In Vivo Biological Investigation of Methanolic Extract of Thymus linearis Whole Plant

Background and purpose: For treatment of pain, pyrexia and inflammation, medicinal plants have exhibited enormous capability. Medicinal plants are used as anti-pyretic, analgesic and anti-inflammatory agents having low gastro intestinal tract disturbing adverse effects. In order to tackle and assure the antipyretic, analgesic and anti-inflammatory response of herbs, scientific backdrop is mandatory. Experimental approach: Abdominal writhing was introduced to experimental mice by intra peritoneal injection (i.p) of 1% acetic acid. Pain is induced thermally by observance the mice on hotplate having temperature around 50°C. For antipyretic activity, pyrexia was induced by 20 ml/kg body weight (b.w) i.p brewer’s yeast injection. Anti-inflammatory activity was examined in mice by Carrageenan. Thymus linearis methanolic extract (TLME) was used at test doses of 100, 200 and 300 mg/kg b. w i.p in mice. Key results: TLME showed reduced writhing in acetic acid method with dose of 100, 200 and 300 mg/kg b. w by 54.00 ± 1.15, 44.00 ± 1.15 and 32.66 ± 0.66 in comparison of control i.e. 66.66 ± 1.76. The latency times in hotplate method was significantly increased from 13.95% to 21.22% at dose of 100 to 300 mg/kg. Antipyretic effect of TLME exhibited 39.70 ± 0.01 protections at 300 mg/kg after 2 h as similar to standard drug Paracetamol 38.57 ± 0.06. Anti-inflammatory effect of TLME was significantly observed by 36.66% and 35.29% respectively at dose of 300 mg/kg. Conclusion and implications: Results of TLME showed that it can be used to treat pyrexia, pain and inflammation. This investigation actively reinforced the ethno pharmacological uses of T. linearis as analgesic, antipyretic and anti-inflammatory agent.


Introduction
As medicine, plants have been used from ancient times [1]. Treatment through medicinal plants has been considered valuable on basis of empiric data of thousands of years [2]. Medicinal agents and their derivatives using nowadays clinically worldwide attributed about 50% of the entire drug while 25% of the total obtained from higher plants [3]. Today as a single chemical entities a large number of medicinally active compound are used from traditional herbal remedies [4]. The utilization of plants as a hotspot for new medication has been remarkably improved in human healthcare. Scientists utilized enthobotanical data as the "intimation to which plants are the prime contender for additionally screening and synthetic investigations". Plants and herbs always are used in various dosage forms such as they can be taken as syrups, ointments, tablets, capsules, tea salves and rubs [5]. T. linearis also contain alkaloids, carbohydrates, sterols, triterpenoids, steroids and tannins [13][14][15]. The crude methanolic extract of whole plant of T. linearis has not been used in in vivo biological investigations. The aim of the present study was to investigate acute toxicity, antipyretic, antinociceptive and anti-inflammatory activity of crude methanolic extract of whole plant of T. linearis to make it rationalized with its folk uses.

Collection of plant material
The whole plant of T. linearis was collected from in August 2011 from Lake Saif-ul-malook situated in Naran valley, Hazara division, Khyber Pakhtunkhwa, Pakistan. Plant sample was identified by Plant Taxonomist Professor Dr. M. Ibrar, Department of Botany, University of Peshawar.

Extraction and fractionation
The whole plant material was macerated for 10 days in 25 liter methanol at temperature of 25-30°C. The soaked plant was vigorously stirred twice daily. A coarse cloth was used for filtration of methanol soluble substances and then it was filtered by filter paper (Whatmann filter paper No.1). Rotary evaporator was used to make concentrate the filtrate. After that fractionation of this crude methanolic extract of whole plant was done with n-hexane, Chloroform, ethylacetate, n-butanol and aqueous respectively [16].

