Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 156765, 11 pages doi:10.

1155/2011/156765

Research Article Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cats Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway
Siddig Ibrahim Abdelwahab,1 Syam Mohan,2 Manal Mohamed Elhassan,2 Nabil Al-Mekhla,2 Abdelbasit Adam Mariod,3 Ahmad Bustamam Abdul,2 Mahmood Ameen Abdulla,4 and Khalid M. Alkharfy5
1 Department 2 Institute

of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia of Biosciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia 3 Food Science and Technology Department, College of Agricultural Studies, Sudan University for Sciences and Technology, P.O. Box 71, Khartoum North, Sudan 4 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 5 Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi arabia Correspondence should be addressed to Siddig Ibrahim Abdelwahab, siddigroa@yahoo.com Received 14 July 2010; Revised 13 October 2010; Accepted 30 November 2010 Copyright 2011 Siddig Ibrahim Abdelwahab et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, -carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for dierent extract/fractions were 126.2 23, 31.25 1.2, 15.25 2.3, 13.56 1.9, 23.0 3.2, and 16.66 1.5 g/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of -carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H2 O2 -induced apoptosis. The current results showed that phenolics may provide health and nutritional benets.

1. Introduction
In the past, herbs often represented the original sources of most drugs, but nowadays, alternative medicines are used widely in all over the world. With increasing recognition of herbal medicine as an alternative form of health care, screening of medicinal plants for biologically active extracts and/or compounds has become an important source of drugs [13], many of them with unique pharmacological and chemical properties. Herbal-derived remedies need a powerful and deep assessment of their pharmacological qualities [4].

Orthosiphon stamineus Benth (family: Lamiaceae) or Misai Kucing (Malay for Cats Whiskers) is commonly used as Java Tea. Java Tea is a medicinal plant, native in South East Asia (Malaysia, Indonesia, and Thailand) and some part of Tropical Australia. It is a herbaceous shrub which grows to a height of 1.5 meter. This plant is best used for treating the ailments or problems of kidney and bladder due to its mild diuretic action. It is used as a remedy for kidney stone and nephritis. It is believed to have antiallergenic, antihypertensiveand anti-inammatory properties. OS is also used for treating gout, diabetes, hypertension, and

2 rheumatism. It is also reported to possess antifungal, and it exhibits considerable antibacterial activity. Java tea/misai kucing is appearing in many products where safe diuretic action is required such as detoxication, water retention, hypertension, weight loss, and for kidney stones. The products appear in form of tablets, capsules, tea sachets, drinks, raw herbs, dried leaves, or extracts [513]. O. stamineus contains several chemically active constituents such as polyphenols, terpenoids, oleanolic acid, and sterols [14]. The polyphenols are the most dominant constituents in the leaf, which has been reported to be eective in reducing oxidative stress by inhibiting the formation of lipid peroxidation products in biological systems [15]. Recently, the diuretic activity of methoxy avonoids isolated from OS was reported [16]. Phenolic compounds such as lipophilic avones, caeic acid derivatives (rosmarinic acid and 2,3-dicaeoyltartaric acid), eupatorine, cichoric acid, sinensetin and methoxy avones were found in O. stamineus [17, 18]. Three highly oxygenated 2,3-secoisopimarane-type diterpenes, named secoorthosiphols A, B, and C, have been isolated from the aerial parts of this plant [19]. The therapeutic benet of medicinal plants is usually contributed to their antioxidant properties [20]. Phenolic compounds possess diverse biological activities such as anti-inammatory, anticarcinogenic, and antiatherosclerotic activities. These activities of phenolic compounds might be related to their antioxidant activities [21]. Other studies showed that there were signicant correlations between phenolic compounds and antioxidant properties of medicinal plants. Therefore, the current study was conducted to investigate the antioxidant and antipoptotic potency of aqueous-methanol extract/fractions of O. stamineus.

