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.
Results
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 β-sitosterol 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%).
1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
AEE showed strong DPPH scavenging activity as indicated by low IC50 (5.2 ± 0.3 μg/mL) and LC90 (9.1 ± 0.27 μg/mL) compared with ascorbic acid (12 ± 3.5 μg/mL).
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 CCl4-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 CCl4-intoxicated rats in a dose dependant manner (P < 0.05; Table 4). Similarly, silymarin significantly improved the liver function parameters compared with CCl4 group (P < 0.05; Table 4).
Effect of N. alba on oxidant status of the liver
Injection of CCl4 resulted in depletion of hepatic GSH content (59.7%), decrease in the activities of SOD and CAT (59.9 and 44.9%, respectively) and decline in TAC of the liver (65.7%) (P < 0.05; Figs. 1, 2, 3 and 4, respectively) compared with control group. Also, CCl4 significantly increased the liver lipid peroxidation product, MDA (249.1%) (P < 0.05; Fig. 5) compared with control group.
The effect of N. alba AEE on the liver content of GSH in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. GSH was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
The effect of N. alba AEE on the liver SOD activity in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. SOD was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
The effect of N. alba AEE on the liver CAT activity in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
The effect of N. alba AEE on the liver TAC in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
The effect of N. alba AEE on the liver content of MDA in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. MDA was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
Treatment of CCl4-intoxicated rats with N. alba (100 and 200 mg/kg) significantly increased the liver content of GSH (55.1 and 143.4%, respectively; P < 0.05; Fig. 1) compared with the CCl4 group. N. alba (100 and 200 mg/kg) significantly enhanced the enzymatic activities of both SOD (79.1 and 111.1%, respectively) and CAT (49.2 and 75.5%, respectively) (P < 0.05; Figs. 2 and 3, respectively) compared with the CCl4 group. Furthermore, TAC of the liver increased significantly (84.1 and 173.5%) by treatment with the N. alba (100 and 200 mg/kg, respectively) (P < 0.05; Fig. 4). On the other hand, treatment with N. alba (100 and 200 mg/kg) significantly decreased liver MDA content (38.2 and 67.6%), respectively, compared with the CCl4 group (P < 0.05; Fig. 5).
Silymarin significantly increased the liver GSH content and TAC (151.7 and 97.3%, respectively) compared with the CCl4 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 CCl4 group (P < 0.05; Figs. 2 and 3, respectively). The hepatic content of MDA decreased significantly (51.8.7%) compared with the CCl4 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 CCl4 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 CCl4 control group (Fig. 6).
The effect of N. alba AEE on the liver content of TNF-α in CCl4-intoxicated rats. Rats were intoxicated with CCl4 (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. TNF-α was determined in the liver homogenate. Data are presented as the mean ± SEM, n = 8. aSignificant difference from control group; P < 0.05. bSignificant difference from CCl4 group; P < 0.05
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 CCl4-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 CCl4-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).
Representative photomicrographs of histopathological examination of the liver. a Liver of control rats (b and c) Liver of rats intoxicated with CCl4 (0.5 ml/kg; P.O.) showing severe feathery degeneration of hepatocytes and lobular necrosis (B) and portal lymphocytic infiltration (c). d Liver of rats intoxicated with CCl4 and treated with N. alba (100 mg/kg; P.O.) showing slight improvement of feathery degeneration of hepatocytes. e Liver of rats intoxicated with CCl4 and treated with N. alba (200 mg/kg; P.O.) showed marked improvement of the histopathological features. f Liver of rats intoxicated with CCl4 and treated with silymarin (100 mg/kg; P.O.)
Immunohistochemical staining of caspase-3
Liver excised from rats injected with CCl4 showed high caspase-3 expression (H score = 60; Fig. 8b) while, caspase-3 was negatively stained in the control group (H score = 0; Fig. 8a). Caspase-3 expression was decreased in CCl4-intoxicated rats treated with low dose of N. alba (100 mg/kg; H score = 20; Fig. 8c). CCl4-intoxicated rats received the high dose of N. alba as well as those received silymarin (Figs. 8d and e, respectively) showed negative staining for caspase-3 (H score = 0).
