Evaluation of Antioxidant, Anti-inflammatory, Analgesic and Antipyretic activities of the Ethanolic Extract along with its Organic soluble fractions of Origanummajorana

Received: 22-Nov-2021;Accepted Date: Dec 06, 2021; Published: 13-Dec-2021

Introduction

Reactive Oxygen Species (ROS) mainly includes various forms of activated oxygen free radicals like superoxide anion radicals (O₂¯) and hydroxyl radicals (OH), non-free radicals species (H₂O₂) and the singled oxygen (1O₂) [1,2]. Lipid Peroxidation accumulation occurs due to excessive generation of ROS induced by various stimuli that initiate the peroxidation of membrane lipids. Variety of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer occurs due to reaction of protein, amines and deoxyribonucleic acid [1] with peroxidation products and their secondary oxidation products such as Malondialdehyde (MDA) and 4-hydroxinonenal [3,4]

Inflammation is the response attributed to cells injury and body tissues through different factors like infections, chemicals, thermal and mechanical injuries [5]. In inflammatory cell, endogenous mediators like histamine, serotonin, bradykinin, prostaglandins are highly abundant and among them prostaglandins are ubiquitous substances that modulate cell and tissue responses involved in inflammation. For the treatment of inflammation related diseases like arthritis, asthma and cardiovascular disease, Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are the mostly used [6]. These drugs have severe adverse effects like gastric lesions for NSAIDs, adverse cardiovascular thrombotic effects for selective cyclooxygenase-2 (COX-2) inhibitors [7], Moreover reactive oxygen species also contribute to several chronic inflammatory diseases like psoriasis, atopic dermatitis and contact dermatitis [8].

In ancient civilization, Natural products have the ability to provide compounds of novel and complex structures that are capable of interacting with biological systems, used various diseases treatment and for body system revitalization. The research on the plant with folkloric use as analgesic, anti-inflammatory agents with antioxidant properties should therefore be considered as a logical research strategy in the exploration of new analgesic and anti-inflammatory drugs [9].

Therefore, the present research study was planned to investigate the possible antioxidant, analgesic, anti-inflammatory and antipyretic activities of Ethanolic extract along with its organic soluble fractions (dichloromethane and ethyl acetate) of the Origanum majorana in different experimental models.

Materials and Methods

Collection of plant

Origanum majorana were collected from surrounding areas of Peddapuram, East Godavari dt., of Andhra Pradesh. The plant authentication was done by Dr. T. RAGHURAM Taxonomist, Maharani College, Peddapuram.

Preliminary phytochemical screening: Preliminary phytochemical screening of Origanum majorana Ethanolic Extract and fractions were performed to test the presence of the active chemical constituents such as alkaloids, flavonoids, tannins, phenolic compounds, saponins, fixed oils and fats [10].

Quantitative phytochemical testing: Aliquots of extract and Fractions were prepared by dissolving 10 mg of extract and fractions in 10 mL of methanol to get 1000 μg/ml.

Estimation of phenolic contents: The phenolic content of the Ethanolic extract and fractions of Origanum majorana (1 mg/ml, aliquots) was determined by using the method Folin-Ciocalteu [11]. 0.5 ml aliquots of extract was mixed with 3 ml Folin-Ciocalteu reagent (1:10 v/v) and the mixture allowed to stand for 5 min. 4 ml of 20% w/v of sodium carbonate solution was added into the mixture tube. The tubes were kept aside for 15 min at 30^°C for colour development. The absorbance was measured at 765 nm by spectrophotometer. Phenolic content was estimated from the calibration curve using standard gallic acid in methanol and the results were expressed as gallic acid equivalent mg/100 mg dry weight of extract.

Estimation of total flavanoids: Total flavonoid content of the Ethanolic extract and fractions of Origanum majorana (1 mg/ml, aliquots) was determined by aluminium chloride method [12]. To 0.6 ml of aliquots of extract,1.8 ml of methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1 M sodium acetate and 3 ml of distilled water were added and left at 30^°C. The absorbance was measured individually after 30 min at 415 nm. Total flavonoid was estimated from the calibration curve using standard quercetin in methanol and the results were expressed as quercetin equivalent mg/100 mg dry weight of extract.

