Antioxidant and Anti-Cancer Potentials of Rheum emodi Rhizome Extracts

The objective of this study was to determine antioxidant and cytotoxic efficacies of methanolic and aqueous extracts of Rheum emodi Wall. ex Meissn. rhizome. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activities, inhibitory effect on lipid peroxidation and Fe3+ reducing antioxidant property have been used to investigate antioxidant properties of the extracts. Cytotoxicity of the extracts was tested on MDA-MB-435S and Hep3B cell lines. Both extracts displayed extensive cytotoxicity to the tested cell lines. The extracts were studied for their ability to protect pBR322 DNA from damage by UV induced photolysis of H2O2. The aqueous extract, though inferior to methanolic extract in its antioxidant potential exhibited efficiency in DNA protection, while the methanolic extract failed to protect the DNA. The amount of total polyphenolics in the extracts was measured by spectrophotometric method. The methanolic extract contained higher polyphenolic contents than aqueous extract. Significant positive correlations were observed (P < .05) between results of phenolic content estimation and that of antioxidant assays. Hence, high-performance liquid chromatography analysis was performed to identify few major phenolic compounds that might be responsible for these therapeutic properties. These results indicate that rhizome of R. emodi possesses antioxidant and cytotoxic activities and therefore have therapeutic potential.


Introduction
Cancer chemoprevention is defined as the use of natural, synthetic or biologic chemical agents to reverse, suppress, or prevent carcinogenic progression [1]. There have been increasing safety concerns over synthetic chemopreventive therapy. Commonly used synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxyltoluene (BHT) have been restricted due to their toxicity and DNA damage induction potential [2,3]. Instead, floral resources have received considerable attention as sources of biologically active substances including antioxidants, anti-mutagens and anti-carcinogens [4].
Rheum emodi Wall. ex Meissn. (Polygonaceae) is a leafy perennial herb distributed in altitudes ranging from 2800 to 3800 m in the temperate and subtropical regions of Himalayas from Kashmir to Sikkim in India [5]. Roots of R. emodi are reported to have antibacterial and antifungal activities [6][7][8][9][10]. In addition several other biological activities such as laxative, diuretic, and in vivo inhibitory effect towards P388 leukemia in mice are also reported [11][12][13].
The aim of this study was to determine the antioxidant and anti-cancer potential of methanolic and aqueous extracts of R. emodi rhizome by various methods including 2,2diphenyl-1-picrylhydrazyl (DPPH) and • OH radical scavenging, Fe 3+ reducing capacity and inhibition of lipid peroxidation in vitro which are rarely reported. Cytotoxicity of the extracts were determined in MDA-MB-435S (human breast carcinoma) and Hep3B (liver carcinoma) cell lines. The protective activity of the extracts was evaluated by UV induced photolysis of H 2 O 2 leading to damage to pBR322 DNA. Total phenolic content estimation was performed, and a few phenolic compounds that might be responsible for the antioxidant property of the extracts were identified by highperformance liquid chromatography (HPLC).

Extraction.
Rhizome powder was serially extracted with methanol and water using a Soxhlet apparatus in a ratio of 1 : 6 [powder (in grams) : solvent (in milliliters)]. The extract obtained was evaporated to dryness at 40 • C under reduced pressure (methanol: 337 mbar, aqueous: 72 mbar in a rotary evaporator (BÜchi, Switzerland). Fifty grams of rhizome powder yielded 24.81 g (percentage extract yield: 49.62% of dry weight) of crude methanolic extract and 3.86 g (percentage extract yield: 7.72% of dry weight) of crude aqueous extract. The samples were stored in a vacuum dessicator at room temperature until further use.

DPPH Radical Scavenging
Activity. Free radical scavenging ability of the extracts was tested by DPPH radical scavenging assay (DRSA) as described by Blois [14]. An amount of 20, 40, 60, 80 and 100 μg of the extracts were taken in test tubes and made up to 0.5 ml with the respective solvents. An amount of 3 ml 0.1 mM DPPH • in ethanol was added to each tube and incubated in dark at room temperature for 30 min. Absorbance was read at 517 nm using a Cary 50 UV-Vis Spectrophotometer (Varian Inc., Australia). Percentage DPPH radical scavenging activity (% DRSA) was calculated using the formula, where A c is the absorbance of the control and A is the absorbance of the extract.

