Collection and extraction of plant material
The fresh aerial parts of Artemisia vulgaris were collected from Mizoram University Campus, Tanhril. These were then cleaned and removed the stalks, branches and stems. Only the leaves were taken and shade dried, and further the dried leaves were chopped and grinded with the help of an electronic grinder. The powder was extracted with 99.8%methanol by cold maceration technique. The liquid extract obtained was filtered by using Whatman filter paper 1 and allowed to evaporate in a rotary evaporator so as to get the plant extract in the crude form. This was collected and stored properly in refrigerator at 4 °C for further used.
Preliminary phytochemical screening
The detection of phytoconstituent present in the methanolic extract of A. vulgaris (MEAV) leaves was carried out by standard methods.
Test for proteins (Xanthoproteic test)
In 2 ml of extract add 2 ml of concentrated HNO3 observation of orange colour indicates the presence of proteins [21].
Test for carbohydrates (Benedict’s test)
In 2 ml of extract add 2 ml of Benedict’s reagent and boiled. Formation of orange red precipitate indicates the presence of carbohydrates [21].
Test for alkaloids (Mayer’s test)
In 2 ml of the extract, drops of Mayer’s reagent were slowly added by the side of the test tube, formation of a white or creamy precipitate indicates the presence of alkaloids [22].
Test for Flavanoids (alkaline reagent test)
2 ml of 2% sodium hydroxide solution was added to 2 ml of the extract. Appearance of yellow colour precipitation indicates the presence of flavonoids [22].
Test for Triterpenoids (Salkowski’s test)
In 2 ml of extract add 1 ml of chloroform followed by a few drops of concentrated H2SO4 on the side of the test tube and shaken well, formation of yellow colour at the lower layered indicates the presence of triterpenoids [22].
Test for Saponins (frothing test)
2 ml of extract was diluted with 10 ml of distilled water in a test tube and shaken for 5 mins, observation of stable foam indicates the presence of saponins [23].
Test for tannins (ferric chloride test)
2 ml of the extract was mixed with few drops of 10% ferric chloride solution, the change of colour into dark blue or green indicates the presence of gallic tannins and catechol tannins [23].
Test for glycosides (Keller-Kilani test)
A mixture of 4 ml of glacial acetic acid and 1 drop of 2.0% FeCl3was added to 10 ml of the extract followed by the addition of 1 ml of concentrated H2SO4. Formation of brown ring between the layers indicates the presence of cardiac glycosides [24].
In-vitro antioxidant assay
DDPH free radical scavenging activity
Radical scavenging activity of methanolic extract of A. vulgaris (MEAV) leaves was determined by Calorimetric assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a source of free radical according to the method of Blois with a slight modification [25, 26]. Stock solutions of MEAV extract were prepared at a concentration of 1 mg/ml and for test sample; it was serially diluted to make the concentrations of 10, 20, 30, 40, 50 and 60 μg/ml respectively.
0.1 mM DPPH solution was prepared by dissolving 3.94 mg in 100 ml of methanol and then 1 ml of this solution was mixed with 150 μl methanol, which was used as control. The above mentioned concentration of MEAV was taken and mixed with 1 ml DPPH solutions to each test tube. After 15 min of incubation, the absorbance of control and test sample was measured at 516 nm in UV- visible spectrophotometer and the compound ascorbic acid was used as a reference. The percentage inhibition and IC50 were calculated. The free radical scavenging activity (% inhibition activity) was calculated using the given equation-
$$ \%\mathrm{inhibition}\ \mathrm{activity}=\frac{\mathrm{Control}\ \mathrm{absorbance}-\mathrm{Sample}\ \mathrm{absorbance}}{\mathrm{Control}\ \mathrm{absorbance}}\times 100 $$
Reducing power by FeCl3
Reducing power of MEAV was measured by method of Oyaizu with slight modifications [27, 28]. Stock solutions of MEAV were prepared at a concentration of 1 mg/ml and the test sample concentrations as 5, 10, 15, 30, 60and 100 μg/ml were made by serial dilution.