Experimental animals: Albino rats weighing between 120-160 g of either sex were used for the study. Animals were housed in colony cages at ambient temperature of 25 ± 20 C, 12 h light/dark cycle and 50 ± 5% relative humidity with free access to food and water ad libitum. To the laboratory environment the animals were acclimatized for at least one week prior to experimentation. Animals were deprived of food except water in overnight and during the period of experimentation. The animal experiments were performed based on the Institutional Ethics Committee (IEC) approval and guidelines REG. No.

Acute toxicity and gross behavioral study: The experimental animals were fasted overnight, divided into groups (n=6) and were orally fed with increasing doses (250, 500, 750, 1000,1250,1500 mg/kg body weight) of Ethanolic extract and fractions of Origanum majorana. The animals were observed during 24 h to find out percentage mortality and gross behavioural changes after administration of the extracts [13-15].

Evaluation of Invitro anti-oxidant activity

Hydroxyl radical scavenging assay: The scavenging ability of the Ethanolic extract and fractions of Origanum majorana of hydroxyl radicals was determined according to the method described by Smirnoff and Cumbes [16] with some modifications. Briefly, individual sample extract/ fractions (1 ml) at different concentrations (50, 100, 300, and 500 μg/ml) were added to the reagent containing 1 mL 1.5 mM FeSO₄, 0.3 ml 20 mM sodium salicylate and 0.7 ml 6 mM H₂O₂ Later on the sample was incubated at a temperature of 37^°C for 1 h and absorbance of the reaction mixture was read at 562 nm.

Scavenging ability on hydroxyl radicals (%)=[(Ao-A1)/ Ao] ×100

Where, Ao indicates the control reaction (containing all reagents except the sample extract) absorbance, and A1 is the sample extract absorbance. Ascorbic acid was used as positive controls.

NO scavenging activity

The scavenging effect of the extract on NO was measured according to the method of Marcocci and colleagues [17]. Briefly, sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS) (pH 7.4) was mixed with different concentrations of the test sample (100-1000 μg/ml) and incubated at 25^°C for 150 minutes. After incubation, nitrite produced from sodium nitroprusside was measured by Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% 1-naphthylethylenediamine dihydrochloride in water). The absorbance was immediately read at 570 nm. Ascorbic acid was used as positive controls.

NO scavenging activity (%)=[(Ao–A1)/Ao] ×100

Where, Ao indicates the control reaction (containing all reagents except the sample extract) absorbance, and A1 is the sample extract absorbance. Ascorbic acid was used as positive controls.

Analgesic activity

Tail-flick method: Analgesia was measured using modified method of D Amour and Smith [18] called as tail flick method. Analgesia is indicated by increase in reaction time. Reaction time is the time between placing the tail of the rat on the radiant heat source and sharp withdrawal of the tail. Minimum cut off time period of about 10 seconds was taken as maximum latency so as to avoid thermal injury to the animals while noting down the reaction time. Animals that showed a mean reaction time outside the range of 5-6 seconds, were discarded. In all the groups, tail-flick test was performed prior to drug administration (at O min) and at 15,30, 60 and 120 minutes after drug administration, and the reaction time at each time interval (test latency) was calculated.

Acetic acid induced writhing method

The writhing model represents a chemical nociceptive test based on the induction of peritonitis like condition in animals by injecting irritant substances i.p. After 30 minutes of drug administration, 0.1 ml of 1% acetic acid solution was injected i.p. Rats were kept individually in glass beakers and were allowed to elapse for a period of about 5 minutes. The animals were then observed for about 10 minutes and the numbers of writhes were recorded. For scoring purpose, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb [19]

Percentage inhibition was calculated using the following formula:

% inhibition= { (Wc- Wt) × 100 } / Wc

Where, Wc=No. of writhes in control group, Wt=No. of writhes in test group

Eddy`s hot plate method: The time of reaction to pain stimulus of the rats placed on the plate, heated at 55^° ± 0.5^°C was recorded at 0,15, 30, 60 and 120 min after the administration of the Ethanolic extract and fractions of Origanum majorana in two different doses (200 mg/kg and 400 mg/kg, b.w) and vehicle were administered orally before 60 minutes. The increase in reaction time against control group was calculated [20].

