Hot Plate (Model–35100, UGO BASILE, ITALY), Electronic Balance (Ohaus manufacturer, Canada), Refrigerator(Butterfly Marketing Ltd, LG), Beakers, Petri dishes & glass wrought, Safety rat handling gloves, Mortar & pestle, Hypodermic Syringes, Holder & test tube, Rotaryevaporator (Eyela n 1000, Tokyo Rikaki Kai Co. Ltd Rotary vacuum, Japan), Glucose kit, Vortex, Centrifuge, Homogenizer, Screw-cap test tubes, Surgical apparatus (forceps, scissors), Micropipette, Incubator, UV-Spectrophotometer, Sonicator.
2.1 MedicinalPlant(Extract)Extract was examined in two concentrations of 250 and 500mg/kg body weight of the animal. 2.2 Reagent, Control & Positive Control
2.2.1PhytochemicalstudyReagents and chemicals-Wagner Reagent,concentrated HCL, 0.1% Ferric chloride,Molishreagent, conc. H2SO4,α-napthol, chloroform.
2.2.2. Analgesic activity1.Control–Distilled water.2.Positive control–Diclofenac sodium (Beximco Pharmaceuticals Ltd. Bangladesh) Administered dose–50mg/kg body weight animal.
2.2.3 Anti-diabetic activity1.Control-Sucrose solution.2.Reagents-NaOH(1N), H2SO4(2N), Ketamine, Ice-cold saline, 80% ethanol.
2.3 Plant ExtractionMethod
2.3.1CollectionThe plant sample of S. Officinalis was collected from an Ayurvedic Institution ‘Back to Nature on June 18, 2012 in the form of fruit shavings. The fruit of the plant was procured and cleansed with water several times to rinse away dirt and undesirable materials. 2.3.2 Drying and grinding The collected fruit was washed with water, separated from undesirable materials and plant parts, partially dried by fan aeration and then fully dried in the oven at below 40ºC for 2 days. The fully dried fruit was then grinded and coarsely powdered. The freshly dried powdered form weighed 1000g and was stored in the refrigerator at +4ºC for a few days.
2.3.3 Cold extraction (Ethanol extraction)542g of powdered plant material was then dissolved in 80% ethanol. The alcoholic mixture was sealed in a conical flask and placed on a shaker for 2 days period to receive occasional shaking and stirring. Subsequently, the homogenized mixture was subjected to coarse filtration by a piece of clean, white cotton material. The primary filtrate underwent a second phase of filtration through Whatman filter paper. The filtrate (Ethanol extract) obtained was evaporated by Rotary evaporator (Eyela n 1000, Tokyo Rikaki kai co.ltd, Rotary vacuum, Japan) at 4 to5 rpm and at 65ºc temperature.It rendered a gummy concentrate of dark brown color, designated as the crude extract ofS.officinalis. Then the crude extract wasdried by freeze drier and preserved at 4ºC. The preserved extract was later subjected tobiological screening and pharmacological experiments.2.4Phytochemical Analysis.
2.4.1StudydesignQualitative phytochemical tests for the identification of alkaloids, flavonoids, steroids, gum and carbohydrates, reducing sugar, saponins, tannin and terpenoids were carried out for the plant extract by the method described by Harborne and Sazada. The freshly prepared extract ofS.officinaliswas qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Wagner reagent, flavonoids with the use of conc. HCl, tannins with0.1% ferric chloride, and saponins with ability to produce suds. Gum was tested using Molishreagents and concentrated sulfuric acid, steroids with sulfuric acid, reducing sugar with the use of -napthol and sulfuric acid and terpenoids with chloroform and conc. HCl. 2.5 AnalgesicActivity.
2.5.1 MicescreeningYoung Swiss-albino mice aged 4-5 weeks, average weight 20-30 gram were used for these studies. They were kept in standard environmental condition for one week in the animal house of the Department of Pharmacy, North-south University, Bangladesh for adaptation after their purchase. The animals were provided with standard laboratory food and tap water ad libitum and maintained at natural day-night cycle. Experimental animals were randomly selected and divided into four groups denoted as group-I, group-II, group-III, group-consisting of 6 mice in each group. The individual weighing was done to adjust individual doses. Here, distilled water was given to group I, 50 mg/kg Diclofenac sodium was given to group II,250 mg/kg and 500mg/kg body weight of the crude extract of S. Officinalis were given to group III and IV respectively.2.5.2 Acetic acid induced writhing test in miceThe analgesic activity of the samples was evaluated using acetic acid-induced writhing method in mice.In this method, acetic acid is administered intraperitoneally to the experimental animals to create pain sensation. As a positive control, any standard NSAIDdrug can be used. In the present study Diclofenac sodium was used to serve the purpose. The plant extract was administered orally in two different doses, 250 and 500 mg/kg body weight of the animal, to Swiss Albino mice after an overnight fast. Test samples and vehicle were administered orally 30 minutes prior to intraperitoneal administration of 0.7% v/v acetic acid solution (0.1ml/10g) but Diclofenac sodium was administered 15 minutes prior to acetic acid injection. Then the animals were placed in individual beakers for observation. Each mouse of all groups were observed individually for counting the number of writhing they made in 15 minutes commencing just 5 minutes after the intraperitoneal administration of acetic acid solution. Full writhing was not always accomplished by the animal, because sometimes the animals started to give writhing but they did not complete it. This incomplete writhing was considered as half-writhing. Accordingly, two half-writhing were taken as one full writhing. The number of writhes in each treated group was compared to that of a control group while Diclofenacsodium (50 mg/kg) was used as a reference substance (positive control). 2.6 Antidiabetic Activity 2.6.1 Experimental animal Both genders of Long Evans rats (Rattus nor vigicus) were selected for the present study and acclimatized under standard conditions. Long Evans rats (male and female), weighing 80-200g of either sex were collected from ICDDRB for the study and were kept in standard environmental condition for weeks in the animal house of the Department of Pharmacy, North-south University, Bangladesh for adaptation after their purchase. The animals were housed under standard laboratory conditions (relative humidity 55-65%,room temperature of25.0 ± 2ºC, and 12 hrs light dark cycle). The animals were fed with a standard diet (pellets)and had free access to filtered water [6].2.6.2Assessment of anti-diabetic activity in different segments of GITPlant extract (500mg/kg) along with sucrose solution (2.5g/kg body weight) were administered orally to 24 hours fasted rats. The Control group was given equal volume of sucrose only. Ketamine hydrochloride was injected intraperitoneally 15 minutes prior to dissection of rats of each hour(30min, 1hr, 2hr & 4hr) to elicit acute anesthetic effect and eventually death. For 30, 60, 180 and 360 minutes respective rats were sacrificed. After sacrificing, the whole GIT was excised into six segments. The segments being–(A)Stomach, (B) Upper 20 cm of small intestine, (C) Middle part of small intestine, (D) Lower 20 cm of small intestine, (E) Caecum and (F) Large intestine. Each segment was then washed with 10 ml of ice-cold saline. The solution was then centrifuged for 15 minutes at3000 rpm. The supernatant was then collected and to this solution, 2N H2SO4(2ml) was added to acidify the solution. These mixtures were then boiled for 2 hours in paraffin oil to hydrolyze the sucrose. After 2 hours, to these mixtures, 1NNaOH was added drop by dropto neutralize the mixture and the pH was set at 6.9-7. Then the concentration of glucose was obtained by the use of the GOD-PAP method and ELISA reader. Blood glucose and the amount of glucose liberated from residual sucrose in the gastrointestinal tract were measured. The gastrointestinal sucrose content was calculated from the amount of liberated glucose.