Identication, Molecular Proling, Docking Studies and determination of In-vivo Anti-Inammatory Potential of Teucrium Stocksianum Bioss Fixed Oil (FO)

Background: Considering the positive and valuable upshots of the naturally occurring ingredients as the complementary treatment for many ailments, this study investigates the Teucrium Stocksianum Bioss Fixed Oil (FO) for the treatment of inammation. Methods: Different plants of the Teucrium genera have been employed occasionally for the management of different disorders. Teucrium stocksianum (Lamiaceae) is traditionally use as antipyretic along with remedy of inammation, diabetes and tumors. It also occupies the capacity to purify the blood. The aim of this study is to determine the chemical constituents and anti-inammatory potentials of Teucrium stocksianum xed oil (FO). Qualitative and quantitative analysis of xed oil were performed via GC-MS (Gas Chromatography coupled with Mass Spectrometer). Preliminary inammation antagonistic activity was calculated in mice using the carrageenan-induced paw edema model while the mechanism behind this inammatory antagonistic effect was determined by employing various inammogens including arachidonic acid, prostaglandins E 2 and leukotriene via paw edema model. Results: The acute toxicity of xed oil was determined at 3, 6 and 10 ml/kg body weight of the mouse. The GC-MS analysis documented 21 various unsaturated and saturated fatty acids elements. The methyl esters of octadecadienoic and linoleic acids were found predominantly at 22.60% and 23.84% respectively. In preliminary screening, FO demonstrated substantial anti-inammatory capacity (67.87%, **P (cid:0) 0.01) in carrageenan induced paw edema model at 3 ml/kg body weight. The activity reached a peak value at the 3 rd hour and continued persistent until the 5 th hour of sample administration. FO displayed dose-dependent inhibition against all inammogens at 3 ml/kg at 3 rd h of the FO administration. These have shown marvelous protection i.e. 64.92%, 68.75%and 58.3% against arachidonic acid, prostaglandin E 2 and leukotriene accordingly, at 3 ml/kg. The arachidonic acid with GLU A: Vander were with GLN A: 329, ASP A: 290, ASP A: 442, ARG B: 520 and ASP A: 285. π-alkyl interactions were also the part of show with bindings between the carbon chain and LEU A: 244, PHE A: 286, ILE A: 365, VAL A: 361, ALA A: 439, LEU A: 288 and LYS B: 441.