Invitro antiinflammatory activity

Protein denaturation method procedure: Test solution (0.5 ml): Made up of 0.45 ml of 5% w/v aqueous solution of Bovine serum albumin and 0.05 ml of test samples of various concentrations (Ethanolic extract and fractions of Origanum majorana of 100 and 200 ug/ml) [21-23].

Product control solution (0.5 ml): Made up of 0.45 ml of distilled water and 0.05 ml of test samples of various concentrations (Ethanolic extract and fractions of Origanum majorana of 100 and 200 ug/ml).

Standard solution (0.5 ml): Made up of 0.45 ml of 5% w/v aqueous solution of bovine serum albumin and 0.05 ml of various concentrations of Diclofenac sodium. All the solutions were adjusted to PH 6.3 using 1N HCl. Incubation of samples were carried out for 20 min at 37^°C. Later, the samples were kept for 3 min at an elevated temperature of 57^°C. 2.5 ml phosphate buffer was added to the above solutions after cooling. The absorbance was recorded at a wavelength of 416 nm using a UV/Visible Spectrophotometer.

% Inhibition of Protein Denaturation=100-[{(O.D of test solution-O.D of product control)/O.D of test control} × 100]

The control represents 100% protein denaturation. The results were compared with Diclofenac sodium.

HRBC membrane stabilization method

Principle: The principle of this method is the stabilization of the human red blood cell membrane from the hypo-tonicity induced membrane lysis.

Preparation of Human Red Blood Cells (HRBC) Suspension

Fresh whole human blood (2 ml) was collected and was mixed with an equal volume of sterilized Alsever solution (0.8% sodium citrate, 2% dextrose, 0.05% citric acid and 0.42% aqueous solution of sodium chloride in). The blood was subjected to centrifugation for 10 min at 3000 rpm. The compacted cells were washed thrice with isosaline solution (0.85%, pH 7.2). The volume of the blood was measured and was reconstituted as a 10% v/v suspension with isosaline.

Procedure (hypotonic solution-induced haemolysis): The reaction mixture (of total volume 4.5 ml) is made up of 2 ml of hyposaline solution (0.25% w/v NaCl), 1 ml of 0.15 M phosphate buffer (pH 7.4) and 1 ml of test solution (Ethanolic extract and fractions of Origanum majorana of 100 and 200 ug/ml) in isosaline. After that, 0.5 ml of 10% HRBC suspension in isosaline was added. For test control, 1 ml of distilled water was used in the place of hyposaline (to produce 100% haemolysis), while the product control was made lacking the RBCs. The mixtures were incubated for 30 min at 37^°C and centrifuged for 20 min at 3,000 rpm. The reference drug was Diclofenac sodium. The haemoglobin content of the suspension was measured as function of absorbance, at 560 nm using a spectrophotometer. Percentage membrane stabilizing activity was calculated as follows, The percentage of HRBC membrane stabilization or protection was calculated by using the following formula:

Percentage Stabilization=Absorbance of control-absorbance of test × 100 Absorbance of control

Invivo antiinflammatory activity

Carragenan induced Paw edema: Edema was induced by injecting 0.1 ml of 1% solution of carragenan in saline to (sub plantar) right hind paw of rats. The Ethanolic extract and fractions of Origanum majorana in two different doses (200 and 400 mg/kg, b.w) and vehicle were administered orally 60 minutes prior to injection of carragenan. The volume of edema of injected and contra collateral paws were measured at 0, 1, 2, 3 and 4 hours after induction of inflammation using a plethysmograph and the percentage of anti-inflammatory activity was calculated. The values are compared with standard drug Diclofenac (10 mg/kg) [24].

