON HYDROGEN PEROXIDE INDUCED INTRACELLULAR REACTIVE OXYGEN SPECIES (ROS) OF HUMAN LYMPHOCYTES 3A) EFFECT OF URSOLIC ACID (20 mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE; 3B) EFFECT OF URSOLIC ACID (40mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE.

All values represent mean ± standard error of samples (in triplicate) from 3 experiments in each group. (*P < 0.05, **P < 0.01: indicates significance of difference between control and ursolic acid treatments).

In the present study, the level of glutathione showed significant decline (56.6% with 100mM of H₂O₂) when lymphocytes were treated with ursolic acid (20μg/ml) for 2 h as shown in Fig.4A. However, when ursolic acid was supplemented in media at higher concentration (40μg/ml) only 28.4 % decrease (P < 0.01) in reduced glutathione was observed in comparison to untreated lymphocytes as shown in Fig. 4 B.

FIG. 4:  EFFECT OF URSOLIC ACID TREATMENT ON GLUTATHIONE PROFILE (NMOL/MG PROTEIN) AFTER EXPOSURE OF LYMPHOCYTES TO VARIOUS DOSES OF HYDROGEN PEROXIDE 3A) EFFECT OF URSOLIC ACID (20mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE; 3B) EFFECT OF URSOLIC ACID (40mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE.

All values represent mean ± standard error of samples (in triplicate) from 3 experiments in each group. (*P < 0.05, **P < 0.01: indicates significance of difference between control and ursolic acid treatments).

Ursolic acid treatment conferred remarkable protection to genomic DNA by mitigating number of AP sites and 8-oxo-dG residues:

In order to explore the extent of DNA damage under various experimental conditions, AP sites were evaluated in control and H₂O₂ exposed lymphocytes in presence of 20 and 40 μg/ml of ursolic acid as shown in Fig. 5. The data were represented as number of AP sites per 100,000 base pairs of genomic DNA. There was no significant difference in AP sites levels between the control and 10 mM exposed lymphocytes (P > 0.05) on co-incubation with 20μg/ml of ursolic acid. The average frequencies of endogenous AP sites in genomic DNA in control ranged from 2.3 to 2.6 AP sites/10⁵ base pairs.

As shown in Fig.5A, lymphocyte DNA revealed significantly elevated levels of purine/pyrimidine free sites after increase in H₂O₂ dose from 10 mM to 100mM in presence of 20μg/ml of ursolic acid. On co-incubation with 20μg/ml of ursolic acid, lymphocytes showed 15.7 ± 0.79 AP sites (P < 0.01; compared to control group) at 50 mM of H₂O₂ and 23.6 ± 1.2 AP sites (P < 0.01; compared to control group) at 100mM of H₂O₂ as shown in Fig.5A. AP sites in genomic DNA were significantly decreased in frequency after co-incubation with 40μg/ml of ursolic acid as shown in Fig.5B. Co-incubation with higher concentration of ursolic acid prevented progressive increase of AP sites. After incubation with 100mM of H₂O₂, the abundance of AP sites was 12.7 ± 0.87 as shown in Fig.5B.

FIG.5: EFFECT OF URSOLIC ACID TREATMENT ON HYDROGEN PEROXIDE INDUCED PURINE/PYRIMIDINE FREE SITES (AP SITES) IN GENOMIC DNA OF HUMAN LYMPHOCYTES 3A) EFFECT OF URSOLIC ACID (20mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE; 3B) EFFECT OF URSOLIC ACID (40 mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE.

All values represent mean ± standard error of samples (in triplicate) from 3 experiments in each group.  (*P < 0.05, **P < 0.01: indicates significance of difference between control and ursolic acid treatments).

Data on lymphocyte 8-oxo-dG residues profile is presented in Fig. 6A and 6B. Lymphocyte 8-oxo-dG level was 19.7 ± 1.3 (ng/ml)/ DNA (mg/ml) in the control. Measurement of 8-oxo-dG residues in H₂O₂ exposed lymphocytes, in general showed significant dose dependent change. However at 10mM of H₂O₂ exposure, the differences were statistically insignificant indicating sufficient protection conferred by 20 μg/ml of ursolic acid. After 25mM of H₂O₂, less pronounced change in 8-oxo-dG levels was observed in lymphocytes co-incubated with 20 μg/ml of ursolic acid as shown in Fig. 6A.  Increase in concentration of ursolic acid to 40μg/ml significantly reduced 8-oxo-dG levels in DNA as shown in Fig. 6B. Maximum increase in lymphocyte 8-oxo-dG levels after 100 mM of H₂O₂ exposure was 71.5 ± 5.3 on co-incubation with 40 μg/ml of ursolic acid (P < 0.01).

