Abstract: Urtica pilulifera is classified as a popular plant used in remedy. The effect of petroleum ether and 20% methanol extracts for different parts (herb, root and seeds) of this plant were determined in albino mice liver pretreated with 200 ppm (0.25 mL/70-80 g mouce) on protein, lipid and phospholipids (PLs) concentrations as well as phospholipid compositions. Dendrogram for SDS-PAGE showed dissimilarity in mice groups between different extracts and parts compared with control group. Lipid and PL concentrations were decreased. Petroleum ether extracts were more effective in lipid concentration while aqueous methanol extracts were more effective for PL concentration. On the other hand, the two extracts increase phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol concentrations. The increase in PLs composition was more pronounced in Pet E. extracts than aqueous methanol extracts. In conclusion, Urtica pilulifera can be used for many therapeutic goals. It can improve liver tissue by increasing protein concentration, reduce lipid in lipidemic liver and remodels phospholipids compositions which can be used in treatment of cancer diseases.
Introduction
Characterization of protein isolates from liver depends on the source of proteins and the compounds affecting it (Zancani et al., 2004). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is currently one of the most commonly used techniques for the characterization and analysis of proteins. It has been extensively used to determine the molecular weights of polypeptide chains and to establish the homogenicity of protein fraction. This method is usually faster than DNA-hybridization method and elaborates phenotypic comparisons between different extracts.
Phospholipids are the major lipid components of cell membrane. Few human diseases have been described to be due to a defect in phospholipids biosynthesis. The benefits of PLs were referred to their dynamic role in the function of membrane protein modulating enzymatic activities which was proved to help normal function and to protect against atherosclerotic heart disease (Yokoyama, 2006). Phospholipids (PLs) are required for providing the essential components of biological membranes and they are important precursors of signaling molecules. One would anticipate that a null mutation in an enzyme required for the biosynthesis of a PL type would be compatible with life, particularly if that mutation resulted in complete elimination of that PL. Phospholipids bilayer does not only surround the protein membrane but it also controls their environments by regulating the fluidity of membranes; furthermore some other functions of biomembranes include their responses to hormones (Hirata and Axelrod, 1980; Prasad and Kumar, 2005). Cellular functions associated with membranes are expected to be modulated by any modification in Pls (Schroeder, 1981; Nakano et al.,1982; Dobrzynska et al., 2005). It was determined that Pls efficaciously prevented or reduced gastric cancer cell peritoneal adhesion formation in numerous studied animals (Jansen et al., 2004).
The objective of the present study is the characterization of proteins in mice liver treated with Urtica pilulifera crude extracts. Petroleum ether and methanol extracts from different parts of the plant were used in this study to determine the relationship between the whole cell protein fingerprints in control and treated livers. Furthermore, the effects of the two extracts on the amount of total lipid and phospholipids and their compositions in liver tissues were also studied. The most abundant species of PLs are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI), so study in PL composition includes these four constituents.
Materials and Methods
All Chemicals are of analytical grade products of Sigma
Animals
Male albino mice (70-90 g).
Experimental Design
The mice were fed with normal diet. The animals were divided into 7 groups
of 7 animals; each three groups were treated with petroleum ether extract from
different parts of Urtica pilulifera (herb, root and seeds), the other
three groups were treated with methanol extract from the same organs of the
plant. The last group was served as control and was treated with saline (0.25
mL saline). The six treated groups were injected with 0.5 mL 200 ppm extract
intraperitoneal for 10 days. Then the animals were sacrificed and livers were
removed for analysis.
Tissue Homogenate
The liver was homogenized in deionized distilled water (1:10) W/V by electric
homogenizer and then centrifuged for 10 min at 3000 rpm in a cooling centrifuge.
The supernatant was used for protein electrophoresis and for protein determination.
Protein Electrophoresis
Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of
the sample homogenates of the seven groups (one control and 6 treated groups)
was carried out as described by Laemmli (1970) using prestained high molecular
weight standered marker (Biored, USA). After electrophoresis, the gel was stained
with coomassie brilliant blue and destained by acetic acid and methanol.
Dice index of similarity was determined in each isolate (Dice, 1945) and a dendrogram was then constructed (advanced American Biotechnology, UPGMA, USA).
Lipid Extracts
Total lipids were estimated in mice liver tissues by the method of Duncan
et al. (1987).
Analysis of Phospholipid Compositions
HPTLC of PC, PE, PA and PI on silica gel was carried out by four fold automated
multiple developments with chloroform-methanol-2-propanol-triethylamine-0.25%
aqueous KCl 60:18:50:36:9, chamber precondition with 0.1 N NH4OH.