healthiness are found from natural resources. Still, human beings have revealed enormous assistance of natural products research in the treatment and cure of almost all types of disease [3][4]. Seventy thousand different plant species are being investigated employing possible natural drugs for the prevention and control of several diseases. Both synthetic and natural materials can be used to produce drugs for different biological purposes [5]. The drugs which are obtained from natural products are invented to be more effective and safer as compared to synthetic drugs. Recently, for the natural product researcher, the anti-in ammatory agent who is attained from medicinal plants has increase magnetism [6][7][8].
The utilization of the NSAIDs (Non-Steroidal Anti-In ammatory Drugs) holds its position as a core approach amongst curative selection for winning the battle against in ammation [9]. Existing studies exposed that in ammation has been related to cancer, diabetes mellitus, coronary disease, atherosclerosis, neurodegenerative disease, asthma and obesity, in addition to wounds, sickness, injuries and swelling [10]. Multiple possibilities of stimuli like Physical and biochemical stimulus canister can prognosis the in ammatory response. Swelling, pain, redness, heat and dysfunction are all common symptoms of in ammation [11]. NSAIDs can be used primarily for in ammation, but due to their prolonged-term use, they produce a lot of side effects which include prompt allergic reactions, gastrointestinal, immunosuppressed system, cardiovascular complications, urinary tract problems and hypersensitivity responses. Mainly NSAIDs are more likely to cause non-speci c COX-2 and COX-1 enzyme inhibition [12][13]. Evidently, because of various side effects, potency, as well as e cacy of the present available NSAIDs and opioids, are not completely helpful [14]. Consequently, it is vital to investigate novel things and compounds for the management and treatment of in ammation.
The family Lamiaceae is one of the most signi cant herbal families, which integrated a broad diversity of the plants having medical and biological applications [15][16]. Lamiaceae included 240 genera and 3500 species around the globe. Reported literature shows that genera of this family exhibit diversi ed biologically active molecules [17]. Some of the species of this genus are also available in Pakistan, i.e. Teucrium stocksianum, Teucrium royleanum, Teucrium scordium and Teucrium quadrifarium [18][19][20][21]. Different oras of this genera have been used traditionally for different disease management. Teucrium polium belongs to this genera is utilized medicinally for management and treatment of atulence, labor pain, analgesia, cough, jaundice, liver abnormalities and abortions [22][23]. Teucrium stocksianumhave also occupied its space in folk remedies for the management of sore throats, cough, fever, foot-warming numbness, diabetes, blood puri er along body coolant [24][25]. Similarly, the T. stocksianum has been explored with antipyretic, analgesic and antiulcer properties [26][27][28]. Fixed and volatile oils have been isolated from this genus showing numerous pharmacological activities, i.e. antimicrobial, antioxidant, anti-in ammatory, antitumor and cytotoxicity [29][30][31]. Generally, different types of fatty acids obtained from plant sources bear diversi ed pharmacological activities like insecticidal activity of Lauric, oleic and linoleic acids [32], fatty acids of Osmium sanctum Linn with anti-in ammatory potential [33], antimicrobial activity of xed oils of Nigella sativa seeds [34]. There are various molecules that have been previously secluded from various species of the Teucrium montanins [35], while from Teucrium montanum compounds A to E were previously isolated [36]. The teucrins A and teucrin E have acquired from the Teucrium chamaedrys [37]. The molecules which were isolated from the T. quadrivium were teucvidin, teu in, epi-teucvidin, teucvidin, teuquadrin B and teuspinin [38]. Coll et al. [9], have been explored the clerodanes, which is called as 11-hydroxyfruticolone, acetyl-hydroxyteucjaponin, β-hydroxyfruticolone, 6-acetylteuc japonin and deacetyl-fruticolone from the T. fruticans. From the Teucrium species various biological potentials have been reported, like anti-seizure [39][40], anti-oxidant [41], antiviral [42] antibacterial and antifungal potential [43] anti-cancer [44] hepato-protective, butarylcholine esterase and acetylcholine esterase [30] and anti-in ammatory potentials [45]. Similarly, Radhakrishnan et al [46] evaluated the anti-in ammatory potential of the aerial part of the crude ethanolic extract of T. stocksianum. Similarly, Mukarram Shah et al [47], explored the crude ethanolic extract and their subfractions for the anti-in ammatory potential and lastly subjected the main active fraction i.e. ethyl acetate for anti-in ammatory mechanism. Likewise, they were also evaluated the anti-in ammatory, analgesic and antioxidant potential of the same plant [38]. Based on the previous literature survey, and explored the crude and subsequent fraction along with essential oil for their anti-in ammatory activity we aimed that determine of chemical composition of xed oil from T.stocksianum using GC-MS. Moreover; the FO extracted from T. stocksianum has not been evaluated yet for anti-in ammatory activity. Therefore, the current study has been arranged to evaluate the xed oil of T. stocksianum for its antiin ammatory activity and authenticate its traditional use against in ammation.

Plant Extract
T. stocksianum plant was collected from District Swat, KPK Pakistan in May. Plant name was con rmed by plant taxonomist Dr. Nasrullah, Botany Department, University of Malakand (UOM), Chakdara Dir, Pakistan. This specimen was placed in the same department with Code (H.UOM.BG.199b) for future indication. Plant material was gloom dried and grounded to powder, using a cutter mill. The coarse powder was macerated in n-hexane at 25 0 C for 2-weeks with mild infrequent shaking. The extract was ltered and then concentrated in controlled conditions (40 o C) using Soxhlet apparatus to get xed oil.

