Profile of bioactive compounds in Nymphaea alba L. leaves growing in Egypt: hepatoprotective, antioxidant and anti-inflammatory activity

Background Nymphaea alba L. represents an interesting field of study. Flowers have antioxidant and hepatoprotective effects, rhizomes constituents showed cytotoxic activity against liver cell carcinoma, while several Nymphaea species have been reported for their hepatoprotective effects. Leaves of N. alba have not been studied before. Therefore, in this study, in-depth characterization of the leaf phytoconstituents as well as its antioxidant and hepatoprotective activities have been performed where N. alba leaf extract was evaluated as a possible therapeutic alternative in hepatic disorders. Methods The aqueous ethanolic extract (AEE, 70%) was investigated for its polyphenolic content identified by high-resolution electrospray ionisation mass spectrometry (HRESI-MS/MS), while the petroleum ether fraction was saponified, and the lipid profile was analysed using gas liquid chromatography (GLC) analysis and compared with reference standards. The hepatoprotective activity of two doses of the extract (100 and 200 mg/kg; P.O.) for 5 days was evaluated against CCl4-induced hepatotoxicity in male Wistar albino rats, in comparison with silymarin. Liver function tests; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT) and total bilirubin were performed. Oxidative stress parameters; malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAC) as well as inflammatory mediator; tumour necrosis factor (TNF)-α were detected in the liver homogenate. Histopathological examination of the liver and immunohistochemical staining of caspase-3 were performed Results Fifty-three compounds were tentatively identified for the first time in N.alba leaf extract, where ellagitannins represent the main identified constituents. Nine hydrocarbons, two sterols and eleven fatty acids were identified in the petroleum ether extract where, palmitic acid and linolenic acids represented the major saturated and unsaturated fatty acid respectively. N.alba AEE significantly improved the liver function, oxidative stress parameters as well as TNF-α in addition to the amelioration of histopathological features of the liver and a profound decrease in caspase-3 expression. Conclusion These results shed light on the hepatoprotective effect of N. alba that is comparable with that of silymarin. The antioxidant activities of N. alba extract in addition to the inhibition of crucial inflammatory mediator, as TNF-α, might be the possible hepatoprotective mechanisms.


Background
Hepatotoxicity is a prevalent problem worldwide. Carbon tetrachloride (CCl 4 ) is a chlorinated hydrocarbon that is commonly used in industries as a solvent and in medicine as a vermifuge. The compound is also found at low levels in ambient air and water [1]. Exposure to CCl 4 is known to result in acute hepatotoxicity in humans and experimental animals. It is widely used in scientific research as a model of hepatotoxicity and to evaluate hepatoprotective agents [2,3]. CCl 4 is converted by cytochrome P450 2E1 to trichloromethyl free radical (CCl 3 •) and trichloromethylperoxy radical (CCl 3 OO•). Both radicals initiate lipid peroxidation and protein deterioration with subsequent damage of the cellular membrane and leakage of intracellular enzymes into the serum. These processes eventually lead to inactivation of the calcium pump with calcium influx and subsequent liver cell death. Moreover, lipid peroxidation and damage of hepatocyte membranes initiated by CCl 4 was reported to be associated with the release of inflammatory mediators such as tumour necrosis factor (TNF)-α from activated hepatic macrophages, which potentiate CCl 4 -induced hepatic injury [3,4].
Nymphaea alba L. (N. alba), known as the European water lily, White Lotus, or Nenuphar, is an aquatic flowering plant of the family Nymphaeaceae. N. alba was widely used in Indian folk medicinal products as an antiseptic, an astringent, radical scavenger, in burning and in insomnia while rhizomes are applied externally as a rubefacient [5]. Previously published studies reported the antioxidant, anti-inflammatory as well as hepatoprotective effect of N. alba flowers [6][7][8]. These effects may result from the phenolic constituents, including ellagic and gallic acid and their methyl and ethyl esters and flavonoids as aglycones of quercetin, kaempferol, isokaempferide, apigenin and their glycosides previously identified in the flowers [9,10]. A recent study on the rhizomes revealed the presence of hydrolysable tannins, glycosylated phenolic acids and flavonoids. The methyl and ethyl gallate as well as pentagalloyl glucose, the main constituents identified, showed powerful cytotoxic activity against liver cell carcinoma [11]. Leaves of the white flowered water lily have been evaluated for their cytotoxic, antiproliferative and anxiolytic activities [12][13][14].
The broad range of traditional uses along with the previous reports concerning the hepatoprotective effect of N. alba flowers [7] as well as other Nymphaea species [15], and the absence of any reports about the phytochemical profile of N. alba leaf, aroused our interest in N. alba as a source of bioactive compounds. This study represents the first detailed chemical investigation of N. alba leaf that demonstrates the presence of a variety of free and conjugated forms of ellagic acid and ellagitannins tentatively identified by high-resolution electrospray ionisation mass spectrometry (HRESI-MS/MS) in the aqueous ethanolic extract.
Hepatoprotective, antioxidant and anti-inflammatory activity of the N. alba leaf extract against CCl 4 -induced hepatotoxicity have also been studied and showed promising results.