FIG. 6:  EFFECT OF URSOLIC ACID TREATMENT ON 8-OXO-DG RESIDUES IN GENOMIC DNA OF HUMAN LYMPHOCYTES 5A) EFFECT OF URSOLIC ACID (20 mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE; 5B) EFFECT OF URSOLIC ACID (40 mg/ml) AGAINST DIFFERENT CONCENTRATIONS OF HYDROGEN PEROXIDE.

All values represent mean ± standard error of samples (in triplicate) from 3 experiments in each group.  (*P < 0.05, **P < 0.01:  indicates significance of difference between control and ursolic acid treatments).

DISCUSSION: Till recently, the use of phytochemicals in mitigating genotoxic stress has focused on enhancing recovery using crude extracts. However, limited numbers of studies have purified bioactive constituents that confer genomic protection. In present study, HPTLC-densitometry methods were successfully applied for the quantification of ursolic acid in O. sanctum. The developed method is simple, precise, specific, sensitive, and accurate, and can be used for standardization and quality control purposes. Ursolic acid is an important bioactive moiety available in plants of O. sanctum, which is involved in protecting genomic DNA.

Studies on role of ursolic acid in protecting DNA against genotoxic agents might provide a surge of interest in the therapeutic possibilities where O. sanctum could be explored to alleviate oxidative injury. To explore the role of ursolic acid in protecting genomic DNA from oxidative stressors such as hydrogen peroxide, we quantified effect of H₂O₂ on DNA strand breaks, intracellular ROS, glutathione levels, purine/pyrimidine free sites and 8-oxo-dG residues. DNA constitutes the primary vital target for cellular inactivation of a living system by oxidative stress; measurement of DNA damage is an important quantitative parameter that is used to detect the harmful effects of environmental oxidants. Generation of reactive species leads to DNA damage that causes activation of many apoptotic pathways.  Therefore, we have investigated the effect of ursolic acid on intracellular ROS level and status of DNA to elucidate potential protective mechanisms.

To explore the role of ursolic acid in protecting genomic DNA from oxidative stressors such as hydrogen peroxide, we quantified effect of H₂O₂ on DNA strand breaks, intracellular ROS, glutathione levels, purine/pyrimidine free sites and 8-oxo-dG residues. Mutagenesis by oxygen free radicals has been demonstrated in mammalian cell systems. The mutations produced are usually the direct result of modifications of DNA by oxygen free radicals. The hypothesis directly linking oxygen damage to mutations is in accord with the plethora of oxygen free radical induced modifications of DNA ^{21, 22}.

In present studies co-incubation with the ursolic acid for 2 h was found to be highly effective at scavenging hydrogen peroxide-induced intracellular ROS generated in lymphocytes. Unregulated intracellular production of ROS is highly toxic to the cell and is countered by various antioxidant defense systems, which can be categorized as enzymatic or non-enzymatic. Direct damage to DNA bases caused by ROS contributes to the development of cancer and the agents that reduce such damage decrease the risk of cancer development. Increased level of cellular antioxidants is known to provide protection against oxidative stress.

Here, in our study, pre-treatment of lymphocytes with the ursolic acid significantly restored the depleted GSH level in the H₂O₂ -treated lymphocytes.  Cellular glutathione content is responsible for the redox status of cell. The rise in the levels of glutathione might be due to ROS scavenging by ursolic acid. H₂O₂ is one of the mutagenic molecules that induce many human diseases including cancer, that is why we explored possibility of ursolic acid protecting against H₂O₂ induced DNA damage in human blood lymphocytes.

The high antigenotoxic activity of ursolic acid at higher concentration indicated dose dependent protection from oxidative stress.  Data presented here shows that co-incubation with ursolic acid conferred remarkable protection to lymphocytes via mitigation of AP sites in genomic DNA. Levels of AP sites in DNA are the consequence of a balance between lesion inductions from H₂O₂ induced radical processes and repair.