Evidence-Based Complementary and Alternative Medicine Each extraction process involved homogenisation of CAME and its fractions in the respective solvent at 13,000 rpm for 15 min followed by sonication at constant temperature of 30 C for 1 h. The CAME and its fractions (hexane fraction HF, ethyl acetate fraction EAF, chloroform fraction CF, nbutanol NBF, water fraction WF) were ltered through lter paper Whatman no. 1. Then solvents were removed by using rotary evaporator (Buchi, Flawil, Switzerland). The yield of each extract and its fractions was measured before kept in refrigerator for further analysis. 2.3. Total Phenolic Content (TPC). TPCs of HF, EAF, CF, NBF, WF, and CAME were determined using Folin-Ciocalteu method [23]. Briey, stock solutions of extract and fraction of OS were prepared in a concentration of 10 mg/mL in methanol. Five microliters of each solution were transferred to 96-well microplate (TPP, USA). To this, 80 L of FolinCiocalteu reagent (1 : 10) were added and mixed thoroughly. After 5 min, 160 L of sodium bicarbonate solution (NaHCO3 7.5%) were added, and the mixture was allowed to stand for 30 min with intermittent shaking. Absorbance was measured at 765 nm using microplate reader (Molecular Devices, Sunnyvale, USA). The TPC was expressed as gallic acid equivalent (GAE) in mg/g extract, obtained from the standard curve of gallic acid. The gallic acid standard curve was established by plotting concentration (mg/mL) versus absorbance (nm) (y = 0.001x + 0.045; R2 = 0.9975), where y is absorbance and x is concentration in GAE (n = 3). 2.4. Total Flavonoid Content. Total avonoid contents (TFCs) were determined by the AlCl3 method [24] using rutin as a standard. The test samples were dissolved in DMSO (10 mg/mL). The sample solution (1 mL) was mixed with one millilitre of AlCl3 (2.0%). After 10 min of incubation at ambient temperature, the supernatant was transferred to 96-well plate. The absorbance of the supernatant was measured immediately at 435 nm using microplate reader (Molecular Devices, Sunnyvale, USA). TFC was expressed as rutin equivalents (REs) in milligrams per gram sample. For the rutin, the curve was established by plotting concentration (g/mL) versus absorbance (nm) (y = 5.6752x 0.0312; R2 = 0.99). Here y = absorbance, and x = concentration (n = 3). 2.5. Free Radicals Scavenging of DPPH. Radical scavenging activity of extract and fractions of OS against DPPH (2, 2diphenyl-2-picrylhydrazyl hydrate, Sigma-Aldrich Chemie, Steinheim, Germany) was determined spectrophotometrically. When DPPH (Violet colour) reacts with an antioxidant compound, which can donate hydrogen, it is reduced and produces a yellow colour. The changes in colour were measured at 517 nm wavelength [25]. Radical scavenging activity of extract/fractions of OS was measured by slightly modied method of Chan et al. [25], as described below. Stock solutions were dissolved in methanol (10 mg/mL). The working solution was prepared using methanol in a concentration of 2000 g/mL. The solution of DPPH in methanol (2.5 mg/mL) was freshly prepared. Five L of

2. Materials and Methods

2.1. Plant Materials. Leaves of OS (Lamiaceae) were collected in July 2009 from the Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia. Authentication was done at the same institute where the voucher specimen under the plant name was deposited. The freshly collected leaves were washed and weighed. After that the parts were cut in small and dried at 5060 C for 5 days. The dried leaves were weighed and then grounded to powder form. The powder form of plants were weighed and kept in an airtight plastic bag at room temperature. 2.2. Aqueous-Methanolic Extraction and Liquid-Liquid Fractionation of Orthosiphon stamineus. Extraction was done according to the method recommended by Mariod et al. [22]. Thirty grams of the dried ground leaves of OS were extracted successively with 60% methanol (3 300 mL) to obtain crude methanolic extract (CAME) at room temperature for two days each, then combined, and concentrated by removing solvent by rotary evaporator (Buchi, Flawil, Switzerland). The obtained CAME (8.27 g) was fractionated using hexane, chloroform, ethyl acetate, n-butanol, and water individually, where the residue from each fractionation step was used to obtain the subsequent fraction as shown in Figure 1.

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O. stamineus leaves

Identication Drying and grinding Dried powder 60% methanol, maceration (3X), ltration, evaporation Aqueous methanolic extract

Extract dissolved in deionised water Liquid-liquid fractionation, hexane Hexane fraction Residues Liquid-liquid fractionation, chloroform Chloroform fraction Residues Liquid-liquid fractionation, ethyl acetate Ethyl acetate fraction Residues Liquid-liquid fractionation, n-butanol Residues

n-butanol fraction

Figure 1: Extraction and fractionation of O. stamineus.