Plant material
Leaves of N. alba L. were collected from El Orman Gardens, Giza, Egypt, in November, 2013 during the flowering stage. Authentication of the plant was performed by Dr. Therese Labib Youssef (consultant in plant taxonomy, Ministry of Agriculture). A voucher specimen (RS006) is deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, October University for Modern Science and Arts, Egypt.

Extraction
The powdered air-dried leaves of N. alba (300 g) were exhaustively extracted with aqueous ethanol (70% v/v) under reflux. After filtration, the aqueous ethanolic extract (AEE) was evaporated to dryness in vacuum at 40°C to yield 33 g.

Identification of Phenolic Compounds of AEE of N. alba by LC-HRESI-MS/MS
The method was performed according to Hassaan et al. [16]. Injection volume was 10 μ L. The solvents were: (A) 2% acetic acid (pH 2.6) and (B) 80% methanol, 2% acetic acid, and pH 2.6. The gradient elution was from 5 to 50% B at 30°C at a flow rate of 100 μ L/min. The ionization technique was an ion spray (pneumatically assisted electrospray). Spectra were recorded in positive and negative ion modes between m/z 120 and 1,500 with capillary voltage, 4000 V and heated dry nitrogen gas (temperature, 200°C) and flow rate 10 L/min. The gas flow to the nebulizer was set at pressure 1.6 bar. For collision-induced dissociation (CID) MS/MS measurements, the voltage over the collision cell varied from 20 to 70 eV. Argon was used as the collision gas. Data analysis software was used for data interpretation. Sodium formate was used for calibration at the end of the LC/MS run. Interpretation for ESI-MS was performed by Xcalibur 2.2 SP1 software from Thermo Scientific (Berlin, Germany).

Gas Liquid Chromatography (GLC) of Unsaponifiable Matter (USM) and Fatty Acid Methyl Ester (FAME)
Powdered air-dried leaves (100 g) were exhaustively extracted with petroleum ether (60-80°C). The petroleum ether extract was filtered and evaporated under reduced pressure. The petroleum ether extract (1 g) was saponified by refluxing with ethanolic KOH (20%) at 60°C for 2 h and then exhaustively extracted with ether. The combined ethereal extracts were washed, dehydrated over anhydrous sodium sulphate, evaporated to dryness and then analysed as unsaponifiable matter (USM) for the hydrocarbon and sterol contents. The saponified extract was acidified with HCl (5 N) and then extracted several times with ether. The combined ethereal extracts were evaporated to dryness, esterified into fatty acid methyl esters (FAMEs) by reflux with MeOH:H 2 SO 4 (50:3) and extracted with ether [17].
The ether extracts of the USM and FAME fractions were analysed by GLC against the available authentic standards. Identification of hydrocarbons, sterols, and fatty acid methyl esters was carried out by comparing retention times of the peaks with those of the available authentic standards. FAMEs were analysed on a 70% Thermo Scientific Trace TR-FAME gas chromatographic (GC) capillary column packed with 70% Cyanopropyl Polysilphenylenesiloxane, 30 m x 0.25 mm id. The injector and detector temperatures were set at 250 and 300°C, respectively. The temperature was increased 70°C to 190°C at a rate of 8°C /min. Nitrogen was used as carrier gas (30 ml/min). USM was analysed on a Capillary HP6890 series, 1.5 m × 4 mm i.d. The injector and detector temperatures were set at 250 and 300°C, respectively. The temperature was increased from 70 to 270°C, at a rate of 10°C /min. Nitrogen was used as the carrier gas (30 mL/min).

