Abstract: Urtica pillulifera extracts were found to exhibit an antioxidant effect on mice. The effect of two doses of aqueous methanolic extract and other two doses of petroleum ether extract of different plant parts was studied in liver homogenates of the animals. The parameters studied were protein, glutathione reduced form, lipid peroxidation level as well as the activities of Glutathione Transferase (GST), Glutathione Reductase (GR), Glutathione Peroxidase (GSHPx) and Superoxide Dismutase (SOD). Methanolic extracts induced greater effect on the measured antioxidant parameters. Among all plant parts the methanolic extract of the herb showed the best effect; where the antioxidant activity enzymes were elevated and the lipid peroxidation was decreased. In conclusion, Urtica pilulifera can be used as natural antioxidant, as a possible food supplement or used in pharmaceutical industry.
Introduction
There is an increasing interest towards medical plants and their active ingradients since 1980's. Some of the underlying motives can be listed as follows; countries lacking developed chemical industries are searching for affordable treatment by using their own plant sources, synthetic drugs show off their dangerous side effects by time but medical plants have generally centuries-long use and little side effects, many commonly used drugs such as steroidal compounds and ergot alkaloids can be purified from plants much more economically compared to synthetic production. Medicinal plants have multiple actions whereas synthetic drugs have usually only one main effect and additional drugs like vitamins are usually needed to prevent side effects of synthetic drugs. Plant-derived drugs do not necessiate such polypharmacy (Aboolenein, 1982).
Urtica pilulifera (UP) is a good plant which is classified as popular plant found in the Palestinian area and in Sinai. Based on number of informants who reported the use of this plant, it can be considered as most important plant. Randal et al. (1999) tested the toxicity of the plant and found that petroleum ether extracted from leaves and roots was completely non-leathal even at doses reaching 12.8 mg kg-1 and considered non toxic, UP dose not have any mutagenic and embyreogenic effects (Graf et al., 1994).
Urtica sp. have a long history of use in the home as an herbal remedy. A tea made from the leaves has traditionally been used as a tonic and blood purifier. The whole plant is antiasthmatic, antidandruff, astringent, depurative, diuretic, galactogogue, haemostatic, hypoglycaemic and a stimulating tonic (Delcourt et al., 1996). An infusion of the plant is very valuable in stemming internal bleeding, it is also used to treat anaemia, excessive menstruation, haemorrhoids and arthritis. In addition, this herb is used to treat rheumatic pain and for colds and cough (Styprekowska and Bieganska, 1980) and skin complaints, especially eczema. The plant is best harvested in May or June as it is coming into flower and dried for later use for medicinal purposes.
However, antioxidant supplements or foods rich in antioxidants may be used to help the human body in reducing oxidative damage by free radicals and active oxygen (Gülçin et al., 2002). Recently, various natural products and their effects on health, especially the suppression of active oxygen species by natural antioxidants from teas, spices and herbs, have been intensively studied (Ho et al., 1994). Therefore, the development and utilization of more effective antioxidants of natural origin are desired for health (Özen and Korkmaz, 2003).
Aqueous infusions of Mediterranean herbs including Urtica species, ex. U. dioica, exhibit antioxidant activity towards iron-promoted oxidation of phospholipids, linoleic acid and deoxyribose (Moron et al., 1979). It was reported that Urtica dioica prevent the damage of rat liver tissue structure (Laurnet, 1981). Urtica dioica herbs are used to treat stomachache in Turkish folk medicine (Grice, 1986) and is used against liver insufficiency (Kanter et al., 2003). Several different principles, including glucopyranosides (Obertreis et al., 1996), glycoprotein (Andersen and Wold, 1978), protein (Tita et al.,1993), flavonol glycosides, carotenoids (Kudritskaya et al., 1986), as well as biologically-active compounds, such as caffeoyl malic acid and caffeoyl quinic acid (Ohkawa et al., 1979), essential oils, formic and acetic acid, histamine, tannins, mucilage, vitamins (A, B1, B2, C, K1, folic and pantothesic acids) (Yagi, 1987) have been identified as contributing to the observed medicinal effects of the plant. The leaves, as well as flowers and roots were used extensively for cancer treatment in Turkey and some reports of positive results exist (Yagi, 1987).
The aim of the present study was to investigate antioxidant activity of the U. pilulifera different extracts from different plant parts and its potential use in pharmaceutical industries as a chemopreventive agent.
Materials and Methods
Chemicals
All chemicals were purchased from local firms and were of the highest purity.
Preparation of Plants
Three plant parts (herb, roots and seeds) were exhaustively extracted using
two solvents; petroleum ether (80-100%) (PE) and 20% aqueous methanolic (AM)
extracts; Pet. ether crude extracts prepared by continuous extraction using
soxhlet apparatus while methanolic extracts were prepared by soaking with shaking
for plant powders of each organ separately, then each extract was filtered to
remove cellular residues. The extracts were concentrated under reduced pressure
till free solvents. The different residues were used for experiment.