this solution were mixed with 100 L of serial dilutions of samples (15.6252000 g/mL) in 96-well plate. The samples were kept in the dark for 30 min at ambient temperature, and then the change in absorption was measured (Molecular Devices Plate Reader, Sunnyvale, USA). Absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured daily. The experiment was carried out in triplicate. Radical scavenging activity was calculated by the following formula: % Inhibition = A B AA 100, AB (1)

the sample extracts and positive control BHA (butylated hydroxyanisole) were added into the plate. An equal amount of methanol was used as negative control. Absorbance was read at 470 nm after incubation for every 20 min until 120 min at 50 C using a microplate reader (Molecular Devices Plate Reader, Sunnyvale, USA). Tests were performed in triplicate. 2.7. Antipoptotic Eect of O. stamineus against H2 O2 Induced Cell Injury 2.7.1. Cell Culture and Drug Treatment. RPMI-1640 medium was used to culture MDA-M231 cells. Cultures were supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 g/mL). Stock cultures of exponentially growing cells were trypsinized and plated (2 105 cells/well) into 96-well plate and incubated at 37 C for 48 h prior to use then washed twice with PRMI-1640 medium and recultured therein. HF, EAF, CF, NBF, WF, CAME, and quercetin (positive control) dissolved in methanol, and later diluted with media, was added to the wells and incubated for 10 min before the addition of hydrogen peroxide (nal concentration 400 mol/L) [27]. After 2 h of incubation, cell viability in every well was assayed. 2.7.2. Assessment of Cell Viability. Cell viability could be quantied using MTT, which yields a purple formazan product in living cells, but not in dead cells or their lytic debris. MTT was dissolved in aseptic PBS to a concentration of 5 mg/mL as a stock solution. MTT was added at the end of incubation to a nal concentration of 0.5 mg/mL

where AB is the absorption of blank sample; AA is the absorption of tested samples. The inhibitory concentration 50% was determined as well as the kinetics of DPPH scavenging reaction. Ascorbic acid was also tested against DPPH as positive control. 2.6. Beta-Carotene Bleaching Assay (BCB). The antioxidant activity (AOA) of the dierent extracts/fractions of OS was evaluated using the -carotene-linoleic acid assay following the method of Amarowicz et al. [26]. An aliquot of 5.0 mg from -carotene was dissolved in 50 mL chloroform. Linoleic acid 200 L and 600 mg Tween-20 were mixed with 1 mL of the chloroform solution. The chloroform was evaporated under vacuum at 45 C for 20 min, then 50 mL oxygenated water was added, and the mixture was vigorously shaken to obtain -carotene-linoleic acid emulsion. Two hundred L of this emulsion were distributed in a 96well microplate. Methanolic solutions (10 L, 2 mg/mL) of

4 and then incubated at 37 C for 4 h, and the resultant formazan product was solubilised using dimethyl sulphoxide and detected using UV-spectrometer at 570 nm. 2.7.3. Microscopic Observation of Cellular Morphology Using Phase-Contrast Inverted Microscope. This analysis examines morphologically whether cell death may be implicated in mediating H2 O2 among MDA-MB231 cells. The EAF was chosen in this analysis, since this fraction showed the most potential results in MTT assay. Morphological appearance of treated cells (EAF + Hydrogen Peroxide) was compared with the control cells (Hydrogen Peroxide) by using the inverted light microscope. Apoptosis was identied using morphological criteria, including shrinkage of the cytoplasm, membrane blebbing, and nuclear condensation, and/or fragmentation. 2.7.4. Colorimetric Assays of Caspase-3. The colorimetric protease assay of caspase-3 provides a simple and convenient means for quantifying the enzyme activity that recognize the amino acid sequence, DEVD (a synthetic tetrapeptide, (AspGlue-Val-Asp), which is the upstream amino acid sequence of the caspase-3 cleavage site), coupled with p-nitroanilide, which is released upon substrate cleavage. This assay was performed using ApoTarget Kit (BioSource International, Inc., Calif, USA). Cells (2 106 ) were treated with hydrogen peroxide with or without EAF and incubated for 72 hours while untreated cells acted as control. The cells were lysed by the addition of 50 L of chilled cell lysis buer and incubated on ice for 10 minutes. The resulting cell lysate was centrifuged for 1 minute at 10,000 xg, and the supernatant was collected. Fifty microliters of 2X reaction buer (containing 10 mM DTT) were added to each sample. Then 5 L of DEVD-pNA (caspase-3 substrate) was added and incubated in the dark at 37 C for 1 hour. At the end of the incubation period, the samples were read at 405 nm in a microplate reader (TECAN, Sunrise, Mannedorf, Switzerland). Data was presented as optical density (n = 3). 2.7.5. Western Blotting. This assay was used to investigate the expression of apoptosis-associated protein, Bcl-2. MDAM231Cells were lysed using CSK buer (10 mM pipes, pH 6.8, 100 mM NaCl, 1 mM MgCl2 , 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethanesulfonyl uoride) containing 0.1% Triton X-100, 1 mM ATP, and proteinase inhibitors (Pharminogen, USA). Cell lysates were centrifuged at 20,000 x g for 30 min, and the protein concentration in the supernatant was determined with Coomassie method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). The membrane was incubated at 37 C for 70 min with primary antibodies in a blocking solution. After being washed with Tris buered saline, the membrane was incubated with conjugated secondary antibodies (Biosyntech, USA). The immunoreacted proteins were detected using a chemiluminescence system (Bio-Rad, USA). 2.7.6. Real-Time Polymerase Chain Reaction. Total RNA was extracted using RNeasy Mini Kit following the manufac-