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
A weighed amount of AEE was dissolved in methanol (100 μg/mL), screened for its free radical scavenging activity using the stable free radical DPPH, and then measured spectrophotometrically. The absorbance was measured at 517 nm and carried out in triplicate [18]. Radical scavenging activity was calculated by the following formula: DPPH scavenging effect (%) = [(A0 -A1)/A0) × 100], where A0 was the absorbance of the control reaction, and A1 was the absorbance of the sample [19]. The concentration of sample required to scavenge 50% of the DPPH was calculated from a graph plotted for the % inhibition against the concentration in μg/mL. Ascorbic acid was used as standard.

Hepatoprotective activity Experimental animals
Eight-week-old male Wistar albino rats (200-220 g) were purchased from the National Institute of Ophthalmology, Egypt. The animals were kept in the animal house, October University for Modern Sciences and Arts (MSA), Egypt. All animals were kept in a pathogen-free facilities under standard laboratory conditions (temperature 25 ± 2°C and 12 h light/12 h dark cycle) with free access to food and water. The animals were housed in groups of four in plastic cages with sawdust bedding. Experimental work was carried out in laboratories at MSA University, Egypt. Procedures involving animals and their care were in conformity with the institutional guidelines (Approval number of ethics committee, MSA University, EC 10 PG10/2011) and in compliance with national and international laws on the care and use of laboratory animals.

Experimental design
Two different doses of N. alba (100 and 200 mg/kg) were tested for their hepatoprotective effect against CCl 4 -induced hepatotoxicity. Doses and route of administration selection were according to previously published studies [12,13]. Hepatotoxicity was induced by injection of a single intraperitoneal (I.P.) dose of CCl 4 (0.5 ml/kg) [20].
A total of 40 rats were randomly divided into five groups ( n Twenty-four hours after the last dose of treatments, blood samples were collected from the retro-orbital plexus. Serum was separated by centrifugation and stored at −80°C . Rats were sacrificed; livers were excised, rinsed in icecold saline and blotted dry. Slices of liver tissue were fixed in 10% neutral formalin for histopathological examination and immunostaining of caspase-3. The rest of the liver tissue was weighed and homogenized in phosphate buffer saline to prepare 10% homogenate and stored at −80°C.

Assessment of biochemical markers of hepatic injury
Biochemical parameters reflecting liver functions such as serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT) and total bilirubin were estimated using commercially available kits, according to the manufacturer instructions (Spectrum, Egypt).

Assessment of oxidative stress in the liver
Liver malondialdehyde (MDA) and reduced glutathione (GSH) contents, catalase (CAT) and superoxide dismutase (SOD) activities and total antioxidant capacity (TAC) were assessed spectrophotometrically using commercial kits supplied by Bio-diagnostic (Bio-diagnostic, Egypt).

Estimation of inflammatory cytokine, TNF-α
TNF-α content was measured in liver homogenate using an ELISA kit (BioLegend ELISA MAX™ Deluxe kit; BioLegend, San Diego, CA, USA). The assay was performed according to the manufacturer's protocol.

Histopathological examination of the liver
Liver specimens in 10% neutral formalin were embedded in paraffin and cut into 4 μm thick sections. Sections were stained with haematoxylin and eosin (H&E) and examined under a light microscope for histological changes.

Immunohistochemical staining for caspase-3 in liver
Caspase-3 expression in the liver was detected by immunostaining of sections prepared from formalin-fixed, paraffinembedded livers using caspase-3 detection kits according to the manufacturer instructions. The intensity of caspase-3 immunostaining was assessed as follows: 0none, 1mild, 2moderate and 3strong. The Immunohistochemical histological score (H-score) was calculated by multiplying the intensity by the percentage of caspase-3 positive cells, creating a range of possible scores of 0-300 [21].