Experimental animals
Swiss albino male mice weighed 25-30 gm were purchased from the NIH (National Institute of Health) Islamabad Capital Territory, Pakistan. They were kept in suitable cages at 25 ± 2°C and in light and dark for 12 hours. All the experimental animals ad libitum were provided with su cient food and water during the accommodation phase. Animals were kept on fasting 18 h before the experiment but the water was available throughout the procedure. Mice were randomly distributed into different experimental groups as (n = 6). The Committee of animal ethics of the University of Malakand KPK accepted the protocols in strict compliance with internationally accepted principles for laboratory animal use and care after conforming to ARRIVE guidelines for the experimental animal studies through letter No; DREC/122 for Control and Supervision of Experiments on Animals standard [48]. After the experimental procedures, the animals were euthanized properly as per the standard procedures using the guideline of the American Veterinary Medical Association (AVMA) for the euthanasia of the animals. Halothane vapors were given to the animals via vaporizer for anesthetic induction and overdose. The overdose and prolong time euthanized the animals [49].

Statistical analysis and calculations
Data were obtained from various experimental groups and Mean ± SEM was calculated as of six mice in every group. The analysis of One-way variance statistical analysis (ANOVA) pursue by post hoc Dunnett's multiple comparison test was applied. P ≤ 0.01 differences and lower values between multiple groups were measured as signi cant values. Almost all in ammogens have shown peak in ammatory reaction at 3 rd hour in right side paw of the control group, followed by decrease edema on 4 th hour. Furthermore, the in ammatory reaction induced via arachidonic acid, prostaglandin E 2 , Leukotrienes and carrageenan give non-signi cant values in presence of the carriers, normal saline, or 10% DMSO in animal, therefore the annotations were pooled [50].

Acute toxicity
Toxicity studied for FO was conducted at 3, 6 and 10 ml/kg body weight of the mouse. Mice were categorized into two sets on a random basis; each contained six animals of either sex and was treated with 3, 6, or 10 ml/kg FO. All experimental animals were under observation for the appearance of any disgusting effect initially for 4 hours. After 24 hours, dead animals were calculated [2].

Extraction of Fixed oil and preparation of FAMEs
The 200 g powder of the plant was extracted with 350ml of the n-hexane in Soxhlet extraction equipment for 6 h. The solvent was regenerated, via an evaporator (rotary) at 42°C. Tri-oro boric acid in ethanol was employed for the generation of the volatile chemical constituent. About 1.6ml of NaOH mixture in 0.5 N methanol was further poured. It was sealed properly and heated for 07 minutes on a water bath instrument. Then compound was chilled and BF 3 solution (in methanol) 2.5 ml was transferred to it. This preserved mockup was warmed for 32 minutes on the water bath and then chilled. Concentrated 6ml NaCl solution was added to esterify solution and then extracted twice through 1 mL of n-hexane. The extracts get were ltered via a membrane lter. Afterward; the prepared samples (1 µl) were injected into GC through an auto-injection system for analysis [51].