Evidence-Based Complementary and Alternative Medicine turers instructions (Qiagen, Germantown, Maryland, USA). RNA concentrations were quantied using a spectrophotometer (Smart Spec, Bio-Rad). RNA quality and integrity were determined via the A260/A280 ratio and agarose gels electrophoresis, respectively. cDNA was synthesized with Revert Aid H Minus M-muLV reverse transcriptase (Biometra, Goettingen, Germany). Real-time RT-PCR was performed using an ABI 7700 Prism Sequence Detection System and TaqMan primer probes (Applied Biosystems, Foster City, CA). The total reaction volume was 20 L : 2 L cDNA, 10 L SYBR Premix ExTaq, 0.4 L of each primer (10 M), and 7.2 L ultrapure water. Cycle parameters were as follows: activation at 95 C for 30 s, 40 cycles of denaturation at 95 for 5 s, and then annealing and extension at 60 C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal control for each sample. PCR products were detected using gel electrophoresis. The primer sequences for Bax were Forward: 5 -TGCTTCAGGGTTTCATCCA-3 Reverse: 5 -GACACTCGCTCAGCTTCTTG-3 The primer sequences for Bcl-2 were Forward: 5 -GGGAGAACAGGGTACGATAA-3 Reverse: 5 -GCTGGGAGGAGAAGATGC-3 The primer sequences for GAPDH were Forward: 5 -GGATTTGGTCGTATTGGG-3 Reverse: 5 -TCGCTCCTGGAAGATGG-3 . 2.8. Statistical Analysis. Experimental values are means SD of the number of experiments indicated in the legends. Signicance was assessed by using the one-way ANOVA followed by multiple comparison test (P < .05 as signicant). Pearson correlation coecient was used to assess the correlation between phenolic content and antioxidant activities.

3. Results and Discussion

3.1. Eciency of Aqueous Methanolic Extraction and Fractionation. Methanol has been recommended for the extraction of phenolic compounds from plant tissues. It is an appropriate solvent due to its ability to inhibit polyphenol oxidase, which could amend antioxidant activity. High methanol extraction eciency has been reported for leaves and owers of some plant species [28]. Leaf powder of OS when extracted previously using aqueous methanolic solvent has shown high phenolic content and good free radical activity. The amount of rosmarinic acid, the principal polyphenol in OS leaf, was conrmed earlier to be higher in aqueous methanol extract, and the amount of this acid was signicantly dierent from the other solvent extracts [9, 12]. The current study provide, for the rst time, a standardized method to obtain rich antioxidant fractions from OS. In this study, amount of extractable compounds (TECs) was calculated as milligram extract in each gram of powdered leaves. As shown in Table 1, the highest TEC was revealed by WF (178.34 mg/g), followed

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Table 1: Total extractable compounds (TEC), total avonoids content (TFC), total phenolic compounds (TPC), and DPPH IC50 (mg/mL) of dierent extract/fractions obtained from O. stamineus (n = 3). Extract/fractions of O. stamineus HF CF EAF NBF WF CAME Total phenolic content (GAE mg/g extract/fraction) 35.507 1.420a 139.813 12.164bd 222.927 7.580
c bd

TEC (mg/g) 4.02 7.75 12.62 72.93 178.34 275.66

Total avonoids content (RE mg/g extract/fraction) 24.52 2.5b 58.11 9.5c 28.83 4.1 14.94 1.9
b a

