Abstract
Ethanol and aqueous extracts obtained from 11 plant species from 11 families selected based on their use in Turkish folk medicine, including Isatis glauca. Aucher x Boiss. subsp glauca. (herb) (Brassicaceae), Tamus communis. L. (herb and root) (Dioscoreaceae), Rumex crispus. L. (herb) (Polygonaceae), Daphne oleoides. Schreber (aerial parts) (Thymeleaceae), Laurus nobilis. L. (leaves and seeds) (Lauraceae), Ranunculus tricophyllus. Chaix (herb) (Ranunculaceae), Anchusa azurea. Miller var. azurea. (herb) (Boraginaceae), Calluna vulgaris. L. (herb) (Ericaceae), Prunella vulgaris. L. (herb) (Lamiaceae), Scabiosa rotata. Bieb. (herb) (Dipsacaceae), and Arceuthobium oxycedri. (DC.) Bieb. (herb) (Loranthaceae), were evaluated for their in vivo. anti-inflammatory and antinociceptive activities. Among the plant extracts studied, the ethanol extracts of Laurus nobilis. seed and Calluna vulgaris. herb and the aqueous and ethanol extracts of Arceuthobium oxycedri. herb displayed significant anti-inflammatory activity using carrageenan-induced hind paw edema model in mice without inducing any gastric damage. The ethanol extracts of Isatis glauca. subsp. glauca., Calluna vulgaris., Laurus nobilis. (seeds) as well as the aqueous and ethanol extracts of Arceuthobium oxycedri. were also shown to possess significant antinociceptive activity in varying degrees against p.-benzoquinone-induced abdominal contraction test in mice.
Introduction
Inflammatory disorders are among the top-ranking ailments worldwide, are characterized by the presence of undesirable inflammation, and contribute to significant restriction of life expectancy and loss of well-being to a vast number of humans. Treatment strategies in the past were nonspecific, mainly based on broad-spectrum immunosuppressant drugs such as corticosteroids (Laudenslager, Citation2004); later, nonsteroidal anti-inflammatory drugs (NSAIDs) became a strong alternative group of agents. All of the NSAIDs are approximately equivalent in terms of anti-inflammatory efficacy but also cause untoward side effects, particularly in the gastrointestinal system, which limits their application in therapy (Basran et al., Citation1982; Peura, Citation2004). There is a need, therefore, for discovering novel compounds that might serve as leads for the development of potent drugs with anti-inflammatory activity.
In our ongoing investigations on the medicinal plants used in Turkish traditional medicine for the treatment of rheumatism and related inflammatory disorders, we undertook the current activity screening study in order to evaluate the traditional use of these plants in terms of scientific validity. The selected plants as the subject of the current study are listed in with their vernacular names and relevant uses in Turkish traditional medicine. The ethanol and aqueous extracts prepared from the mentioned plants were tested in mice for anti-inflammatory activity using the carrageenan-induced hind paw edema model and for antinociceptive activity using the p.-benzoquinone- induced abdominal contractions test.
Table 1.. Traditional use of plant materials in Turkish folk medicine.
Materials and Methods
Plant material
Plant material was collected (depending upon the climatic characteristic of the collection site between April and July 2004) from different localities in Turkey. Voucher specimens were authenticated by Prof. Dr. Hayri Duman of the Department of Biology, Faculty of Science & Art, Gazi University, and were deposited in the herbarium of the Faculty of Pharmacy, Gazi University (Ankara, Turkey). Collection sites, parts used, and herbarium numbers for all plant material are given in .
Table 2.. The collected plant parts, collection sites during collection, and percentage yields of EtOH and H2O extracts.
Preparation of plant extracts
All plant material was dried under shade and powdered to a fine grade by using a laboratory scale mill. The plant parts and the extract yields (w/w) are given in . The extracts were prepared as given below.
Ethanol (EtOH) extract: Dried plant material (10 g) was extracted with 96% EtOH at room temperature two times (× 200 mL). The combined ethanol layers were evaporated to dryness in vacuo. to give crude EtOH extract.
Aqueous (H2O) extract: Dried plant material (10 g) was extracted with distilled water at room temperature two times (× 200 mL). The combined aqueous layers were lyophilized to give the crude H2O extract.