Visualization by spraying with 1) 1,6-diphenyl-1,3,5-hexatriene and after intermediate
drying with 2) molybdenum blue reagent was carried out according to Dillmer
and Lester. Quantification was performed by densitometry at 370/>460 nm.
Visual detection limits of down to 10 ng were obtained (Muthing and Radloff,
1998).
Statistical Analysis
Data are expressed as mean±SD and statistically analyzed using analysis
of variance (ANOVA) and Least Significant Difference (LSD) post hock test.
Results
Liver proteins in treated mice had revealed 7 to 9 protein bands in Pet E extracts in the different parts and 9 to 12 protein bands in ME of molecular weight varying from 192 to 7.1 KDa had also be obtained (Table 1). A protein band of 173.53 KDa was present only in herb petroleum ether extract treated mice liver tissue homogenates after 10 days of injection while 4 bands appeared in mice liver homogenates treated with root and seed extracts (192, 125.9, 115.36 and 9.26 KDa). In mice liver treated with herb methanol extract, there are four protein bands that are different from those obtained from control group; three bands look like those found in liver mice homogenates treated with root and seeds of the Pet E. extracts (125.9, 115.36 and 9.26 KDa) and only one band looks like that found in root Pet E. extract treated mice liver homogenates. Furthermore, there are other four protein bands that appeared in root and seed ME treated mice liver homogenates that are not found in control liver (148.5, 40.1, 22.6 and 9.26 KDa).
Figure 1 illustrates the cluster analysis on the basis of similarity coefficient values and average likage method of groups formed by SDS-PAGE protein patterns.
Effect of U. pilulifera extracts on lipid and phospholipid concentrations in mice liver tissue is presented in Table 2. It was found that all extracts decreased lipid and phospholipids concentrations except in seed ME where lipid concentration increased. The phospholipid compositions in liver tissues were represented in Table 3. The data showed that all determined types of PL, whish are more dominant in Pet E extract, increased.
For simplicity, it was of interest to present the obtained data of phospholipid compositions (mg/gt) in terms of percentage compositions (Table 4). The PL composition ratio R in the liver of mice is expressed by this equation:
| (1) |
| Table 1: | Effect of Urtica pilulifera extracts on mice liver protein profile |
| Table 2: | Effect of Urtica pilulifera extracts on lipid and phospholipid concentrations of mice liver tissues |
| Table 3: | Phospholipid composition levels (μg g-1 tissue) in mice liver tissues injected with Urtica pilulifera extracts |
| Each reading is mean of seven samples | |
| Table 4: | Phospholipid composition percents in mice liver tissues injected with Urtica pilulifera extracts |
Discussion
The effect of Urtica pilulifera extracts on protein, lipid and phospholipids (PL) levels depend on two main factors that are; the type of the used solvent and the plant part which was extracted (herb, root or seeds). Injecting the animals with petroleum ether and methanol extracts of Urtica markedly affected all parameters in mice liver. The petroleum ether (Pet E) extracts induced higher change than that of the methanol extracts. This effect may be due to its higher selectivity towards certain biological components. Although methanol is more polar and hence it extracts the polar compounds from the plant tissue, some of these components antagonize with each other; consequently its effect is comparatively low.
SDS-PAGE analysis indicates the multiple differences in protein expression as mutants between different plant extracts which may lead to physiological change in the animal life (Plosch et al., 2004).
By cluster analysis of tissue homogenate pattern of the basic similarity coefficient values and average linkage method, the proteins analysis showed different similarities. The occasional appearance of protein fractions of high molecular masses (192, 173.53, 148.5 125.9 and 115.36 KDa) and others of lower masses (60, 40.1, 22.6, 18.2 and 9.26 KDa) in liver tissue homogenates of mice treated with U pilulifera extracts (Table 1) may be due to gene expression of certain protein masses (Riehemann et al., 1999).
| Fig. 1: | Dendrogram showing similarity based on protein patterns lanes (1-7) |
These results were coincided with Mahmoud et al. (2006) who found that injection of the petroleum ether and hydro alcoholic extracts of Urtica pilulifera to mice showed a marked effect on protein synthesis, some hepatic biotransformation enzyme systems and antioxidant enzymes. Accordingly, there were alterations in the SDS-PAGE separated protein profile in mice liver homogenates ascertained through comparing the identity index of treated and control mice (Fig. 1).