DPPH IC50 (g/mL) 126.2 23 31.25 1.2 15.25 2.3 13.56 1.9 23.0 3.2 16.66 1.5

183.250 14.110

160.327 15.552bd 189.073 49.15

bd

13.20 4.2a 17.56 5.5

a

Values with dierent superscript letters within the same column are statistically dierent (P < .05).

by NBF (72.93 mg/g), EAF (12.62 mg/g), CF (7.75 mg/g), and HF (4.02 mg/g), respectively. The TECs were obviously varying between the fractions of OS leaves and increase gradually with the increasing of the solvents polarity. 3.2. Total Phenolic and Flavonoids Contents. Since plant phenolics represent one of the major groups of compounds behaves as primary antioxidants or free radical scavengers [29], it was reasonable to determine their total amount in the extract/fractions of OS using Folin-Ciocalteau method. The TPC was expressed as gallic acid equivalent in mg/g, GAE. The content of the total phenolic compounds of CAME and its dierent fractions (HF, EAF, CF, NBF, and WF) is shown in Table 1. Results in this table showed that EAF showed the highest TPC followed by CAME, NBF, WF, CF, and HF, respectively. TPC of ethyl acetate fraction (EAF) is signicantly (P < .05) higher in comparison to the dierent extract/fractions obtained from OS. Ethyl acetate is often used as an extraction solvent with a signicant selectivity in the extraction of low-molecular-weight phenolic compounds and high-molecular-weight polyphenols [30]. On the other hand, Conde et al. have reported that ethyl acetate allowed the highest phenolic content and the selective removal of nonphenolic compounds [31]. Flavonoids are the most common and widely distributed group of plant phenolic compounds, which usually are very eective antioxidants [32]. In this study, total avonoids content (TFC) of OS was determined (Table 1). The TFC was expressed as rutin equivalent in mg/g, RE. The results showed that the avonoids content was in the order of the CF >EAF> HF > CAME > NBF > WF with the values of 58.11 9.5, 28.83 9.1, 24.52 2.5, 17.56 5.5, 14.94 1.9, and 13.20 4.2 mg RE/g extract, respectively. From our results, the chloroform fraction (CF) gave the highest TFC. There was a signicant dierence (P < .05) of the TFC in CF, HF, and EAF compared with the amounts in NBF, WF, and CAME. Among the various solvents used for the fractionation, chloroform and hexane have the lowest polarity; therefore, the amounts of lipophilic avonoids were higher, as expected. This indicates that the lipophilic avonoids are major phenolic compounds present in OS.

It can be observed that the content of phenolics in the extracts/fractions of OS correlates with their antiradical activity (e.g., correlation coecient between data of DPPH assay and total phenolic compounds is 0.832, P < .001), conrming that phenolic compounds are likely to contribute to the radical scavenging activity of these plant extract/fractions. Detailed examination of phenolic composition in plant extract/fractions of OS is required for the comprehensive assessment of individual compounds exhibiting antioxidant activity. A previous study had found that phenolic compounds contributed signicantly to the antioxidant capacity of the medicinal herbs as there was a direct relationship between antioxidant activity and total phenolic content [33]. 3.3. DPPH Scavenging Activity Test. The DPPH assay has been widely used to test the free radical scavenging ability of plant materials and pure compounds [22]. The DPPH is a stable free radical (purple coloured), which is reduced to ,diphenyl--picrylhydrazine (yellow coloured) when reacting with an antioxidant agent. Antioxidants interrupt free radical chain oxidation by donating hydrogen from hydroxyl groups to form a stable end-product, which does not commence or proliferate further oxidation of lipids in human body [28]. The data obtained from this assay is commonly reported as IC50 , which is the concentration of antioxidant required for 50% scavenging of DPPH radicals in the specied time period. The DPPH scavenging activities of extract/fractions of OS are shown in Table 1. The IC50 values for dierent extract/fractions were 126.2 23, 31.25 1.2, 15.25 2.3, 13.56 1.9, 23.0 3.2, and 16.66 1.5 g/mL for HF, CF, EAF, NBF, WF, and CAME, respectively. As depicted in Table 1, a correlation was observed between the TPC and IC50 when the TPC level was high; the IC50 was low which indicates high antioxidant activity. This is due to the high TPC present in the leaves of OS that act as free radical scavengers. It also observed that there is a positive linear relationship (P < .05) between DPHH inhibition and the polyphenolic content of extract/fractions of OS, whereby polar extract/fractions have shown the higher DPPH scavenging activities. The ndings

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1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 20 40 60 Time (min) 80 100 120