Pharmacological procedures
Animals
Male Swiss albino mice (20–25 g) were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health (Ankara, Turkey). The animals were acclimatized to the animal room conditions for 2 days and were maintained on standard pellet diet and water ad libitum.. The food was withdrawn on the day before the experiment, but animals were allowed free access to water. A minimum of six animals was used in each group. Throughout the experiments, animals were processed according to the suggested international ethical guidelines for the care of laboratory animals.
Preparation of test samples for bioassay
All the extracts were administered in standard doses of 250 and 500 mg/kg after suspending in 0.5% sodium carboxymethyl cellulose (CMC) suspension in distilled water. The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of the dosing vehicle. Either indomethacin (10 mg/kg) or acetylsalicylic acid (ASA) (100 and 200 mg/kg) in 0.5% CMC was used as reference drug.
Antinociceptive activity
p.-Benzoquinone-induced abdominal constriction test (Okun et al., Citation1963) was performed on mice for determination of antinociceptive activity. According to the method, 60 min after the oral administration of test samples, the mice were intraperitoneally injected with 0.1 mL/10 g body weight 2.5% (w/v) p.-benzoquinone (PBQ; Merck, Darmstadt, Germany) solution in distilled H2O. Control animals received an appropriate volume of dosing vehicle. The mice were then kept individually for observation and the total number of abdominal contractions (writhing movements) was counted for the next 15 min, starting on the fifth minute after the PBQ injection. The data represent the average of the total number of writhes observed. The antinociceptive activity was expressed as percentage change from writhing controls. Aspirin (ASA) at 100 and 200 mg/kg doses was used as the reference drug in this test.
Anti-inflammatory activity
The carrageenan-induced hind paw edema model was used for determination of anti-inflammatory activity (Yesilada & Küpeli, Citation2002). The difference in footpad thickness between the right and left foot was measured with a pair of dial thickness gauge calipers (Ozaki Co., Tokyo, Japan). Mean values of treated groups were compared with mean values of a control group and analyzed using statistical methods. Sixty minutes after the oral administration of test sample or dosing vehicle, each mouse was injected with freshly prepared (0.5 mg/25 µL) suspension of carrageenan (Sigma, St. Louis, MO, USA) in physiologic saline (154 nM NaCl) into subplantar tissue of the right hind paw. As the control, 25 µL saline solution was injected into the subplantar tissue of the left hind paw. Paw edema was measured every 90 min during 6 h after induction of inflammation. The difference in footpad thickness was measured by a gauge calipers (Ozaki Co., Tokyo, Japan). Mean values of treated groups were compared with mean values of a control group and analyzed using statistical methods. Indomethacin (10 mg/kg) was used as the reference drug.
Acute toxicity
Animals employed in the carrageenan-induced paw edema experiment were observed during 24 h and morbidity or mortality was recorded, if any occurred, for each group at the end of the observation period.
Gastric-ulcerogenic effect
After the antinociceptive activity experiment, mice were sacrificed under deep ether anesthesia and stomachs were removed. Then, the abdomen of each mouse was opened through the greater curvature and examined under a dissecting microscope for lesions or bleedings.
Statistical analysis of data
Data obtained from animal experiments were expressed as mean standard error (±SEM). Statistical differences between the treatments and the control were evaluated by ANOVA and Student-Newman-Keuls post hoc. tests. p < 0.05 was considered to be significant (*p < 0.05; **p < 0.01; ***p < 0.001).
Results and Discussion
Eleven plant species selected based on ethnobotanical information compiled from Turkish folk medicine, as listed in , have been evaluated for their in vivo. anti-inflammatory and antinociceptive activities. Two kinds of extracts with ethanol and water were prepared from each plant material, and their inhibitory effects in the p.-benzoquinone-induced writhing test for the assessment of antinociceptive activity and in the carrageenan-induced hind paw edema model in mice for assessment of anti-inflammatory activity were studied. Results of both assays are presented in Tables and , respectively.
Table 3.. Effect of the materials against p.-benzoquinone–induced writhings in mice.
Table 4.. Effects of the extracts against carrageenan-induced paw edema in mice.
Among the plant extracts studied, the ethanol extracts of Laurus nobilis. (seed) and Calluna vulgaris. (herb) and the aqueous and ethanol extracts of Arceuthobium oxycedri. (herb) showed significant anti-inflammatory and antinociceptive activity in mice without inducing any gastric damage, whereas the ethanol extract of Isatis glauca. subsp. glauca. had only antinociceptive activity.