Hydroxyl Radical Scavenging
Activity. • OH radical scavenging activity (HRSA) of the extracts was estimated by the method of Klein et al. [15]. An amount of 50, 100, 150 and 200 μg of the extracts were taken in test tubes. An amount of 1 ml iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of 0.018% EDTA and 1 ml of 0.85% (v/v) DMSO (in 0.1 M phosphate buffer, pH 7.4) were added followed by 0.5 ml of 0.22% (w/v) ascorbic acid. The tubes were capped tightly and incubated on a water bath at 85 • C for 15 min. Post-incubation, the test tubes were uncapped and ice-cold trichloroacetic acid (17.5% w/v) was added in each immediately. An amount of 3 ml Nash reagent (7.5 g of ammonium acetate, 300 μl glacial acetic acid and 200 μl acetyl acetone were mixed and made up to 100 ml with distilled water) was added to all the tubes and incubated at room temperature for 15 min. Absorbance was measured at 412 nm. Percentage hydroxyl radical scavenging activity (%HRSA) was calculated by the following formula: where A c is the absorbance of the control and A is the absorbance of the extract.

Thiobarbituric Acid Assay.
The assay was performed as described by Halliwell and Gutteridge [17], in which the extent of lipid peroxidation was estimated from the concentration of malondialdehyde (MDA), a thiobarbituric acid reactive substance (TBARS), which is produced due to lipid peroxidation. A 6-week-old female Wistar albino rat weighing ∼150 g was sacrificed under ethereal anesthesia and its liver was excised. 10% (w/v) liver homogenate was prepared in Dulbecco's phosphate buffered saline (PBS) (Ca 2+ /Mg 2+ -free) (pH 7.4), and centrifuged at 503 g for 15 min to obtain a clear supernatant. An amount of 50, 100, 150, 200 and 250 μg of the extracts were taken in test tubes and were evaporated to dryness at 80 • C. An amount of 1 ml 0.15 M potassium chloride was added to the tubes followed by 0.5 ml of rat liver homogenate (10% w/v in PBS). Peroxidation was initiated by the addition of 100 μl of 2 mM ferric chloride. After incubating the tubes for 30 min at 37 • C, the peroxidation reaction was stopped by adding 2 ml of icecold HCl (0.25 N) containing 15% TCA and 0.38% TBA. The tubes were kept at 80 • C for 1 h, cooled and centrifuged at 3144 g. The absorbance of the supernatant, containing TBA-MDA complex was read at 532 nm. Percentage inhibition of lipid peroxidation (%LPI) was calculated using the formula, where A c is the absorbance of the control and A is the absorbance of the extract. This experiment was performed according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes" (and its appendix) with the approval of the institutional animal ethical committee (PSGIMSR/27.02.2008).

XTT Assay.
XTT assay was performed on MDA-MB-435S (grown in L-15 medium) and Hep3B (grown in MEM medium) cell lines as described by Weislow et al. [18]. A total of 6 × 10 3 cells were seeded on 96-well plates and were supplemented with 200 μl of the respective culture media for a period of 24 h. The media were then substituted by 200 μl of fresh media containing varying concentrations of the extracts (15.625, 31.25, 62.5 and 125 μg/ml). The plates were incubated at 37 • C for 24 h, after which, media was removed and fresh media added. An amount of 50 μl XTT reagent prepared in respective media (0.6 mg/ml) containing 25 μM of PMS was then added to all the wells, and the plates were incubated in dark humid conditions at 37 • C for 4 h. After incubation, the orange colored complex formed was read at 450 nm using a Dynex Opsys MR Microplate Reader (Dynex Technologies, VA, USA) with a 630 nm reference filter. Wells containing cells without extract treatments served as the control. Wells containing only culture medium and XTT reagent served as the blank. Percentage cytotoxicity of the extracts was calculated by using the formula: where A c is the absorbance of the control and A is the absorbance of the sample.

DRSA.
Both extracts showed a concentration dependent scavenging of DPPH • radicals. Methanolic extract was found to be more active radical scavenger than aqueous extract. Results were plotted as %DRSA and also expressed as AAE in micrograms (Figure 1).

HRSA.
The ability of the extracts to quench • OH radicals can be related to the prevention of lipid peroxidation, and it seems to be a good scavenger of active oxygen species, thus reducing the rate of chain reaction. Figure 2 shows %HRSA of the two extracts. Hydroxyl radical has been implicated as highly damaging in free-radical pathology, capable of damaging almost every molecule found in living cells [22]. The extracts have shown a dosage-dependent increase in inhibition of • OH radicals.