Representative photomicrographs of immunohistochemical staining of caspase-3. a Liver of control rats, (b) Liver of rats intoxicated with CCl4 (0.5 ml/kg; P.O.), (c) Liver of rats intoxicated with CCl4 and treated with N. alba (100 mg/kg; P.O.), (d) Liver of rats intoxicated with CCl4 and treated with N. alba (200 mg/kg; P.O.), and (e) Liver of rats intoxicated with CCl4 and treated with silymarin (100 mg/kg; P.O.)
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 [9–11], 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. Peak 1 with a precursor ion at m/z 481 was identified as HHDP-glucose [28, 29]. The presence of HHDP was supported by the formation of m/z 301 in the negative ionization and 303 in the positive.
Peak 31, shows m/z at 633 [M-H] and fragment ions at m/z 301 [M-H-152-180], 589 [M-H-44], 481 [M-H-152] was tentatively identified as a galloyl-HHDP-glucose (corilagin) isomer, agreeing with Fischer et al., [30] and Barros et al. [31]. Peak 32 shows m/z at 783 [M-H] and fragments at m/z 481 [M-H-302] and 301 [M-H-302-180] was identified as pedunculagin (Bis HHDP hexoside), the release of one HHDP molecule yielded 481 (peak 1) [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 35 shows m/z at 649 [M-H] and fragments at m/z 603 [M-H-44], 631 [M-H-18], 469 [M-H-180], 451 [M-H-44-152] and 301, which was identified as lagerstannin C (galloyl HHDP-gluconic acid). Peak 9 presents m/z at 799 [M-H] and fragments at m/z 755 [M-H-44], 497 [M-H-302] and 301 [M-H-302-196] and identified as lagerstannin A (Bis-HHDP-gluconic acid). Peak 37 shows m/z at 949 [M-H] and fragments at m/z 647 [M-H-302], 905 [M-H-44], 629 [M-H-44-152], 477 [M-H-302-170], 301 [M-H-302-170-176]. This peak was tentatively identified as Lagerstannin B (flavogalloyl-HHDP-gluconic acid) [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 phyllanthusiin U, B, C and chebulagic acid detected with [M-H] at m/z 924, 969, 925 and 953, respectively and previously identified in Phyllanthus urinaria [36, 37]. Peak 28 with a pseudomolecular ion [M-H] at m/z 933.06 and fragment ions at m/z 915, 631, 451 and 301 were in agreement with the fragmentation pattern attributed to castalagin [38]. The release of one HHDP or ellagic acid moiety (302 Da) from castalagin generated Peak 33 with an [M-H] at m/z 631, showing the typical ellagic acid fragments at m/z 299 and m/z 271 and tentatively identified as castalin [28, 30].
The free ellagic acid was confirmed by its MS data and MS/MS fragmentation (peak 5), having m/z at 301 [M-H] in the negative mode and fragment ions at m/z 275, 257, 247, 229 and 185 [38]. Peaks 17, 48, 49 were tentatively identified as the glycosylated forms of ellagic acid with [M-H] at m/z 463, 433 and 447, respectively, showing the characteristic fragments of ellagic acid at m/z 301 and 275 in addition to the characteristic losses of a hexosyl, pentosyl and rhamnosyl residue, so, identified as ellagic acid hexoside, pentoside and rhamnoside respectively [30]. Peak 42 presents m/z at 469 [M-H] and a fragment at m/z 425, was tentatively identified as valoneic acid dilactone, a compound that often occurs in plants containing ellagitannins [39]. While peak 12 represented a dimer of valoneic acid dilactone with [M-H] at m/z 939 and main fragments at 469 and 425.
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 fragment ions at m/z 569 [M-H-44], 461 [M-H-152] and 299 [M-H-152-162] [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 [44–46]. N. alba was shown as a potent radical scavenger with low IC50 (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 CCl4-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 CCl4. 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 CCl4-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, anti-apoptotic and antioxidant [48]. Our results showed that silymarin protects against CCl4-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 CCl4-intoxicated rats denoting the correlation between CCl4 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 CCl4 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 CCl4 [51, 52]. Our results showed an obvious disturbance in oxidant-antioxidant balance of the liver subjected to CCl4 where injection of CCl4 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 CCl4-injected group. The oxidant-antioxidant status of the liver excised from CCl4-intoxicated rats was significantly improved by treatment with N. alba in a dose-dependent 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 CCl4-intoxicated 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].