L. nobilis., laurel, is a spice plant widely distributed in the Mediterranean area, and the seed oil is used against rheumatic pain in Turkish folk medicine as well as in Europe. Previously, the oil obtained from laurel fruit was also reported to be used for treatment of furuncles, sprains, bruises, and rheumatism (Simic et al., Citation2003). However, the anti-inflammatory activity of laurel has not been evaluated thus far. Our results indicated that the ethanol extract of the seeds possesses a remarkable anti-inflammatory (29.1–37.3%) and antinociceptive (34%) activity at a dose of 500 mg/kg. On the other hand, extracts prepared from the leaves were also studied for comparison and were found completely devoid of any activity.
Calluna vulgaris. is used as a diuretic and urinary antiseptic in Turkish folk medicine (Baytop, Citation1999) and against rheumatic pain elsewhere (Baquar, Citation1989; Kumarasamy et al., Citation2002). Several biological activity studies performed on this plant revealed the acetone extract from C. vulgaris. flowers to possess inhibitory activity both on lipoxygenase and cyclooxygenase, and ursolic acid was reported as one of the active components (Najid et al., Citation1992). Later, Tunon et al. (Citation1995) reported in vitro. anti-inflammatory activity both through inhibition of prostaglandin synthesis and platelet activating factor (PAF)-induced exocytosis. In the current study, the ethanol extract of the herb had a notable in vivo. anti-inflammatory activity in the ranges 34.2–38.7%, which is comparable with the reference drug indomethacin. Moreover, the ethanol extract possessed antinociceptive activity having 37.9% inhibition at 250 mg/kg and 47.0% at 500 mg/kg doses, which are quite comparable with ASA.
Arceuthobium oxycedri. is a semiparasitic plant that grows on Juniperus oxycedri., and the shape and color of the plant looks very similar to the host plant. In Anatolia, this plant is believed to be effective against a wide range of diseases; from inflammatory to infectious (Fujita et al., Citation1995; Yesilada et al., Citation1995; Honda et al., Citation1996). Both the ethanol and aqueous extracts of the plant exhibited significant anti-inflammatory effect in the ranges 28.9–34.2% and 21.1–30.5%, respectively. The plant also showed a potent antinociceptive activity; for the aqueous extract, 34.2% at 500 mg/kg dose, and for the ethanol extract, 28.6% at 250 mg/kg and 39.9% at 500 mg/kg doses. Our results supported the folkloric use of the plant as no such study has been carried out on this plant previously.
In Turkish folk medicine, Isatis. sp. is known to be used for wound healing and against constipation (Baytop, Citation1999). In Iran, another species of the plant, I. cappadocia., has also been reported to be used against rheumatism, asthma, eczema, fever, headache, and wound healing. Rezaeipoor et al. (Citation2000) showed that the plant possesses in vivo. suppressive effect on humoral primary immune response at the dose of 250 mg/kg, but stimulated secondary immune responses at the dose of 500 mg/kg. In the current study, the activity of a Turkish species, I. glauca. subsp. glauca., was studied, and the ethanol extract demonstrated a significant antinociceptive activity (37.2%) at 500 mg/kg, as well as a medium anti-inflammatory activity (24–28% inhibition).
Daphne oleoides. is another medicinal plant used to treat rheumatism and lumbago in folk medicine in Turkey. In an activity screening study on a number of plant extracts used for treatment of various inflammatory conditions, D. oleoides. subsp. oleoides. of Turkish origin was found to be the most effective against interleukin-1α and TNF-α (Yesilada et al., Citation1997). Then, through in vitro. activity-guided fractionation procedures assessing the inhibitory effects on inflammatory cytokines, two diterpenes and one coumarin derivative were isolated and chemically defined as the main principles that supported its folkloric usage (Yesilada et al., 2001). In the current study, ethanol extract showed some in vivo. anti-inflammatory and antinociceptive activity at a higher dose, although this was not significant. Several other Daphne. species were also previously reported to be used against some inflammatory conditions; for instance, D. tangutica. is the main component of a formulation in Chinese traditional medicine used for treatment of rheumatoid arthritis (Taniguchi et al., 1988; Hong et al., Citation2002; Chen et al., Citation2004).
Ranunculus. species are used in Turkish folk medicine against rheumatic pain, in particular to drain the pus from the inflamed joint through applying the fresh flowering herb after pounding on the joint for about 20 min (Yesilada et al., Citation1995; Baytop, Citation1999). In the current study, ethanol extract of R. tricophyllus. herb showed weak anti-inflammatory and antinociceptive activity.