Here, we show that co-incubation of lymphocytes with ursolic acid plus hydrogen peroxide prevented progressive increase of AP sites. Our studies have clearly shown that ursolic acid mediated ROS scavenging might have protected DNA. Results of present studies suggested that exposure to hydrogen peroxide in absence of ursolic acid might lead to drastic increase in 8-oxo-dG levels in genomic DNA of lymphocytes.

Ursolic acid supplementation, which resulted in scavenging of intracellular ROS, lowered 8-oxo-dG level in lymphocytes. Similar to other purine/pyrimidine based lesions 8-oxo-dG is converted to AP site by action of a specific glycosylase. 8-oxo-dG levels are well-established marker for damage to purine and pyrimidine bases in DNA. Our data showed that H₂O₂ induced increase in 8-oxo-dG levels in lymphocytes was significantly abolished by protective effect of higher doses of ursolic acid.

Recent work has shown that dietary triterpenes supplementation protects by reducing oxidative stress. Therefore, mitigation of H₂O₂ induced abasic sites in genomic DNA by ursolic acid might be partly attributed to anti-oxidant potential of this bioactive constituent derived from O. sanctum.  DNA strand breaks are the most critical lesions induced by oxidative stress, which can lead to genomic instability and cell death when misrepaired or unrepaired. The results of the present study demonstrate that ursolic acid is highly effective at protecting lymphocytes from the hazardous effects of hydrogen peroxide, as made evident by the decrease in the ROS and AP sites.

Based on these experiments we hypothesize that stringent ROS scavenging is performed by ursolic acid so that DNA repair is not impaired under oxidative stress conditions.  Taken together, our work suggests that opportunities exist for the development of phytochemicals based protective agents that mitigate ROS and reduce DNA damage. For new therapies to emerge continued progress will be needed to understand the complex regulatory mechanisms that govern ursolic acid mediated DNA protection. The results of the present study indicate that ursolic acid from O. sanctum possess a remarkable capacity to protect cells from oxidative stress and DNA damage.

CONCLUSIONS: Our results indicated that ursolic acid can be utilized as possible chemo-preventive phytochemical agent against oxidative stress inducing agents. Another important finding showed that anti-oxidant effect of ursolic acid is exhibited in dose-dependent manner. Pre-dominant mechanism governing antioxidant effect of ursolic acid on genomic DNA involved: reduction in intracellular 8-oxo-dG levels, mitigation of oxidative strand breaks and alleviation of purine/pyrimidine free sites. Moreover, higher level of natural antioxidant glutathione was maintained in response to ursolic acid treatment

ACKNOWLEDGEMENTS: The authors wish to thank Dr. Gadge, Principal, Sterling Institute of pharmacy for his guidance and support in carrying out this work. The authors also wish to thank TIFR NMR Facility and Ms. Mamata V. Joshi for her guidance.

DECLARATION OF INTEREST: The authors report no conflict of interest.

REFERENCES:

