Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI)-Induced Cellular and DNA Toxicity

Hexavalent chromium Cr(VI) is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI)-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae), commonly known as Henna. The extracts showed significant (P < .05) potential in scavenging free radicals (DPPH• and ABTS•+) and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI)-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma) cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05) positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI)-induced oxidative cell damage.


Introduction
Hexavalent chromium [Cr(VI)] is the most toxic and mutagenic heavy metal in biological systems [1]. It exists as oxo-species such as CrO 3 and CrO 2− 4 , which are robustly oxidizing [2], leading to excessive cytotoxicity that in turn may cause dermal damage, gastrointestinal bleeding, renal failure, intravascular hemolysis, liver damage, coma and even death [3]. Cr(VI) is transported into cells through the sulfate transporter [4], and leads to alteration of signal transduction pathways [5], cell transformation [6], and increases the risk for developing cancer [7]. Simultaneously, it is also known to inhibit cell proliferation/cell cycle [8], thereby inducing growth arrest, accompanied by the generation of reactive oxygen species (ROS) that presumably triggers oxidative damage to DNA [9] and consequent apoptosis [10]. Oxidative damage is associated with the generation of free radicals in cells exposed to Cr(VI) ion, and a propensity of cells to develop mutations in response to Cr(VI)-induced oxidative damage has been reported [11,12].
Sources of Cr(VI) toxicity are broadly classified into occupational and non-occupational exposure types. Highest occupational exposures to Cr(VI) occur during chromate production, welding, chrome pigment manufacture, chrome plating and spray painting. Non-occupational sources of exposure include food, air and water [7].
Various compounds with differential antioxidant properties are found in floral resources which are considered to have high potential in context of therapeutic approaches to encounter and prevent free radical damage as that caused by Cr(VI) toxicity. Lawsonia inermis Linn. (Lythraceae), commonly known as Henna, is a popular skin and hair coloring agent in many parts of the world. In addition, it is traditionally used as a medicinal plant [13] by diverse groups of tribal/ethnic people [14][15][16]. Lawsonia inermis is used as an antirheumatic and antineuralgic agent [15], and also has potential as an antidiabetic drug [16]. There is evidence of the plant having wound healing properties [17]. Furthermore, treatment with hydroalcoholic extract of L. inermis (in vivo) has been proved to increase levels of cellular antioxidant enzymes such as glutathione reductase, superoxide dismutase and catalase [18].
This study was aimed at evaluating the effects of aqueous and methanolic extracts of L. inermis on induced oxidative 2 Evidence-Based Complementary and Alternative Medicine toxicity in MDA-MB-435S (human breast carcinoma) cells, along with an estimation of the compositions of these extracts and their respective antioxidant potential.

ABTS
where A c is the absorbance of the control and A is the absorbance of sample. LPI of the extracts were compared with that of BHT and expressed in BHT equivalence. The experiment was performed with the approval of the institutional animal ethical committee (PSGIMSR/27.02.2008) and was in accordance with the "Principles of Laboratory Animal Care" (NIH publication #85-23, revised in 1985) [23].

Test for Inhibition of Cr(VI)-Induced DNA Damage by the Extracts.
Efficiency of the extracts as potential DNA protectors against Cr(VI)-induced genotoxicity was tested by treating pBR322 plasmid DNA with CrO 3 in presence of UV radiation (8000 μW cm −2 ). 1 μL aliquots of pBR322 (200 μg mL −1 ) were taken in four polyethylene microcentrifuge tubes. 50 μg of each extract was separately added to two individual tubes-S M (sample with methanolic extract) and S A (sample with aqueous extract). The remaining two tubes were kept without addition of any extract, and served as controls. 5 μL of 10 μM CrO 3 was added to both S M and S A and in one of the control tubes (now designated C Cr ). The other control tube was left without addition of CrO 3 and was designated as C 0 . C Cr , S M , and S A tubes were then placed directly on the surface of a UV transilluminator (300 nm) and irradiated for 15 min at room temperature. All DNAs (C 0 , C Cr , S M , and S A ) were run on 1% agarose gel (stained with 10 μg μL −1 ethidium bromide solution) and photographed on Lourmat Gel Imaging System (Vilbar, France).