Antipyretic assay: The phenomena of increase in tempertaure was induced by injecting 15% suspension of Brewer’s yeast (Saccharomyces cerevisiae), following a standard method [25]. Into the rectum, a thermister probe was inserted 3-4 cm deep to record the basal rectal temperature. A subcutaneous injection of 10 ml kg-1 of 15% w/v Brewer’s yeast suspended in 0.5% w/v methylcellulose solution was given to animals and at 19 h after injection, the rats rectal temperature was recorded. Immediately the Ethanolic extract and fractions of Origanum majorana were administrated at doses of 200 and 400 mg kg-1, b.wt. Paracetamol was used as standard drug. Before extract/fractions or vehicle or paracetamol administration the Rectal temperature of all the rats was recorded at 19 h after injection, again at 1 h interval upto 5 h, after yeast injection.

Statistical analysis

Data were analyzed by Graphpad INSTAT^® version 3.0 software and presented as mean ± S.E.M. values. The statistical tests used were one-way analysis of variance (ANOVA) followed by Dunnet‘s multiple comparison test. The levels of statistical significance ranged from p<0.05.

Results

Preliminary and quantitative phytochemical screening

The results imply that extracts contains alkaloids, flavonoids, saponins, carbohydrates, proteins and Amino acids which are the main phytochemical groups with biological activities. The results of Quantitative Phytochemical screening were tabulated (Table 1).

Table 1: Quantitative Phytochemical Determination of Ethanolic extract and fractions of Origanum majorana.

The results of antioxidant activity were expressed in terms of IC50 values using different antioxidant methods. The calculated IC50 values using Nitric oxide and Hydroxyl radicals method for Origanum majorana ethyl acetate fraction are 21.42 μg/ml and 28.70 μg/ml and for ascorbic acid it is 33.11 and 1.69 μg/ml. The results are indicated in Table 2. From the results obtained it may be postulated that Origanum majorana extract/fractions reduces the radicals to the corresponding hydrazine when it react with the hydrogen donor in the antioxidant principles. Free radical scavenging activity of the Origanum majorana is concentration dependent. Lower IC 50 value reflects better protective action.

Table 2: Effect of Ethanolic extract and fractions of Origanum majorana against NO and hydroxyl radical.

Acute toxicity studies

No toxicity or death was observed in the experimental rats when they are subjected to toxicity study. To establish the safety of the Ethanolic extract and fractions of Origanum majorana administered to both male and female rats. There were no significant toxic signs or death during the the entire observation period. The Ethanolic extract and fractions of Origanum majorana did not exhibit any mortality up to the dose level of 1500 mg/kg.

Analgesic activity

The present study investigated the analgesic and effect Ethanolic extract and fractions of Origanum majorana in rats. In the analgesic study the extract produced significant analgesic effects in the 3 models of pain employed.

Tail flick method

In tail flick method Rats are treated with ethanolic extract/fractions of Origanummajorana doses (200 mg/kg and 400 mg/kg orally) significantly inhibited nociception in rats. Paracetamol 100 mg/kg significantly inhibited pain perception at 30 mins by 75.0% (p<0.05). Whereas Origanum majorana Ethyl acetate fraction 200 mg/kg at 30 mins by 56.8% and 400 mg/kg at 30 mins by 61.8%. The results are tabulated in Table 3.

Table 3: Effect of Ethanolic extract and fractions of Origanum majorana in Rats Using Tail Flick Method.

Eddy’s hot plate method

In Eddy’s Hot Plate method rats are treated with ethanolic extract of Origanum majorana doses (200 mg/kg and 400 mg/kg orally) significantly inhibited noceception in rats. Tramadol 5 mg/kg significantly inhibited pain perception at 60 mins by 83.3%. Origanum majorana Ethyl acetate fraction 200 mg/kg at 60 mins by 61.8% and 400 mg/kg at 60 mins by 74.3%. The results are tabulated in Table 4.

Table 4: Effect of Ethanolic extract and fractions of Origanum majorana in Rats Using Eddy’s Hot Plate Method.