Evidence-Based Complementary and Alternative Medicine compounds. Ethyl acetate is often used as an extraction solvent with a signicant selectivity in the extraction of lowmolecular-weight phenolic compounds and high-molecularweight polyphenols [30]. Recently ethyl acetate extract has been observed to possess the highest total phenolic content, DPPH scavenging ability, and antioxidant activity (in carotene bleaching assay) among the extracts of Pereskia grandifolia [35]. 3.5. Eect of OS on Hydrogen Peroxide Induced Apoptosis 3.5.1. Cell Viability. To examine the eect of OS on oxidant-induced apoptosis, epithelial cells (MDA-M231) were pretreated with or without EAF and exposed to H2 O2 (400 mol/L). Incubation of cells with H2 O2 leads to the decrease of formazan production compared with the untreated one, which can be seen from the decrease of optical density at 570 nm, and it means that cell viability in H2 O2 -treated wells was much lesser than the untreated one. However, when cell is incubated with extract/fractions of O. stamineus (HF, CF, EAF, NBF, WF, and CAME) prior to H2 O2 exposure, cell viability was higher than that of control (Table 2). Among the tested samples, EAF and NBF showed signicant (P < .01) protection against hydrogen peroxide induced apoptosis. Nevertheless, all extract/fractions of the plant under this investigation have shown signicant (P < .05) in vitro free radical inhibition. This result is in agreement with the previous study by Zhang and Yang [21] who reported that avonoids extracted from the radix of Scutellaria baicalensis exhibited inhibitory activity towards H2 O2 induced cell injury. This conrms that protecting cells from antioxidants such as H2 O2 is a vital bioprocess. However, hydrogen peroxide is made by quite a few enzymes in the body. In particular, some enzymes breaking down certain amino acids and fatty acids (D-amino acid oxidase and acylCoA oxidase) make signicant amounts of hydrogen peroxide. Since hydrogen peroxide can be damaging to normal tissue, these enzymes are kept inside specialized organelles inside cells called peroxisomes [36]. Since EAFs were the most cytoprotective fraction of OS against H2 O2 induced cell injury, all downstream experiments were conducted to assess cell death between control and treated cells. 3.5.2. Phase-Contrast Microscope. Recent studies have suggested that hydrogen peroxide, a reactive compound formed endogenously in the breakdown of superoxide, may intercede the initiation of apoptosis in various cell types in response to outer stimuli. Apoptosis, a type of programmed cell death, is an active process and a way of eliminating a cell from an organism [37]. Morphological assessment of hydrogen peroxide treated-MDA-M231 using inverted microscope demonstrated that suspected apoptosis had occurred due to exhibition of morphological features changes (Figure 3). Morphological changes associated with apoptotic cell death induced by H2 O2 were characterized by the presence of shrunken cells with surface blebbing and nuclear condensation. However, these cellular events were clearly prevented when the cells were preincubated with EAF. Conrmation of

Absorbance at 470 nm

HF EAF CF NBF

WF CAME Control BHT

Figure 2: Eect of extract/fractions of O. stamineus on oxidation of -carotene/linoleic acid at 50 C. BHA: butylated hydroxyanisole, CAME: crude aqueous-methanolic extract, HF: hexane fraction, CF: chloroform fraction, NBF: n-butanol fraction, EAF: ethyl acetate fraction, and WF: water fraction.

of the current study are in an agreement with those of Akowuah et al. [9] who indicated that the extracts of OS are free radical inhibitors and primary antioxidants that react with free radicals and the polar extracts have the highest free radical scavenging activity. However, the current study investigated for the rst time the antioxidant activities of OS fractions. 3.4. -Carotene Bleaching (BCB) Assay. In the BCB assay, the oxidation of linoleic acid produces peroxyl free radicals due to the abstraction of hydrogen atom from diallylic methylene groups of linoleic acid [34]. The free radical then will oxidise the highly unsaturated -carotene. The presence of antioxidants in the sample will reduce the oxidation of -carotene by hydroperoxides. Hydroperoxides formed in this system will be decomposed by the antioxidants from the extracts. Thus, the degradation rate of -carotene depends on the antioxidant activity of the extracts. Eect of cat whiskers (OS) extracts/fractions (CAME, HF, CF, WF, NBF, and EAF) on oxidation of -carotene is shown in Figure 2. It was clear that the presence of antioxidants in OS reduced the oxidation of -carotene by hydroperoxides. There were signicant dierences (P < .05) between the dierent extracts/fractions, control, and BHA eect as shown in Figure 2. NBF, EAF, WF, and CAME fractions were better in their eect on reducing the oxidation of carotene than CF and HF fractions. The degradation rate of -carotene clearly depends on the antioxidant activity of OS. There was a correlation between the degradation rate and the bleaching of -carotener, where the extract with the lowest -carotene degradation rate exhibited the highest antioxidant activity (Figure 2). The high antioxidant activities of n-butanol fraction and ethyl acetate fraction tested using -carotene model may be correlated with their high phenolic content. These ndings are in good agreement with those of Matthaus [29] who found that extraction with polar solvents gave the highest amounts of phenolic