Test for Inhibition of Cr(VI)-Induced Cytotoxicity by the Extracts: XTT Assay.
Inhibition of Cr(VI)-induced cytotoxicity by the two extracts at various concentrations was tested by the method of XTT-formazan dye formation [24]. MDA-MB-435S cells cultured in L-15 (Leibovitz) cell culture medium (with 10% serum) were seeded (200 μL, 6 × 10 3 cells/well) in a 96-well plate and allowed to grow for 24 h at 37 • C. After incubation, medium was removed from all wells. 200 μL fresh medium was added to the control wells. Cells in each test well were treated with 10 μM CrO 3 (prepared in medium) along with different extract dosages (0, 125, 250, 500, and 1000 μg). Cells in both control and test wells were re-incubated for 24 h maintaining the same conditions. After the treatment incubation period, medium in each well was substituted by 200 μL of fresh medium followed by the addition of 50 μL of XTT (0.6 mg mL −1 ) containing 25 μM PMS. The plate was further incubated for 4 h in the same conditions. Absorbance was measured at 450 nm (with a 630 nm reference filter) in a Dynex Opsys MR Microplate Reader (Dynex Technologies, VA, USA). Percentage cytotoxicity was calculated by the following formula: where A C is the mean absorbance of the control wells and A T is the mean absorbance of test wells with a particular extract dosage.

Antioxidant
Potential. DPPH • is a stable free radical whose absorbance (λ max = 517 nm) decreases when antioxidants donate protons to DPPH • [19]. Quantitative analysis  revealed strong DPPH • radical scavenging ability in both the aqueous and methanolic extracts. Figure 1(a) shows the mean (±SD at P < .05) values of percentage DPPH •scavenging for different dosages of the two extracts along with Trolox equivalence. Figure 1(b) depicts the percentage scavenging of ABTS •+ radical by the two extracts and along with Trolox equivalence (in μg). Both extracts showed high antioxidant property, which was in qualitative congruity to the results of the DPPH assay. Quantitatively, however, radical scavenging efficiency was considerably higher in the ABTS assay in comparison to DPPH assay.
Results of the FRAP assay (Figure 1(c)) showed that the aqueous extract was a stronger Fe 3+ -reductant than the methanolic extract. Moreover, both extracts showed considerably lesser antioxidant potential in FRAP in comparison to both DPPH and ABTS assays.
All three radical-scavenging assays showed significant mutual positive correlation at P < .05. Coefficient of determination (R 2 ) values between DPPH and ABTS, ABTS and FRAP, and DPPH, and FRAP are 0.96, 0.98, and 0.99, respectively. Intracellular and membrane lipids, when subjected to considerable oxidative stress, lose a hydrogen atom from an unsaturated fatty acyl chain, thus initiating lipid peroxidation which propagates as a chain reaction. This leads to the generation of diverse peroxides and cyclic endoperoxides which consequently form malondialdehyde (MDA). On reacting with TBA, MDA produces a pink chromogen with highest absorbance at 532 nm, thus providing an estimate of LPI [22]. Figure 1(d) shows a dose-dependent inhibition of lipid peroxidation (by both extracts) along with BHT equivalence (in μg).

Inhibition of Cr(VI)-Induced DNA Damage. Cr(VI)
causes DNA damage involving adducts, breaks, and crosslinks and inhibits repair of DNA damage induced by UV [27]. This suggested that both extracts showed varying degrees of potential in inhibiting DNA damage due to Cr(VI)-UV exposure, the aqueous extract being a better DNA-protector than the methanolic.

Inhibition of Cr(VI)-Induced Cytotoxicity.
Live cells metabolically reduce XTT to a soluble product XTTformazan, which can be estimated spectrophotometrically as a measure of cell viability [24]. Figure 3 shows the protective effects of the aqueous and methanolic extracts on Cr(VI)induced cytotoxicity in MDA-MB-435S cells as determined by XTT assay. Cells treated with only Cr(VI) were subjected to severely high toxicity. However, cytotoxicity was effectively mitigated in Cr(VI)-treated cells by action of aqueous and methanolic extracts of L. inermis in a dosage-dependent pattern. hydroxyl and other subconstituents, including their functional derivatives [29]. Variations in the quantity of total phenolics in the two extracts are presented in Figure 4. Quantitative estimation proved that both extracts have considerably high constitutions of phenolic compounds that increase with extract dosage. Total phenolic content, which  was expressed in GAE in μg, demonstrated a much enhanced phenolic composition of both extracts than the gallic acid standard.

Phenolic
Phenolic compounds have been found to encounter heavy metal-induced stress in plants [30]. Further, phenolic compounds are also known for their antioxidant, antimutagenic and anti-tumor activities [31]. To check whether the phenolic content of the two extracts can be accredited for their antioxidant potential and their property to inhibit Cr(VI)-induced cytotoxicity, correlation and regression analyses were performed. Total phenolic content of both extracts showed significant and strong positive correlation (P < .05) with free radical scavenging efficiency (DPPH, ABTS and FRAP assays), potential of LPI (TBA assay) and property of inhibition of Cr(VI)-induced oxidative cytotoxicity ( Figure 5).

