Part 1 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.

Hepatotoxicity is a prevalent problem worldwide. Carbon tetrachloride (CCl₄) 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 CCl₄ 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]. CCl₄ is converted by cytochrome P450 2E1 to trichloromethyl free radical (CCl₃∙) and trichloromethylperoxy radical (CCl₃OO∙). 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 CCl₄ was reported to be associated with the release of inflammatory mediators such as tumour necrosis factor (TNF)-α from activated hepatic macrophages, which potentiate CCl₄-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 [6–8]. 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 [12–14].

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 CCl₄- 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.

Liquid chromatography coupled with High-resolution electrospray ionisation mass spectrometry (LC-HRESI-MS/MS)

LC-HRESI-MS/MS was performed on a Bruker Micro-TOF-Q Daltonics (API) Time-of-Flight mass spectrometer (Bremen, Germany), coupled to a 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with a high performance autosampler, binary pump, and PDA detector G 1314 C (SL). Chromatographic separation was performed on a Superspher 100 RP-18 (75 × 4 mm i.d.; 4 $\mu$m) column (Merck, Darmstadt, Germany).

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 $\mu$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 $\mu$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:H₂SO₄ (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 Polysilphenylene-siloxane, 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 CCl₄-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 CCl₄ (0.5 ml/kg) [20].

A total of 40 rats were randomly divided into five groups ($n$ = 8). Group I (Control): received vehicles. Group II (CCl₄): received CCl₄ (0.5 ml/kg; I.P.) once. Group III (N. alba low dose): received CCl₄ (0.5 ml/kg; I.P.) + N. alba extract (100 mg/kg; P.O.) 24 h after CCl₄ for 5 days. Group IV (N. alba high dose): received CCl₄ (0.5 ml/kg; I.P.) + N. alba (200 mg/kg; P.O.) 24 h after CCl₄ for 5 days. Group V (Silymarin): received CCl₄ (0.5 ml/kg; I.P.) + silymarin (100 mg/kg; P.O.) 24 h after CCl₄ for 5 days. Treatments were given at 10 a.m. 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 ice-cold 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, paraffin-embedded livers using caspase-3 detection kits according to the manufacturer instructions. The intensity of caspase-3 immunostaining was assessed as follows: 0 – none, 1 – mild, 2 – moderate and 3 – strong. 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].