The degree of dissimilarity of protein bands obtained from mice treated with different extracts showed variations in the type and/or the concentration of the compounds that occurred in the different extracts of each part of the plant as well as among the different solvent extracts. The identified compounds in the methanol U. pilulifera herb extracts are also present in Pet E extracts of root and seeds but of different concentrations (Saeed and Omer, 1994; Mahran et al.,1994). This can explain the high similarity of protein bands in liver mice treated with root and seeds Pet E extracts and those of herb methanol extract of the same plant.
The change of protein bands in liver homogenates of mice injected with petroleum ether extracts of roots and seeds may be due to high sterol components in these parts, whereas in herbs no change was detected since herbs contains low sterol components (Drsata, 2002; Durak et al., 2004).
Alteration in protein’s localization might affect its function as well as lipid metabolism and phospholipids transferring and composition (Komatsu et al., 2004; Ma et al., 2004). It was observed that lipid concentration as well as phospholipids in mice liver of all groups are generally decreased (Table 2). The decrease in lipid concentration in liver tissue is correlated with the previous finding of different authors.
Fekete et al. (1990) found that when animal fat diet was mixed with PetE extract of sunflower, the lipid concentration of the animal decreased. The authors concluded that vegetable oil modified the fatty acid composition of blob lipid due to the presence of polyunsaturated fatty acids.
Asai et al. (1999) found that spice extracts has the ability to prevent the deposition of triacylglycerol in the mice liver.
Han et al. (2005) found that Z. offianale Rosce inhibited the hydrolysis of triolein emulsified with phosphatidylcholine by pancreatic lipase in vitro and it reduced the elevation of plasma triacylglycerol levels after oral administration. They also suggested that this extract might inhibit intestinal absorption.
Most of proteins of PL biosynthesis seem to be constitutively expressed (Vance, 1989). Alteration in PL synthesis occurs mainly by changes in the supplied substrates in addition to changes in enzymes activities. This fact can be explained through a change in protein bands (Table 1). The decrease in phospholipids may be due to the presence of polysaccharides extracted with aqueous methanol (Yu and Zhang, 1995) and/or steroid compounds separated by Pet E (Jung et al., 2004); which differ in their concentrations according to the type of the plant parts and the used solvent. It was found that when the S180 cell treated with polysaccharide extracted from Achyranthes bidentata, the sialic acid increased and PL decreased after 24 h. It is also well known that sterol functions to modulate the physical properties of membrane PLs. It is undoubted that there is an appropriate interaction between specific sterols and the particular phospholipids found in a given biological system and a small fraction of it may have an essential regulatory function distinct from PL's role modulating membrane lipid physical properties.
Injection with leaves and root Pet E extracts affect phosphatides concentration, in mice liver tissues, more than those of other extracts. This may be due to the possibility that minor components which are present in herb and root extract, decreased the PL concentrations more than other extract parts.
Furthermore, phospholipids composition levels in mice liver treated with Pet E extracts were higher than those treated with ME (Table 3). The presence of a parochial amount of active compound/s may enhance remodeling of PL constituents (Rujanavech and Silbert, 1986). Moreover, excess of choline, free and combined amino acids in plant extracts might help CDP- choline pathway for the production of PC and/or PE (Vance and Vance, 2005). Phosphatedylcholine plays a role in cholesterol clearance in blood being hydrolysable by PLase-A2 controlling potent lipolysis. The increase level of it, in the liver tissues of treated mice, was in parallel with the absence of 30 KD proteins (Ishigami et al., 2004). This protein is responsible for translocating PC from liver to the luminal side of the canalicular membrane where bile salts extract the PC in the form of micelles into bile (Ruetz and Gros, 1995; Vance and Vance, 2005). The increase in PE also helps in synthesis of PC which in turn help in decrease lipid concentration (Muller et al., 2004).
All iLps exhibit a net negative charge that is determined by both the apoprotien and lipid constituents of the lipoprotein particle thus, their individual charge varies quantitavely (Sparks et al., 1992). The primary anionic lipid lipoprotein particle is PI (Chauhan et al., 1998). It normally affects lipoprotein metabolism both by controlling interfacial interactions and uniquely regulating intracellular signaling pathways. PI increased the remodeling ability of PL molecules provided for macrophage recognition and phagocytosis at any stress (Fabisiak et al., 2000). PE was further proved to be precognitive marker of appoptic cells preceding the development of other biochemical steps of apoptotic machinery (Shvedova et al., 2002; Xie et al., 2005). Increased Phosphatidic Acid (PA) may be due to enhance phospholipids enzymatic activity.
In conclusion, Urtica pilulifera can be used for many therapeutic goals. It can improve liver tissues by increasing protein concentration, reduce lipid level in lipidemic liver and remodels phospholipids compositions which may help in treatment of cancer diseases.