Determination of Phenolic Compounds: HPLC Analysis.
Due to the diversity and complexity of natural phenolic compounds, it is hard to characterize every compound and elucidate its structure [13]. The major types of phenolic compounds in the two extracts of L. inermis were determined by HPLC analysis. A library of the analytical characteristics (λ max , retention time, determining λ, slope and limit calibration) of more than 100 phenolic standards established by Sakakibara et al. [26] was used as the reference for compound identification. Table 1 shows the major phenolic compounds in the two extracts.

Discussion
Aqueous and methanolic extracts of L. inermis showed considerable antioxidant potential in all the analytical studies. The results of the DPPH, ABTS and FRAP assays were in congruity with those previously reported [13], and additionally, proved a dosage-dependent increase in antioxidant potential over different ranges with distinct extract-specific efficiencies. The differential scavenging activities of the extracts against DPPH • , ABTS •+ , and Fe 3+ radicals may be referred to the different mechanisms of the radical-antioxidant reactions in these assays. The stoichiometry of reactions between the antioxidant compounds in the extracts and the DPPH • , ABTS •+ , and Fe 3+ radicals is distinctively dissimilar, which may be inferred as a reason for the difference in scavenging potential. The diversity in radical scavenging shown in these assays may also be due to factors like stereoselectivity of the radicals or the differential solubility of the extracts [32] in the three testing systems. Therefore, the considerable difference in antioxidant efficiency for both extracts among the three models is justified.   A previous study [33] reported that aqueous solution of commercially available L. inermis powder did not show any prominent inhibition of lipid peroxidation (i.e., no decrease in the level of MDA in test samples in comparison to an untreated control group) in the liver cells of female Swiss albino rats. The results of the present study, however, showed considerable difference in results, and found both extracts efficient in this precinct. Treatment by both extracts showed a significant (P < .05) dosage-dependent increase in the capacity to inhibit lipid peroxidation in the rat liver cells. The reason for this difference in results might be attributed to the presence of toxic substances like para-phenylenediamine (PPD), nickel, cobalt, and so forth. in commercially available L. inermis powder [34] which was used for the previous study [33]; and hence it might not have inhibited lipid peroxidation.
pBR322 DNA was protected by both extracts against 10 μM Cr(VI) in presence of UV radiation. Although such a protective potential is extensively dependent on the dosage of both Cr(VI) and the extracts, it is evident that both aqueous and methanolic extracts demonstrated variable magnitudes of DNA protection against Cr(VI)-induced oxidative stress which completely damaged DNA in absence of any extract (i.e., in C Cr ).
A steady decline was noted in the magnitude of Cr(VI)induced cytotoxicity in MDA-MB-435S cells with increasing dosage of the extracts in the XTT assay. The aqueous extract proved to be a considerably better cyto-protective agent in comparison to the methanolic extract at 125, 250, and 500 μg dosages. At 1000 μg, however, both extracts showed almost identical levels of protection to the cells, with the aqueous extract minutely ahead of its counterpart. ROS causes oxidative damage to genomic DNA, mtDNA, lipids (lipid peroxidation) and proteins, and leads to cellular dysfunction and/or cell loss through energy deficit and apoptosis [10,35]. Cr(VI), being an efficient generator of ROS [9], thereby causes considerable cytotoxicity to cells through free radicalmediated oxidative cascades [11,12]. Both aqueous and methanolic extracts of L. inermis have demonstrated effective potential in free radical scavenging and lipid peroxidation inhibition, by virtue of which they may have been able to counter the oxidative stress generated in MDA-MB-435S cells by Cr(VI).
Phenolic content of both extracts showed significant correlation with free radical scavenging, LPI and cytoprotective potential against Cr(VI)-induced toxicity. HPLC analysis revealed presence of a variety of phenolic compounds in both extracts which might have been responsible for their effective therapeutic potentials as reported by this study. However, it is interesting to note that the diversity of phenolic compounds in the aqueous extract is colossally more in comparison to that of the methanolic extract.
In conclusion, it can be inferred that aqueous and methanolic extracts of L. inermis have high antioxidant potential (by virtue of their diverse phenolic constituents) which simultaneously inhibits Cr(VI)-induced oxidative toxicity to MDA-MB-435S cells and pBR322 DNA. Hence, the tested extracts inhibit the oxidative damage pathway induced by Cr(VI), and thereby prevent cell death ( Figure 6). The plant can serve as a prospective source of natural phenolics and other metabolites which could prove to be precursors for designing effective drugs against heavy metal toxicity.