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(a)

(b)

(c)

(d)

(e)

Figure 3: Inhibition of hydrogen peroxide-(H2 O2 -) triggered apoptosis using ethyl acetate fraction (EAF) of O. stamineus. Epithelial cells were pretreated with or without EAF and later exposed to H2 O2 . Cells were viewed under phase contrast microscopy. (a) MDA-MB-231 control cells; (bd) cells pre-treated with dierent concentrations of EAF and incubated with hydrogen peroxide; (e) cells treated with hydrogen peroxide.

1.2 Optical density (405 nm) 1 0.8 0.6 0.4 0.2 0 EAF (31.25 g/mL) + hydrogen peroxide EAF (62.5 g/mL) + hydrogen peroxide EAF (125 g/mL) + hydrogen peroxide Hydrogen peroxide Control cells Bcl-2 -actin

Figure 4: The colorimetric assay of caspase-3 in MDA-M231 cells treated and untreated with H2 O2 and EAF for 72 hours. Cells were cultured in RPMI 1640 (75 mL ask) media maintained at 37 C and 5% CO2 . ANOVA showed a signicant dierence (P < .05) between treated and untreated cells in the activity of caspase-3.

EAF (31.25 g/mL) + hydrogen peroxide

EAF (62.5 g/mL) + hydrogen peroxide

Figure 5: Eect of EAF on hydrogen peroxide- (H2 O2 -) triggered apoptosis. Cells were treated with hydrogen peroxide with or without EAF, incubated for 72 hours while untreated cells acted as control. After incubation, cells were harvested and used western blotting analysis.

Hydrogen peroxide

Control

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15 12 9 6 3 0 EAF (125 g/mL) + hydrogen peroxide Hydrogen peroxide EAF (31.25 g/mL) + hydrogen peroxide EAF (62.5 g/mL) + hydrogen peroxide Control

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4 3.5 3 2.5 2 1.5 1 0.5 0 Hydrogen peroxide

Relative quantity of Bcl-2 mRNA

Relative quantity of Bax mRNA

EAF (31.25 g/mL) + hydrogen peroxide

EAF (62.5 g/mL) + hydrogen peroxide

EAF (125 g/mL) + hydrogen peroxide

Bcl-2
(a)

Bax
(b)

Figure 6: Bcl-2 and Bax mRNA expression in MDA-M231. Cells were treated with EAF and then incubated with H2 O2 . After incubation, cells were harvested and used real-time RTPCR analysis. (a) Bcl-2; (b) Bax. Normalization relative to GAPDH was performed. Results presented in bar graph are the means SD of three independent experiments.

Oxidative injury

Cell membrane

Orthosiphon stamineus Benth

Blc2/Bax Caspase-3

Apoptosis

Figure 7: Hypothetical scheme of the cytoprotective properties of Orthosiphonstamineus against free radical induced cell death.

apoptosis in the current study was performed using western blot, caspase-3 assay, and gene expression. 3.5.3. Colorimetric Assay of Caspase-3. Exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and activation of its associated proteins such as capsase-3. However, H2 O2 -induced apoptosis was associated with activation of caspase-3 [38, 39]. Since EAFs were the most cytoprotective fraction against H2 O2 induced cell injury, an in vitro colorimetric assay of caspase-3 was conducted to assess cell death between control and treated cells. As shown in Figure 4, H2 O2 signicantly (ANOVA, P < .05) stimulated caspase-3, the hallmark enzyme of cell death. The level of

this enzyme is higher in H2 O2 -reated MDA-M231 cells as compared to untreated cells. EAF reduced remarkably (P < .05) the level of caspase-3 in a dose-dependent manner. We suggest that EAFs prevent apoptosis in epithelial cells by inhibiting caspase-3 during oxidant injury. 3.5.4. Eect of EAF Mitochondrial Apoptosis. To examine whether the antipoptotic eect of EAF is due to intervention with Bcl2/Bax regulated apoptosis, proteins and mRNAs of epithelial cells were studied using western blot and qRT-PCR, respectively. Bcl-2 protein (Figure 5) and mRNA (Figure 6(a)) levels were signicantly decreased in cells exposed to H2 O2 compared to levels in control cells