Statistical analysis
Data from animal work are expressed as the mean ± standard error of the mean (SEM). Comparisons between different groups were carried out by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test. The level of significance was set at p < 0.05. Graphpad software instat (version 2) was used to carry out statistical analysis.

Phytochemical investigation HRESI-MS/MS analysis of N. alba AEE
The chemical constituents in N. alba AEE were identified and characterized in both negative and positive ESI modes. The retention times and fragmentation patterns of the identified compounds are listed in Table 1. Compounds were tentatively identified based on matching their masses and fragmentation pattern with the literature information and ChemSpider. MS fragmentation interpretation is not discussed except when of special interest. Table 1 outlines 53 compounds, among them 42 hydrolysable tannins, three simple phenolic acids, and eight flavonoids were detected.

Estimation of USM and FAME
Nine hydrocarbons, two sterols and eleven fatty acids were identified in N. alba AEE. The percentage content of individual hydrocarbons and fatty acids are summarized in Tables 2 and 3. The percentage of identified hydrocarbons was estimated as 94.9% while the sterol content represented 5.03%. n-Tetracosane was determined as the major hydrocarbon (59.6%), n-tetratriacontane was detected as the second most abundant hydrocarbon (18.96%) while βsitosterol was of significant percentage (3.5%). By comparison with the FAME standards, the percentage of the saturated fatty acid represents 49.3% where the major saturated fatty acid was palmitic acid (40.8%) while the major unsaturated fatty acids were linolenic acid (24.5%), linoleic acid (16.8%) and palmitoleic acid (8.5%).

Hepatoprotective activity Effect of N. alba on liver function parameters
Serum level of liver functions parameters; ALT, AST, GGT, ALP and total bilirubin were significantly increased in CCl 4 -intoxicated rats compared with normal level (P < 0.05; Table 4). Treatment with N. alba (100 and 200 mg/ kg) resulted in significant decrease of ALT, AST, GGT, ALP and total bilirubin compared with CCl 4 -intoxicated rats in a dose dependant manner (P < 0.05; Table 4). Similarly, silymarin significantly improved the liver function parameters compared with CCl 4 group (P < 0.05; Table 4). Silymarin significantly increased the liver GSH content and TAC (151.7 and 97.3%, respectively) compared with the CCl 4 group (P < 0.05; Figs. 1 and 4, respectively). The activities of SOD and CAT improved significantly (71 and 73.8%, respectively) by treatment with silymarin compared with the CCl 4 group (P < 0.05; Figs. 2 and 3, respectively).  The hepatic content of MDA decreased significantly (51.8.7%) compared with the CCl 4 group (P < 0.05; Fig. 5).

Effect of N. alba on hepatic content of TNF-α
Hepatic content of TNF-α was significantly increased in CCl 4 group compared with control rats. Treatment with N. alba (100 and 200 mg/kg) resulted in significant decrease of TNF-α in a dose dependant manner (Fig. 6). Silymarin significantly improved the hepatic content of TNF-α compared with CCl 4 control group (Fig. 6).

Histopathological examination of hepatic tissue
Livers excised from a control group showed a normal architecture of hepatocyte lobules with normal central and portal areas and normal hepatocytes (Fig. 7a). The liver samples from CCl 4 -intoxicated rats showed severe feathery degeneration and necrosis of hepatocytes (Fig. 7b).
The portal area showed severe infiltration by lymphocytes and dilation of the central vein (Fig. 7c). Rats treated with low dose of N. alba (100 mg/kg; P.O.) for 5 days showed slight improvement of the histopathological features of the liver compared with the CCl 4 -intoxicated group (Fig. 7d). However, a high dose of N. alba (200 mg/kg; P.O.) and silymarin showed marked advances in the liver features and disappearance of the feathery degeneration of hepatocytes ( Fig. 7e and f, respectively).