GC-MS analysis of FAMEs
Fixed oil was analyzed using GC coupled to MS and auto-injector. Separation of the component was carried out in a column with a length of 35 m, 0.35 mm internal diameter, 0.240 µm thickness. Helium (He) was used as a carrier gas. Other parameters for chromatography were 250°C, 110 kilopascals and 1.8 min as temperature, pressure and solvent cut time of interface.1 µl of sample and standard were administered at 250°C. After that, the column was operated primarily for 4 minutes at 85°C and then increased to 185°C, at an increased rate of 15°C/min. Column remained sustained at 185°C, for 15 min with a residual rise at the rate of 2.5°C/min to 220°C withhold there for 5 minutes. All the constituents got recognized by relating the spectrum acquired with instrument library standard spectra [52].
Fixed oil anti-in ammatory potential in mice Initially, stocksianum xed oil anti-in ammatory effect was determined in mice of both sexes, weighing 25-30 g. The animals have divided arbitrarily into ve groups as 1-5with 06 (n = 6) mice of either sexing each group [53]. Normal saline as a negative control was utilized in Group 1 in 10 ml/kg of body weight. Group 2-4 were treated with 1, 2 and 3 ml/kg, the body weight of FO correspondingly, while group 5 treated with 100 mg/kg Aspirin (positive control), After 30 min of the treatment, all of the groups received 0.05 mL (1% w/v) subcutaneous single injection of carrageenan suspension in right-hand paw sub planar area of the test animals. An initial reading of the in ammation was taken immediately with Plethysmometer (LE 7500 plan lab S.L) after the injection of carrageenan after 01 to 05 hours. The mean volume of the paw of each group 2-5 was observed at different time intervals then comparing with the mean paw volume of group animals of negative control [54]. Edema Percentage inhibition was measured by the formula Inhibition = (A verage -T ested ) / A × 100 Whereas "A" indicates the control group's average in ammation while "T" indicated the tested group's paw volume.
Fixed oil anti-in ammatory mechanism Mice were randomly distributed in different experimental groups. Preliminary the animals received a single intraperitoneal injection of any vehicle like 0.9% normal saline or 10% DMSO or 100 mg/kg of caffeic acid that is a lipoxygenase inhibitor or 100 mg/kg of aspirin or xed oil of T. stocksianum as 3 ml/kg. After half hour of the treatments, in ammation was injected in the right-hand paw of the mice with 0.1 ml of sub plantar injection of 0.5 % w/v arachidonic acid or 0.01 µg/ml of prostaglandin E 2 or 0.1 ml Leukotriene (0.1 µg/ml). Edema in terms of every animal paw bulk got determined before, instantly (0 h) and then for four hours (1, 2, 3 and 4 h) at 01 h interval after injection of the in ammogens [47].

Docking Studies
Computational studies were carried out by Autodock Vina 1.1.2 interrelated Pyrex-0.8. To get a better understanding of the mechanism by which xed oils inhibit in ammation, the structures of enzymes were downloaded from Protein data bank as reported in our previous studies [55]. Crystal structure of Cyclooxygenase enzyme COX-2 was obtained as PDB code 1CX2 and Crystalline structure of lipoxygenase enzyme 5-LOX was acquired as 3O8Y. The structures were modi ed and puri ed through Discovery studio visualizer and stored in PDB format which was further prepared for docking by converting them into PDBQT format.
All the components of xed oils were assessed and those were selected for docking purposes which were obtained in good quantity after GC-MS studies. Palmitic acid attained in 2.77 % in the case of saturated oils and linoleic acid in 23.84% was nominated due to higher values. Structures of interested components ligands were obtained from PUB Chem and saved in PDB format.

Results And Discussions
Teucrium is a rich genus composed of 350 species. Multiple biological accomplishments have been described through this genus. T. royleanum exhibits antioxidant and antimicrobial potentials. The ethyl acetate extract is reported with excellent enzyme inhibition activity against butyrylcholine and acetylcholine . Numerous scientists have reported xed oils and essential oils from different species of Teucrium. The essential oils of T. polium and T. stocksianum contain strong anti-nociceptive properties [26].

Composition of Fixed oil
In the current study, the chromatogram obtained from GC-MS analysis highlighted the presence of twentyone (21) different fatty acids; out of which 14 are saturated. Details of saturated and unsaturated acids are given in Table 1 and Table 2 respectively. Results of all data collected from GC-MS.

Acute toxicity study
During the acute toxicity study, there was no evidence of mortality and behavioral change in the experimental mice. Considering the results of acute toxicity, a dosage regimen of 1, 2 and 3 ml/kg from xed oil could be nominated as the safe dose. Detail of the dosage regimen to animals are given in Table  3 [58].