Control

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Table 2: Eects of extract/fractions of O. stamineus on cell viability. Treatment 500 g/mL HF + Hydrogen Peroxide CF + Hydrogen Peroxide EAF + Hydrogen Peroxide NBF + Hydrogen Peroxide WF + Hydrogen Peroxide CAME + Hydrogen Peroxide Quercetin + Hydrogen Peroxide Hydrogen Peroxide control Control cells

Concentrations of extract/fractions of O. stamineus 250 g/mL 125 g/mL 22.37 1.56 20.61 0.94 43.03 2.65 24.81 0.09 100.00 16.51 84.82 3.98 66.03 0.01 37.92 0.99 35.20 13.12 23.88 1.51 52.64 7.56 37.53 1.65 95.45 3.72 15.86 0.23 100

62.5 g/mL 23.10 3.18 20.66 1.56 56.43 5.98 29.90 4.00 23.41 5.49 26.27 1.86

25.90 0.95 70.81 10.24 100.00 27.75 100.00 8.69 65.62 10.70 91.31 5.32

Cell viability after 2 h exposure in 400 mol/L H2 O2 was assayed by MTT. Cells were preincubated with dierent tested samples (62.5, 125, 250, and 500 g/mL) for 10 min before the addition of 400 mol/L H2 O2 . After 2 h incubation, MTT was added to a nal concentration of 0.5 mg/mL and then incubated at 37 C for another 2 hrs; the resultant formazan product was extracted for 4 h with DMSO and detected by UV-spectrometer at 570 nm. Values are expressed as means SD (N = 3). P < .05, P < .01 versus hydrogen peroxide control.

(Figure 6(a)). In contrast, Bcl-2 levels were signicantly increased from control by various concentration of EAF in a dose-dependent manner (Figure 6(a)). Bax mRNA levels were signicantly higher in the H2 O2 group. EAF group has higher Bax expression compared to control group (Figure 6(b)). Many genes are involved in the regulation of apoptosis. Bcl-2 is an antipoptotic regulator, while Bax is a proapoptotic regulator. The balance between Bcl-2 and Bax expression plays an important role in sustaining cell morphology and function. Several studies have shown that Bcl-2 overexpression can disrupt regulation of the proapoptotic proteins Bax [38, 39]. Moreover, an increase in Bcl-2 expression prevents cytochrome c release from the mitochondria, thereby inhibiting activation of caspases, such as caspase-9 and caspase-3, and preventing apoptosis [39]. Other studies have demonstrated that Bcl-2 can act as a mitochondrial membrane channel protein [40]. Thus, cells are active when Bcl-2 is overexpressed and die if Bax is hyperexpressed [41]. In the present study, we found that expression of Bcl-2 was downregulated by H2 O2 . These alterations of Bcl-2 protein expression were restored by EAF. Our ndings suggest that Bcl-2 is involved in mediating the antipoptotic eects of EAF in H2 O2 -exposed MDA-M231.

provide health and nutritional benets. On the other hand, epidemiological studies have shown a relationship between high dietary intakes of phenolic-rich foods and reduced risk of cardiovascular disease and cancer whereby, the production and development of many diseases are closely related to oxidative damages caused by free radicals. So it becomes one of investigating hotspots that safe and eective antioxidants in plants are searched.

Conict of Interests
Authors state that there is no conict of interests.

Acknowledgment
Authors are very grateful to the sta of Institute of Bioscience, Universiti Putra Malaysia for their technical assistance.

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4. Conclusion
Despite the wide spread use of medicinal uses of OS as a remedy for kidney stone, nephritis, gout, diabetes, hypertension, and rheumatism, evidence from in vitro previousbiological studies is not sucient [11]. The current paper provides strong evidence about the antioxidant properties of Orthosiphon stamineus and its fractions as well as their use as cytoprotective against free radical induced cell death. Antipoptotic eect of this plant is suggested to be through the intervention in Bcl-2-mediated apoptotic pathways. The results obtained support the ecacy of natural phenolics from plant origin in oering protection against oxidative injury and highlight the fact that phenolic-rich foods may

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