Animals
Male swiss albino mice (8-9 weeks old), bred in the animal house of National
Research Center, were used. The animals were maintained under normal conditions
(12 h light/dark cycle) and were fed with pelleted standard laboratory mice
feed and tap water.
Experimental Procedure
A total of 49 mice were used, divided into the following groups: Group I
(n = 7) was injected interpretoneal with saline, daily for 10 days. This group
was designated as a control group. Group II, III and IV (each of 7 animals)
were treated with 50 ppm (0.25 mL/mouce) of petroleum ether extract of U.
pilulifera (herb, root and seed) through interpretoneal injection daily
for 10 days. Group V, VI and VII (each of 7 animals) were treated with 50 ppm
(0.25 mL mouce) of methanolic extract of herb, root and seed through interpretoneal
injection daily for 10 days. This treatment is repeated with high concentration
(200 ppm).
Preparation of Subcellular Fractions from the Liver
Mice were killed by cervical dislocation. Liver homogenates were prepared
in ice-cold bidistilled water. The homogenate was centrifuged at 3000 rpm in
cooling centrifuge (-4°C) for 10 min. The supernatant was used for determination
of NP-SH and different enzymatic activities.
Determination of NP-SH Group
The level of sulfhydryl groups was determined using 5, 2-dithiobis-2-nitrobenzoic
acid (DTNB) according to Neugebauer et al. (1995).
Determination of Lipid Peroxidation (LPO)
LPO of liver microsomes was determined spectrophotometrically as the number
of nmoles of malondialdehyde (MDA) formed per mg protein at 532 nm (Oktay et
al., 2003).
Assay of Glutathione S-transferase (GST)
It was assayed using 1-chloro-2, 4- dinitrobenzene (CDNB) as substrate (Habig
et al., 1974). The substrate was previously dissolved in the least amount
of absolute ethyl alcohol. The formation of conjugated derivatives was followed
spectrophotometrically at 340 nm as a function of time.
Determination of Protein
Microsomal and cytosolic proteins were determined using the method of Bradford
(Bradford, 1976).
Assay of Glutathione Reductase (GR)
GR was assayed spectrophotometrically with slight modification according
to Calberg and Mannervik (1974).
Assay of Glutathione Peroxidase (GPHPx)
GSH peroxidase activity was determined as described by Lebedev et al.
(2001).
Assay of Superoxide Dismutase (SOD)
Measurement of SOD activity was carried out spectrophotometrically (Folhé
and Otting, 1984).
Statistical Analysis
Results are expressed as mean±SD. Statistical analysis was determined
using ANOVA following Dunnet’s t-test. Values showed a significant difference
from those of control groups. The SPSS 10.0 (Statistical Program for Social
Sciences) was used for statistical analysis (Campbell, 1989).
Results
Antioxidant Activity of Aqueous Methanolic and Petroleum Ether Extracts
at 50 ppm
Table 1 and 2 indicate that all six
U. pilulifera extracts significantly enhanced the protein synthesis in
mice liver and enhanced glutathione reductase activity but the glutathione transferase
activity reduced than control group using aqueous methanolic extracts of herb
and roots. Superoxide dismutase activity was significantly enhanced by using
all extracts.
Antioxidant Activity of Aqueous Methanolic Extracts at 200 ppm
All estimated protein concentration and antioxidant parameters were enhanced
specially with herbal extract, the protein concentrations measured in liver
homogenate of mice treated with 200 ppm aqueous methanolic extracts were enhanced
by 25.97, 75.86 and 78.7% for herb, root and seeds, this increment of protein
synthesis was in parallel with induction of enzyme levels.
Table 3 shows that, glutathione concentrations determined in mice liver homogenate were significantly increased specially in case of mice treated by herbal extracts (299.2, 84.2 and 22.5% for herb, root and seeds, respectively). Glutathione transferase activity was elevated by 285.76, 118.69 and 181% using herbs, roots and seeds extracts, respectively. The glutathione reductase activity were enhanced with animal treated by herb extract only (36.69%) whereas root and seeds extract were reduced by 24.88 and 20.69%, respectively. Glutathione peroxidase has the same manner which induced by herbal extract (68.3%) and reduced by the other extracts; root and seeds (-40.14 and -43.66%, respectively). The Superoxide Dismutase (SOD) levels were increased by (232.8%) using extract of herb while the other extracts (roots and seeds extracts) caused lowered increments by 31.25 and 64.84%, respectively.
| Table 1: | Effect of Urtica pilulifera methanolic extracts at 50 ppm on antioxidant parameters in mice liver homogenates |
| Table 2: | Effect of Urtica pilulifera petroleum ether extracts at 50 ppm on antioxidant parameters in mice liver homogenate |
| Conc: concentration, G: Glutathione, P: Protein, SOD = Superoxide
dismutase, F: ratio of one way ANOVA. p: value of ANOVA significant <0.05 |
|
| Table 3: | Effect of Urtica pilulifera methanolic extracts at 200 ppm on antioxidant parameters in mice liver homogenate |
| Table 4: | Effect of Urtica pilulifera petroleum ether extracts at 200 ppm on antioxidant parameters in mice liver homogenate |
| Conc: concentration, G: Glutathione, P: Protein, SOD = Superoxide
dismutase, F: ratio of one way ANOVA. p: value of ANOVA significant <0.05 |
|
The enhancement of antioxidant parameters caused high reduction in lipid peroxidation and the highest reduction was caused by aqueous methanolic herb extract which represented 81.8% followed by roots and seeds extract (69.86% and 58.39%, respectively).