Acetic acid induced writhing response

In Acetic acid induced writhing responses method, rats are treated with ethanolic extract/Fractions of Origanum majorana doses (200 mg/kg and 400 mg/kg orally) shows significantly inhibited nociception in rats. Tramadol (5 mg/ kg) showed 94.38% protection against acetic acid induced writhings in rats. Origanum majorana Ethyl acetate fraction was found to be 68.53% at 200 mg/kg and 83.14% at 400 mg/kg (Table 5)

Table 5: Effect of Ethanolic extract and fractions of Origanum majorana In Rats Using Acetic Acid Induced Writhing Responses

Anti-inflammatory activity

Invitro studies: Protein denaturation method: Invitro Anti- inflammatory effect by Protein denaturation method, maximum percentage inhibition of Origanum majorana Ethylacetate fraction was found to be 89.54% at 200 ug/ ml. Diclofenac sodium showed the maximum inhibition 93.79% at 20 ug/ml concentrations. The results are tabulated in Table 6

Table 6: Invitro Anti Inflammatory Effect of Ethanolic extract and fractions of Origanum majorana By Protein Denaturation Method

HRBC Membrane stabilization method

Invitro Anti-inflammatory effect by HRBC Membrane Stabilization method, maximum percentage inhibition of Origanum majorana Ethylacetate fraction was found to be 69.87% at 200 ug/ml. Diclofenac sodium showed the maximum inhibition 73.70% at 20 ug/ml concentrations. The results are tabulated in Table 7

Table 7: Invitro Anti Inflammatory effect of Ethanolic extract and fractions of Origanum majorana by HRBC Membrane Stabilization Method

All the values are expressed aas mean ± SEM ,n=3

InVivo studies

Carrageenan induced Paw Oedema in rats: Invivo anti-inflammatory effect of ethanolic extract of Origanum majorana in Carrageenan induced paw oedema in rats. It was observed that rats are treated with ethanolic extract/Fractions at 200 mg/kg and 400 mg/kg showed significant anti- inflammatory activity and caused a significant inhibition in the percentage increase Carrageenan induced rat paw oedema when compared with standard Diclofenac sodium. The results are tabulated in Table 8.

Table 8: InVivo Anti Inflammatory effect of Ethanolic extract and fractions of Origanum majorana in Carrageenan Induced Paw Oedema In Rats

Effect of Ethanolic extract and fractions of Origanum majorana on Brewer’s induced pyresia in rats and the results are tabulated in Table 9.

Table 9: effect of Ethanolic extract and fractions of Origanum majorana on Brewer’s induced pyresia in rats

Discussion

Phenolic Compounds (flavonols, flavonoids, tannic acid, anthocyanins, phenolic acid, etc.) possess an scavenging activity against free radicals [26] and exhibit a wide range of therapeutic potentials like anti-allergenic, anti-atherogenic, anti-inflammatory, anti-microbial, anti-thrombotic, cardioprotective and vasodilatory effects [27-30]. Antioxidant Activity is mainly attributed to the beneficial effects derived from phenolic compounds [31] which are major determinants of food [32] and could therefore acts as natural source. These play an vital role in inducing the cellular antioxidant system and increase approximately 50% cellular glutathione concentration. In the modulation of γ-glutamylcysteine synthase in both cellular antioxidant defenses and detoxification of xenobiotics, Flavonoids plays an important role [33]. Ethylacetate fraction of Origanum majorana, the highest amount of total phenolic and flavonoid content was found to be 12.31 ± 0.21 mg g-1 plant extract (in GAE) and 33.21 ± 0.19 mg g-1 plant extract (in quercetin equivalent), respectively. The results are tabulated in Table 1.

To assess the natural antioxidants efficacy either as pure compounds or as plant extract, a wide variety of invitro methods have been developed in which antioxidant compounds act by several mechanisms. Antioxidants scavenge the reactive oxygen species or reduce the free radicals formation [34].

In fact, the phenolic compounds radical scavenging capability is mainly due to their hydrogen donating ability/ number of hydroxyl groups present which in turn is closely related both to the chemical makeup and spatial conformation [35]. In the present study, this possibility is supported by the estimation of total polyphenols and flavonoids [36] which were found to be present in high concentration in the Origanum majorana test extract/fractions.