FRAP.
Fe 3+ reducing activity of the two extracts were determined by FRAP assay. The methanolic extract showed higher reducing power in comparison to the aqueous extract for all tested dosages. Figure 3 shows %Fe 3+ reduction by both extracts along with AAE in micrograms.

TBA Assay.
Both extracts were capable of preventing formation of MDA in a dosage-dependent manner. The   methanolic extract was observed to be a significantly better inhibitor of lipid peroxidation (at P < .05) compared with the aqueous extract for all tested dosages. Figure 4 shows %LPI with their corresponding BHT equivalences (in micrograms). Significant correlations (P < .05) were observed between the following: (i) %LPI and %DRSA, (ii) %LPI and %HRSA and (iii) %LPI and %Fe 3+ reductions ( Figure 5) for both methanolic and aqueous extracts for all dosages. This infers that both extracts differentially inhibit lipid peroxidation by virtue of their varying degrees of free radical quenching potential.

XTT Assay.
Both the extracts demonstrated considerable cytotoxicity in both cell lines, thereby indicating the presence of anti-cancer metabolites. Table 1 [21].
• OH) scavenging efficiencies, %LPI and %Fe 3+ reductions ( Figure 7). These results suggest a probable paramount role that the polyphenolic constituents of the extracts might play in free radical neutralization and lipid peroxidation inhibition.

HPLC Analysis.
Due to the diversity and complexity of natural phenolic compounds, it is difficult to characterize every compound present in the crude extract to elucidate its structure [23]. Major types of phenolic compounds were determined in the two extracts of R. emodi 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. [21] was used as a reference for compound identification. Table 3 shows the phenolic compounds identified in the methanolic extract of R. emodi rhizome along with the respective retention times (R t ). Both aqueous and methano-lic extracts also contained unknown compounds evident from the HPLC data whose characterization is in prospect.

Discussion
Aqueous and methanolic extracts of R. emodi showed promising antioxidant activity in all the experimental models used. Both extracts were found to have a dosage-dependent increase in their antioxidant potentials with varying degrees of efficiencies. The differential scavenging activities of the extracts may be attributed to the varying mechanisms of radical scavenging in these assays. The extracts were observed to be good scavengers of hydroxyl radical, which is involved in DNA crosslinkings and strand breaks, and is considered to be one of the quick initiators of lipid peroxidation [24]. The ability of the extracts to quench hydroxyl radicals might directly relate to the prevention of lipid peroxidation. It can be inferred that the extracts might 8 Evidence-Based Complementary and Alternative Medicine prevent reactive radical species from damaging biomolecules such as lipoproteins, polyunsaturated fatty acids, DNA, amino acids, proteins and sugars in biological systems [25]. Both extracts showed concentration-dependent cytotoxicity when tested in each of the two cancer cell lines. According to the American National Cancer Institute, the IC 50 value to consider a crude extract promising for development of anti-cancer drug(s) is lower than a limit threshold of 30 μg/ml [26]. The extracts can thus serve as potential source for anti-cancer compounds. The aqueous extract, on the other hand, although has lower potential as a cytotoxin, shows considerable degree of DNA protection against oxidative damage, while its methanolic counterpart holistically lacks this property. These differences can be attributed to the presence of differential protective metabolites eluted out in the two solvents, and also due to factors like stereoselectivity and/or solubility of the two extracts [27]. Both extracts were therefore found to have promising potential towards the development of drugs that might be used to target tumors for chemoprevention/chemotherapy to check neoplastic growth and malignancy.
A significant (P < .05) positive correlation was extrapolated between the results of the assay for estimation of total phenolic content and those investigating other therapeutic parameters. In view of this, HPLC analysis was performed to identify some of the major phenolic compounds in both extracts. However, we accomplished in identifying major polyphenols only in the methanolic extract.
The antioxidant, cytotoxicity and DNA protection abilities of the extracts render them suitable to be considered as a source for the development of anti-cancer drugs (Figure 8). In tumor cells, ROS is produced extensively, which thereby increases levels of certain growth factors and enzymes like metalloproteinases (MMPs) which promote angiogenesis, and also elevates the risk of metastasis and development of secondary tumors [28]. Antioxidant properties of the extracts might therefore prevent progression of cancer; while the cytotoxic potential, on the other hand, might be used against cancer cells, thereby directing them towards apoptosis and cell death. DNA protection property might hold good in inhibiting secondary mutations in progressive tumor tissues.

Conclusion
Rhizome of R. emodi might be a potential source for anticancer metabolites which can be mustered for the development of effective cancer drugs. Isolation and characterization of compounds from R. emodi rhizome extracts are in prospect.