Discussion
The hyphenated HPLC-MS technique is an important method used for identifying complex mixtures, especially the phenolics in the crude extracts or the fraction found in the plant, either by using standard compounds (cochromatography) or by comparing mass spectra obtained with the literature (tentative identification) [22]. A chemical characteristic of the order Nymphaeals, which includes the family Nymphaeaceae, is the occurrence of significant amounts of gallic acid and ellagic acid [23]. Ellagitannins attracted considerable attention because of their vast structural diversity and biological activity, including antioxidant, antiviral and antitumor activity [24,25]. N. alba flowers and rhizomes are known for their high phenolic content [9][10][11], while leaves have never been well studied before. In this study, N. alba AEE was demonstrated as a very rich source of phenolic compounds where, hydrolysable tannins were the main polyphenols identified (forty-two compounds) distinguished by their characteristic fragment ion spectra yielding losses of galloyl (m/z 152), gallate (m/z 170) and esters of hexahydroxydiphenic acid (HHDP) residues (m/z 302), while the common loss of 44 amu indicates the presence of a free carboxylic group (COOH) [26,27].
A total of twenty-six ellagitannins were tentatively identified. HHDP and a polyol and in some cases, gallic acid represent the majority of Nymphaea constituents.    [32]. Two compounds with m/z 951 were detected at different retention time (Peak 38 and 50) which significantly differ in their fragmentation pattern, indicating the presence of isomeric structures which is common with ellagitannins. They were tentatively identified as granatin B and geraniin respectively [33,34].
Three lagerstannins previously identified in Lagerstroemia speciosa were tentatively identified with the presence of gluconic acid. The common loss of 44 amu indicates the presence of a free carboxylic group [35]. Peak  [30,35].
Analysis of peaks 11, 27, 30 and 47, yielded intense product ions resulting from the loss of the HHDP and/or galloyl moiety. These peaks were tentatively identified as Table 4 Effect of N.alba AEE on CCl 4 -induced changes in the liver function parameters in rats. Rats were intoxicated with CCl 4 (0.5 ml/kg; I.P.) and treated with N. alba (100 and 200 mg/kg; P.O.) and silymarin (100 mg/kg; P.O.) for 5 days. ALT, AST, total bilirubin, GGT and ALP were measured Data are presented as the mean ± SEM, n = 8. a Significant difference from control group; P < 0.05. b Significant difference from CCl 4 group; P < 0. Gallic acid and its derivatives were also tentatively identified where gallic acid appeared at peak 53 while peak 39 was tentatively identified as dehydrated tergallic acid with a pseudomolecular ion [M-H] at m/z 613 and Fig. 3 The effect of N. alba AEE on the liver CAT activity in CCl 4intoxicated rats. Rats were intoxicated with CCl 4 (0.5 ml/kg; I.P.) and treated with N. alba (100 and 200 mg/kg; P.O.) and silymarin (100 mg/ kg; P.O.) for 5 days. CAT was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. a Significant difference from control group; P < 0.05. b Significant difference from CCl 4 group; P < 0.05 Fig. 4 The effect of N. alba AEE on the liver TAC in CCl 4 -intoxicated rats. Rats were intoxicated with CCl 4 (0.5 ml/kg; I.P.) and treated with N. alba (100 and 200 mg/kg; P.O.) and silymarin (100 mg/kg; P.O.) for 5 days. TAC was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. a Significant difference from control group; P < 0.05. b Significant difference from CCl 4 group; P < 0.05  [29,30].
Flavonoids have also been detected where Peak 8 shows m/z at 505 [M-H] and daughter ion at m/z 301 [M-acetyl hexoside], was tentatively identified as quercetin 3-O-acetyl hexoside that was previously identified in the Nymphaea species [40]. Peak 31 has an [M-H] ion at m/z 289 and base peak at m/z 245 [M-H-44] was tentatively identified as catechin or epicatechin by Pérez-Magariño et al., [41].
Beside the phenolic content N. alba extract appeared also as rich source of fatty acid. Essential fatty acids (EFAs) such as linolenic, linoleic and oleic acids help to raise HDL cholesterol, supporting cardiovascular, reproductive and immune systems. N. alba extract contains several essential fatty acids as linoleic (16.78%) and linolenic acid (24.45%), and, therefore, has a potential nutritional value in agreement with Eromosele and Eromosele, [42]. In addition, N. alba provided a rich source of β-sitosterol (5%), which is reported to reverse the impairment of the glutathione/oxidized glutathione ratio induced by phorbol esters in macrophage cultures with the increase in manganese superoxide dismutase and glutathione peroxidase activities and the decrease in catalase activity [43].