On the other hand, Prunella vulgaris. is used against rheumatism, colds and cardiac disorders in Turkish folk medicine (Yesilada et al., Citation1993). In a previous study, an herbal combination (SKI 306X) consisting of Clematis mandshurica., Trichosanthes kirilowii., and Prunella vulgaris., which was used in Chinese traditional medicine for the treatment of various inflammatory disorders (i.e. lymphadenitis and arthritis), was studied for its in vitro. effects as an anti-inflammatory, analgesic, antiarthritic, blood-microcirculation enhancer, as well as for inhibition of cartilage degeneration enzyme activities on proteoglycan degradation in cartilage explant culture and in collagenase-induced rabbit ostheoarthritis model (Choi et al., Citation2002). The results revealed that SKI 306X inhibited proteoglycan degradation in a concentration-dependent manner, and it also showed a protective effect on the knee joint of rabbit from ostheoarthritis-like changes with prophylactic administration. In another study, the aqueous extract of P. vulgaris. was shown to possess strong antioxidant potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates (Liu & Ng, Citation2000). However, the plant did not show any in vivo. significant anti-inflammatory or antinociceptive activity in the current study.
Rumex crispus. is reported to treat headache, used for wound healing, and used to promote maturation of abscess in folkloric medicine in Turkey (Yesilada et al., Citation1995). Besides, a literature survey revealed that several other Rumex. species have also been traditionally used in the treatment of eczema, wound healing, swelling, tonsillitis, leprosy, and hemorrhoids in Ethiopia or as analgesic and antipyretic in India and to treat burns and to remove pain from the back and lumbar region in Bangladesh (Ghosh et al., Citation2003; Rouf et al., Citation2003; Gabrie et al., 2004). In a previous study of anti-inflammatory activity of two Rumex. species using cell viability and measurement of PGE2 concentration methods, the results verified the root extract of R. abyssinicus. to inhibit PGE2 synthesis, whereas the leaf extract of R. nervosus. showed no inhibition at all (Getie et al., Citation2003). In another study, anti-inflammatory activity of R. patientia., a plant used for wound healing and anti-inflammatory properties in Turkish folk medicine, was studied against carrageenan, histamine, dextrane, serotonin, formaldehyde-induced edema, cotton pellet granuloma, and Kabak test models in rats, and the extract was found to possess significant anti-inflammatory activity, which was concluded to be dependent on its rich anthraquinone and tannin content (Süleyman et al., Citation1999). However, our experiments demonstrated that both the aqueous and ethanol extracts of R. crispus. displayed no remarkable anti-inflammatory or antinociceptive activity.
In a previous report, the ethanol extract from Tamus communis. roots was shown to possess a significant inhibitory effect on granuloma formation when tested on the cotton pellet granuloma model in rats (Capasso et al., Citation1983; Mascolo et al., Citation1987). However, neither aqueous nor ethanol extracts prepared from both roots and aerial parts of this plant showed any remarkable anti-inflammatory or antinociceptive activities in the current study.
Another plant material, Anchusa azurea. var. azurea., reported to be used in common colds as a diaphoretic (Baytop, Citation1999), was also found to be ineffective in anti-inflammatory and antinociceptive activity tests.
Scabiosa. species are used against constipation and for wound healing in Anatolia. Thus far, no anti-inflammatory or antinociceptive activity for the plant has been reported. In our study, the ethanol extract of S. rotata. revealed a weak antinociceptive and anti-inflammatory activity.
In conclusion, our experimental results indicated that among the 11 plant species reported to be used traditionally against inflammatory conditions or pains of different origin, only four, Laurus nobilis. (seed), Calluna vulgaris., Arceuthobium oxycedri., and Isatis glauca. subsp. glauca., were found to possess significant anti-inflammatory and/or antinociceptive activities that confirmed the folkloric use of these plants. Among the other plants studied, anti-inflammatory and antinociceptive activities of Ranunculus tricophyllus., Scabiosa rotata., and Daphne oleoides. were not significant, but a weak activity (around 20% inhibition) was observed, whereas Anchusa azurea. var. azurea., Rumex crispus., and Prunella vulgaris. showed only medium antinociceptive activity. However, neither roots nor aerial parts of Tamus communis. were found completely active. Although results of the pharmacological studies did not support the folkloric use for some of the studied plants, this might be due to the methodology employed or to dosage. Hence, other models for inflammatory activity assessment should be studied before regarding any as ineffective.
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