Preliminary Anti-in ammatory Potential via carrageenan induction in paw edema
Carrageenan is a non-speci c in ammogen, commonly employed for preliminary evaluation of antiin ammatory potential of the test samples. It develops in ammation in two phases, in the early phase chemical mediators, histamine and serotonin are released while the later phase is mediated by another class of mediators that mainly includes prostaglandin E 2 and leukotriene [59]. In preliminary antiin ammatory screening, the FO of T. stocksianum has shown marked anti-in ammatory activity in a dose-dependent manner. The results are show in Table 4. Test sample displayed, 23.07%, 38.91% and 67.87 % inhibition against carrageenan induced paw edema test, at a dose of 1, 2 and 3 ml/kg at 3 rd h of the test sample administration as elaborated in Figure 1.

Possible anti-in ammatory Mechanism of Fixed oil
There are multiple causes of in ammation like it might be initiated by trauma, infection, pollutants, or burn [60]. When any sort of damage occurs to the cytoplasmic membrane, it initiates a cascade of biological reactions, initially, it releases phospholipids which are enzymatically (Phospholipase A 2 ) converted to arachidonic acid. Arachidonic acid is converted to prostaglandins and leukotriene (metabolites), by cyclooxygenase and lipoxygenase respectively, which are strong pro-in ammogens [61].
To determine the involvement of the actual mediator in the anti-in ammatory activity of the xed oil, paw edema in mice was induced with arachidonic acid, prostaglandin E 2 and leukotriene mediators (Table 5).
FO (3 ml/kg, I/p) and caffeic acid (100 mg/kg) exhibited profound protection, 64.92, and 69.43% respectively at 3 rd h against arachidonic acid induced paw edema. While the reference standard drug aspirin remained non-signi cant and could only produce 08.2% protection against edemogen shown in Table 6 and Figure 2.
The test sample also exhibited signi cant 68.75 and 58.3% anti-in ammatory activity against PGE 2 (prostaglandin E 2 ) and LT (leukotriene) correspondingly, with a calculated dose of 3 ml/kg of body weight of the mouse at 3 rd h of test sample administration, depicted in Table 7 and Figure 3.
A literature survey revealed that linolenic acids exhibit strong anti-in ammatory activity (Singh et al., 1997). It is a precursor for Gamma Linolenic acid (GLA), while GLA is metabolized to dihomogamma linolenic acid (DGLA). DGLA is further metabolized by lipoxygenase and cyclooxygenases to produce leukotriene of series 3 and prostaglandins of series 1 (eicosanoids) (Singh et al., 1997). The GC-MS spectra of the xed oil of T. stocksianum have revealed that the test sample is composed of a signi cant quantity (21.16%) of Linolenic acid (Table 2)  To support the anti-in ammation mechanism by components of xed oils, docking studies were performed and binding interactions were analyzed for the components which were obtained in a higher ratio i.e. Palmitic acid in the case of saturated oil and linoleic acid in the case of unsaturated oil. Both chemical moieties were docked inside the binding pocket of the cyclooxygenase enzyme (COX-2). The results of 2D interactions are elaborated on in Figure 4.

Conclusion
Based on the above observed results, it could be summarized that xed oil of T. stocksianum contained the anti-in ammatory potential via both inhibitions of lipoxygenase and cyclooxygenase alleyways of arachidonic acid. However, noteworthy activity was pragmatic through inhibition of prostaglandins. These observations strappingly sustained the traditional natural plant usage in the management of different in ammatory situations. Thus, it's understandable through binding a nity parameters that a large number of molecules persists the ability to inhibit COX-2 synergistically, thus preventing in ammation.

Declarations Ethics approval and consent to participate
The Committee of animal ethics of the University of Malakand KPK accepted the protocols in strict compliance with internationally accepted principles for laboratory animal use and care after conforming to ARRIVE guidelines for the experimental animal studies through letter No; DREC/122 for Control and Supervision of Experiments on Animals standard.

Consent for publication
Not applicable

Availability of data and materials
The data is available on request from corresponding authors on reasonable request.

Competing interests
All other authors declare that they have no competing interests.