Antioxidant Activity of Petroleum Ether Extracts at 200 ppm
Table 4 shows that liver protein concentration in mice
treated by petroleum ether extract were significantly increased by 132.76, 120.97
and 64.7% for herb, roots and seeds extracts respectively. The glutathione concentrations
were enhanced by 66.8, 80 and 126.8% for herb, roots and seeds extract treatments,
at the same time the glutathione transferase activities were induced by 169.57,
104.35% for herb and roots extracts treatments while it decreased by seeds extract
treatment (-10.14%). The glutathione reductase activities were enhanced by different
levels; 59.92% for herb extract treatment, 25.15% for roots extract treatment
and 10.22% with seeds extract treatment. On the other hand, glutathione peroxidase
has high significant increase by 187.72% when animals treated by petroleum ether
extract of herb, but roots and seeds petroleum ether extract did not have the
same effect which caused enhancement by 34.04 and 15.09%, respectively.
Data of superoxide dismutase activity in liver tissue of treated animals showed an increment using herb petroleum ether extract (26.32%) whereas they decreased by 78.95 and 52.63% in animal treated by roots and seeds pet. ether extracts. Herb petroleum ether extract has a strongest effect on antioxidant parameters, it inhibited lipid peroxides production (-31.2%) but the other two extracts did not have the same activity, they enhanced lipid peroxides by 12 and 52.8% for roots and seeds extracts, respectively.
Discussion
In recent years, natural bioactive compounds have been used as chemo preventive agents to inhibit hazardous effects of xenobiotics different sites. Xenobiotic detoxification is controlled by the liver as the main organ.
In this study, application of the petroleum ether and hydro alcoholic extracts of U. pilulifera to mice showed a marked effect on protein synthesis, some hepatic biotransformation enzyme systems and antioxidant enzymes. The intraperitoneal injection of U. pilulifera extracts (0.735 and 2.94 mg kg-1 day-1 for 10 days) had discernible effects on the tested parameters.
Protein concentration was increased by different extracts in the order of herb>root >seed by using PE extracts and take the opposite direction by using methanol extracts (Table 1 and 2). This may be due to presence of a procial amount of active compounds that enhance protein synthesis. This process also differs according to content of active compounds in different part of plants which affects some hepatic biotransformation and antioxidant enzymes. The enhancement of protein synthesis is also dose dependent (Table 1 and 3). The decrease of lipid peroxidation by U. pilulifera suggests a role of cytoprotection as well as prooxidant and peroxisomes induced membrane dangerous.
The findings are coincided with Özen and Korkmaz (2003) who used hydroalcoholic herb extract of U. dioica by 50 mg and 100 mg kg-1day-1. Table 1 and 2 showed that UP has a high antioxidant activity although it was used in low concentration, this indicate a high reducing power activities of the plant. The antioxidant activity of putative antioxidants have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging (Samur et al., 2001).
GST, which plays an important role in conjugating the metabolites resulting from the action of cytochrome P450, with endogenous ligands (reduced glutathione) favoring their elimination from the body of organisms, is a critical detoxification enzyme (Hartman and Shankel, 1990). It has already been reported that glutathione S-transferase induces protection against various cytotoxic, mutagenic and carcinogenic chemicals (Ketterer, 1988). The study showed that GST activity increases (in common) by using different extracts and this increase differs in its magnitude according to dose and parts of plants. The antioxidant enzymes in mice liver is enhanced with using leaf extracts (PE and AM) but decrease in roots and seeds. This may be due to quinching activity of different parts of plants and hydrogen donating ability of separate compounds (Gülçin et al., 2004). The phytochemical investigation of the plants showed the presence of flavonoids, coumarins, some sterols, alkaloids and hydrocarbons by aqueous menthanol extract meanwhile sterols, fatty acids and terpenes were extracted by petroleum ether. Flavonoids, phenolic acids and some terpenes possess antioxidant activities in different mechanisms which gave the induction of different antioxidant estimated parameters.
On the basis of the results of this study, it is clearly indicated that U. pilulifera has a powerful antioxidant activity against various oxidative systems in vivo moreover, it can be used as accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical industry. In addition, free radicals have been demonstrated to be a contributing factor in the tissue injury and modulation of the pain, phenolic compounds which present in plants appear to be responsible for the antioxidant activity of extracts. Some studies have revealed that the antioxidants melatonin and β-carotene potentiate the antinociceptive responses (Ruiz-Larrca, 1997).