Oxidative stress plays a crucial role in the development of the aging process and some chronic diseases [21]. The antioxidant potential of medicinal plants is attributed to the redox properties of the phenolic compounds and there are several reports that correlate the total phenolic content to the antioxidant activity [44][45][46]. N. alba was shown as a potent radical scavenger with low IC 50 (5.2 ± 0.3 μg/mL) compared with ascorbic acid. This high radical scavenging activity suggests the ability of N. alba to reduce oxidative stress.
In this study, the hepatoprotective effect of N. alba AEE against CCl 4 -induced hepatotoxicity was demonstrated for the first time in a dose-dependent manner. This protection was reflected biochemically by the significant improvement in serum levels of ALT, AST, ALP and GGT, indicating the ability of N. alba AEE to protect hepatocytes against the deleterious effects of CCl 4 . Furthermore, the significant decrease in the serum level of bilirubin indicated that bilirubin was taken up into the liver as a function of a healthy hepatocyte. The hepatoprotective effect of the extract against CCl 4 -intoxication was further supported by histopathological examinations which showed considerable improvement of the histopathological features of the liver with N. alba treatment.
Silymarin is a unique flavonoid complex that has been reported to possess strong hepatoprotective properties and commonly used in experiments as a reference hepatoprotective substance [47]. Silymarin has a broad array of in vitro and in vivo activities such as anti-inflammatory, antiapoptotic and antioxidant [48]. Our results showed that silymarin protects against CCl 4 -induced hepatotoxicity as reflected by the significant improvement in the liver enzymes and bilirubin as well as enhancement of the histopathological features of the liver which was in agreement to previous studies [46,47]. The protective effect of a high dose of N. alba (200 mg/kg) against hepatotoxicity is comparable with the effect observed with silymarin (100 mg/kg) which indicates a strong hepatoprotective property of the high dose of N. alba.
Caspase-3 is a protein that plays a vital role in apoptosis [49]. In the present study caspase-3 was extensively expressed in the liver excised from CCl 4 -intoxicated rats denoting the correlation between CCl 4 induced hepatotoxicity and the high level of apoptosis of the hepatocytes as previously reported [50]. N. alba decreased the level of caspase-3 expression while the effect of the high dose of N. alba is similar to that of silymarin as both drugs showed negative staining for caspase-3. Consequently, the protective effect of N. alba extract against CCl 4 is mediated, in part, by inhibition of apoptosis through caspase-3 dependant pathway.
Oxidative stress has been shown to play a pivotal role in liver injury induced by CCl 4 [51,52]. Our results showed an obvious disturbance in oxidant-antioxidant balance of the liver subjected to CCl 4 where injection of CCl 4 increased the degree of lipid peroxidation as indicated by the significant increase in MDA level in the liver homogenate. Both non-enzymatic and enzymatic antioxidant defence mechanisms were deteriorated in CCl 4 -injected group. The oxidant-antioxidant status of the liver excised from CCl 4 -intoxicated rats was significantly improved by treatment with N. alba in a dosedependent manner. These findings imply a profound in vivo antioxidant effect of N. alba. These results are consistent with the studies documented by Khan and Sultana, [12,13], who reported that N. alba extract suppresses chemically-induced oxidative stress and kidney damage in Wistar rats.
The strong antioxidant activity of silymarin has been documented previously in several studies [47,48]. In the present study the antioxidant activity of the high dose of N. alba is comparable to the antioxidant activity of silymarin.
TNF-α is an important inflammatory mediator that has been shown to be involved in diverse pathological processes and in our study, TNF-α is elevated significantly in the CCl 4intoxicated group, which was previously reported [51,52]. Treatment with N. alba resulted in a significant decrease in the hepatic content of TNF-α, which is comparable, in its high dose, with silymarin. This result indicates a profound anti-inflammatory effect of N. alba which was in agreement with that reported in models of acetic acid-induced vascular permeability and cotton pellet-induced granuloma. In both models, N. alba exhibited an anti-inflammatory effect in a dose-dependent manner, which can be comparable with that of diclofenac sodium [6].