Here, 2 ml of above concentration were mixed thoroughly with 2.5 ml of phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide solution. These mixtures were warm up in the water bath at a constant temperature of 50 °C for 20 min and then allowed for cooling, added 2.5 ml of 10% trichloroacetic acid to the mixture and centrifuged for 10 min at 3000 rpm. After centrifugation, 2.5 ml of supernatant part was taken and mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride and incubated for 10 min. Then, absorbance at 700 nm was measured in spectrophotometer. Control value was also recorded with same procedure without the MEAV. The standard compound Ascorbic acid was used as a reference compound.
Test animals
The Swiss albinos male mice weighing between 18 and 25 g obtained from the animal house of department of zoology, Mizoram University were used for testing of the present study. These animals were kept in a well-ventilated room at a maintained temperature of 25 ± 2 °C with 12:12 h light/dark cycle in polypropylene cages containing sterile locally procured paddy husk as bedding. The animals were fed with commercially available food pellets and water ad libitum till the completion of the experiment. Animals were adapted in the laboratory environment 15 days earlier to initiation of studies.
All the investigation protocols followed in the experiment were revised by the Institutional animal ethical committee and followed accordingly under the guidelines of CPCSEA (2003) [29]. The present research was approved by the Institutional Animal Ethical Committee, Mizoram University, India. (Approval No. MZU-IAEC/2018/10).
Acute toxicity test
The acute toxicity study of the MEAV was carried out for the selection of dose according to the OECD guidelines [30]. The extract was administered orally at increasing doses of 500, 1000 and 2000 mg/kg and the observation was made for 48 h for its behavioral changes, any toxicity and mortality. No mortality and other acute toxicity effect were found. Therefore, the doses for the present investigation were selected at 200 and 400 mg/kg body weight for analgesic evaluation [15, 31].
In-vivo analgesic activity
Acetic acid induced writhing
The In-vivo analgesic activity was carried out by acetic acid induced writhing method [32]. Swiss albino mice were categorized in 4 groups with 6 animals in each groups and proper marking was done individually as test group I and II, standard group and control group. The test groups were received with MEAV at a dose of 200 and 400 mg/kg b. wt. respectively. Similarly, indomethacin of dose 10 mg/kg b. wt. used as a standard drug and normal saline as control were given orally with the help of a gavage 30 min before the administered of 0.7% (v/v) acetic acid (0.01 ml/g) intraperitoneal injection to induce the writhing. The writhes count (number of abdominal constrictions) were observed and counted for 30 min for every single animal in each group.
Percentage of protection was expressed using formula:
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{protection}=\left(\mathrm{A}-\mathrm{B}\right)/\mathrm{A}\times 100 $$
Where, A = No. of writhes in control,
B = No. of writhes in test.
Tail immersion methods
Tail immersion methods in mice were employed to detect the analgesic activity [33, 34]. The experiment was carried out on the basis of observation that the pain relieve drugs was capable of lasting the time taken for the reflex withdrawal of tail by mice when immersed in warm water at a temperature of 55 °C.
According to divided groups, the standard group received the drug indomethacin at the dose 10 mg/kg b. wt., the test group treated with MEAV at the dose of 200 and 400 mg/kg b. wt. respectively and normal saline in control were administered orally. After the administration of the test and standard drug, the lower part of the tail was marked up to1-2 cm and dipped into the water bath at 55 °C temperature. The reaction time of the deflexion of tail by the mice were observed and recorded at 0, 10, 15, 30 and 60 min respectively.
Statistical analysis
The data obtained in in vitro antioxidant activity were expressed as mean ± standard deviation of mean (SD) and the statistical significance of in-vivo analgesic activity was analyzed by one way analysis of variance (ANOVA) followed by post hoc Duncan multiple comparison tests.