In vivo biological screening
Acute toxicity activity: In acute toxicity assay the mice were treated with 500, 1000, 2000 mg/kg body weight of crude methanolic extract of T. linearis. Normal saline was used as negative control given through oral route. The LD 50 was measured for 2 days and animal weight was observed on daily basis for seven days.

Antinociceptive activity
Writhings induced by acetic acid: Albino mice of both sexes having weight 19-23 gms were selected for antinociceptive activity. The whole plant crude extract in particular doses of 100, 200 and 300 mg/kg body weight were given through oral route to selected mice and after 30 minutes of that 3% acetic acid about 0.34 ml was injected intraperitoneal to induce writhings in animals. These writhings or stretching of hind limb along with abdominal constrictions were seemed after 5 min of acetic acid administration intraperitoneal. The anti-nociceptive effect of crude methanolic extract was compared with diclofenac sodium which was used as positive control.

Hot plate method:
The crude methanolic extract of T. linearis was given in a dose of 100, 200 and 300 mg/kg and after 30 min of dose administration mice were placed on hot plate. The temperature of hot plate was set at 50-55°C. The latency time was observed at 0, 30, 60, 90, 120 and 180 min by licking the hind paw or by jumping of mice. Percent analgesic effect was measured using the following formula: Test Latency Control Latency % Analgesic Effect 100 Cut off Time Control Latency

Anti-inflammatory activity
The crude methanolic extract of T. linearis whole plant was evaluated for its anti-inflammatory assay by carrageenan induced paw edema. After 30 min of test doses 100, 200 300 mg/kg body weight of methanolic extract the 0.1 ml of freshly prepared 1% carrageenan suspension was injected sub-plantar of the hind paw of mice. The edema induced by carrageenan and the antiinflammatory effect of extract was measured after 0, 1, 2, 3, 4 and 5 hours. The normal saline was used as negative control and diclofenac sodium in a dose of 10 mg/kg body weight was used as positive control.

Antipyretic activity
The antipyretic effect of crude methanolic extract of T. linearis whole plant was tested in selected mice by inducing pyrexia against Brewer's yeast. About 2 ml/kg body weight of 20% w/v aqueous suspension of Brewer's yeast was given subcutaneously to mice. The rectal temperatures were measured before and after 24 hours of Brewer's yeast administration. The rectal temperatures of extract treated mice was examined after 1, 2, 3, 4,5 h by administration of 100, 200 and 300 mg /kg body weight of extract of T. linearis. The Salisylic acid was used as standard about 150 mg/kg body weight.

Results and Discussion
The crude methanolic extract of T. linearis showed dose dependent significant antinociceptive response at the dose of 300 mg/kg.  1 and 2, Figures 1 and 2. In case of hotplate method the latency time was significantly increased from 13.95% to 21.22% at the dose of 100 to 300 mg/kg. The analgesic effect was observed significant after 90 min of extract administration given in Table 3 and Figure 3. In antipyretic activity the maximum antipyretic effect of TLME was observed at 2 nd hour which remained significant up to 4 th hour of extract administration as shown in Table 4 and Figure 4. The anti-inflammatory effect of TLME was found to be significant (p˂0.01) at dose of 200 and 300 mg/kg as given in Table 5 and Figure 4.

Conclusion
Thymus linearis contains essential oils thymol and p-cymene as main elements [17]. Phytochemical analysis of crude extract of plant showed presence of flavonoids, sterols, steroids and triterpenoids. These essential oils have already been reported to have analgesic, anti-inflammatory and antipyretic activities by inhibition of COX enzyme by a mechanism same like non-steroidal anti-inflammatory drugs [18,19]. Therefore, the analgesic, antiinflammatory and antipyretic activities of Thymus linearis may be due to these compounds. 15.33 ± 0.88*** TLME 100 54.00 ± 1.15 *P<0.05, **P<0.01, ***P<0.001 Table 2: Analgesic effect of TLME at 100, 200, 300 mg/kg in acetic acid induced test.