1. Iovdijova A, Bencko V: Potential risk of exposure to selected xenobiotic residues and their fate in the food chain – Part I: Classification of Xenobiotics. Annals of Agricultural and Environmental Medicine 2010; 17: 183–192.
2. Kelkel M, Jacob C, Dicato M and Diederich M: Potential of the Dietary Antioxidants Resveratrol and Curcumin in Prevention and Treatment of Hematologic Malignancies. Molecules 2010; 15(10): 7035-7074.
3. Saxena S, Gautam S and SharmaA: Comparative Evaluation of Antimutagenicity of Commonly Consumed Fruits and Activity-Guided Identification of Bioactive Principles from the Most Potent Fruit, Java Plum (Syzygium cumini). Journal of Agricultural and Food Chemistry 2013; 61 (42):10033–10042.
4. Liu J R, Dong H W, Chen B O, Zhao P and Liu R H: Fresh Apples Suppress Mammary Carcinogenesis and Proliferative Activity and Induce Apoptosis in Mammary Tumors of the Sprague−Dawley Rat. Journal of Agricultural and Food Chemistry 2009; 57: 297–304.
5. Kaur P, Kaur S, Kumar N, Singh B and Kumar S: Evaluation of antigenotoxic activity of isoliquiritin apioside from Glycyrrhiza glabra L. Toxicol. in Vitro 2009; 23, 680-686.
6. Oh M M, Carey E E and Rajashekar C B: Environmental stresses induce health promoting phytochemicals in lettuce. Plant Physiology and Biochemistry. 2009; 47: 578-583.
7. Lee J H, Khor T  O,  Shu L,  Su Z  Y,  Fuentes F  and A N  Kong: Dietary phytochemicals and cancer prevention: Nrf2 signaling, epigenetics, and cell death mechanisms in blocking cancer initiation and progression. Pharmacology & Therapeutics 2013; 137(2): 153-171.
8. Yang E J, LeeW, Ku S K, Song K S, Bae J S: Anti-inflammatory activities of oleanolic acid on HMGB1 activated HUVECs.Food and Chemical Toxicology 2012; 50(5):1288–1294.
9. Ghosh J, Das J, Manna P and Sil P C: Arjunolic acid, a triterpenoid saponin, prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection. Free Radical Biology and Medicine 2010; 48(4): 535-553
10. Yin M C and Chan KC: Nonenzymatic antioxidative and antiglycative effects of oleanolic acid and ursolic acid. Journal of Agricultural and Food Chemistry 2007; 55: 7177-7181.
11. Bauer M, Goldstein M, Christmann M, Becker H, Heylmann Dand Kaina B: Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress. Proceedings of the National Academy of Sciences of The United States of America 2011; 108 (52):21105-
12. Dizdaroglu M, Jaruga P: Mechanisms of free radical-induced damage to DNA. Free Radical Research 2012; 46(4): 382-419.
13. Swalwell H, Latimer J, Haywood R M, Birch-Machin M A: Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells. Free Radical Biology and Medicine 2012; 52 (3): 626–634.
14. Stacey M M, Vissers M C, and Winterbourn C C: Oxidation of 2-Cys Peroxiredoxins in Human Endothelial Cells by Hydrogen Peroxide, Hypochlorous Acid, and Chloramines. Antioxidants & Redox Signaling 2012; 17(3): 411-421.
15. Fujii S and Akaike T: Redox Signaling by 8-Nitro-cyclic Guanosine Monophophate: Nitric Oxide and Reactive Oxygen Species-Derived Electrophilic Messenger. Antioxidants & Redox Signaling: 2013; 19(11): 1236-1246.
16. MadisonA L,  Perez Z A,  To P, Maisonet T, Rios E V,  Trejo Y, Paniagua C O,  Reno A , and  Stemp  E D A: Dependence of DNA–Protein Cross-Linking via Guanine Oxidation upon Local DNA Sequence As Studied by Restriction Endonuclease Inhibition. Biochemistry 2012; 51 (1): 362–369.

17. Niles J C, Wishnok J S and Tannenbaum S R: Peroxynitrite-induced oxidation and nitration products of guanine and 8-oxoguanine: structures and mechanisms of product formation. Nitric Oxide 2006; 14: 109-121.
18. HammM L,  Crowley K A , Ghio M,  Lindell M A M,  McFadden  E J, Silberg J S L  and Weaver A M: Biochemical Investigations into the Mutagenic Potential of 8-Oxo-2′-deoxyguanosine Using Nucleotide Analogues. Chemical research in Toxicology 2012; 25 (11): 2577–2588.
19. Moron M S, Depierre J W and Mannervik B: Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 1979; 582:67-78.
20. Lowry O H, Rosebrough N J, Farr A L and Randall R J: Protein measurement with the Folin phenol reagent.The Journal of Biological Chemistry 1951; 193: 265-275.
21. Valko M,Izakovic M, Mazur M, Rhodes C J and Telser J: Role of oxygen radicals in DNA damage and cancer incidence. Molecular and Cellular Biochemistry 2004; 266: 37-56.
22. Ziech D, Franco R, Pappa A, Panayiotidis M I: Reactive Oxygen Species (ROS)––Induced genetic and epigenetic alterations in human carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2011; 711: 167–173.
    

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    How to cite this article:

    Batra A and SastryVG: Extraction of Ursolic Acid from Ocimum Sanctum: Modulatory Effect of Ursolic Acid on Hydrogen Peroxide Induced DNA Damage in Human Lymphocytes. Int J Pharm Sci Res 2015; 6(5): 2042-51.doi: 10.13040/IJPSR.0975-8232.6(5).2042-51.

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