In the biological system, Hydrogen peroxide is present at low concentration and is catalyzed in to water and oxygen. Hydroxyl radicals are generated during this process and considered as the most damaging species in the biological system which exhibits cellular injuries by causing damage of proteins, DNA and lipids [37]. In pathophysiological process, hydroxyl radical is considered as a most damaging species and it is able to damage almost each molecule of biological system and may be responsible for the development of carcinogenesis, mutagenesis and cytotoxicity [38]. The Hydroxyl radicals are formed by the reaction between hydrogen peroxide and the ferrous that would reacts with 2-deoxyribose. By the addition of thiobarbituric acid, this reaction can be blocked that would produce red color if the malonaldehyde produced as a result of the reaction between the free radical and 2-deoxyribose. Origanum majorana test extract/fractions when added to the reaction mixture vigorously scavenged the hydroxyl radicals and inhibited the degradation of 2-deoxyribose. In air, water, human body, plants, microorganisms and food, Hydrogen peroxide occurs naturally at a very low concentration and is quickly decayed into water and oxygen creating hydroxyl radicals (OH) that begins lipid peroxidation and consequently induce DNA damages [39]. Origanum majorana Ethyl acetate fraction significantly scavenged hydrogen peroxide that can be suggested to the presence of phenolic compounds that could contribute electrons to hydrogen peroxide, thus neutralizing it into water.

Nitric oxide (NO) is synthesized from amino acid L-arginine by vascular endothelial cells, phagocytes and certain cells of the brain. Because of its unpaired electron, Nitric oxide is classified as a free radical and displays an important reactivity with certain types of proteins and other free radicals. The NO toxicity becomes adverse when it reacts with superoxide radical, forming a highly reactive peroxynitrite anion (ONOO−) [40]. From sodium nitroprusside, the nitric oxide generated reacts with oxygen to form nitrite. The nitrite ions diazotize with sulphanilamide acid and couple with naphthyl ethylenediamine, forming pink colour [41]. Antioxidants donate protons to the nitrite radical and due to this the absorbance is decreased. To measure the extent of nitrite radical scavenging, the decrease in absorbance was observed [42]. Origanum majorana test extract/fractions exhibited good action against nitrite radicals (Table 2).

Three anti-nociceptive models; acetic acid-induced writhing reflex, tail flick and hot plate models were used to evaluate the analgesic activity of Origanum majorana test extract/fractions since analgesic drugs testing involves measuring nociception and the reaction of animals to painful stimuli [43] The stimulus may be thermal (tail flick or hot plate tests), chemical (acetic acid-induced writhing or formalin tests) [44]. Acetic acid-induced writhing reflex is a model of visceral pain used for screening analgesic drugs [45] and chemicals like phenylquinine and acetic acid could induce writhing reflex in laboratory animals [46].

Glacial acetic acid Intraperitoneal injection produced abdominal writhing by stimulating the chemo sensitive nociceptors [47]. This phenomena is indicated by percent reduction in the number of abdominal constrictions [48]. In the present study, the reference drug and Origanum majorana test extract/fractions significantly decreased the mean number of abdominal constrictions or writhes in an dose dependent manner. Ethylacetate fraction exhibited better cativity compared to ethanolic extract and dichloromethane fraction (Table 5). Acetic acid-induced writhing model causes pain sensation by triggering inflammatory response and such pain stimulus leads to arachidonic acid release from tissue [49]. The analgesic effect of Origanum majorana test extract/fractions may be mediated through peripheral pain mechanism and or through suppression of prostaglandin pathway by inhibiting prostaglandin synthesis, a peripheral mechanism of pain inhibition [50].

The tail flick and hot plate models have been used to study centrally acting analgesics [51]. In these models, sensory nerves sensitise the nociceptors and the involvement of endogenous substances such as prostaglandins are less [52]. From the results obtained it was observed that, Origanum majorana test extract/fractions exhibited significant analgesic activity by prolonging the response time to termal stimuli (Tables 3 and 4). Protein Denaturation is a phenomena where proteins loses their tertiary structure and secondary structure by application of external stress or compounds like strong acid or base, a concentrated inorganic salt, an organic solvent or heat. During the process of denaturation many of the biological proteins lose their biological function and this is an major cause of inflammation.. Ethanolic extract/Organic Soluble Fractions of Origanum majorana were effective in inhibiting heat induced albumin denaturation. Maximum inhibition of 89.54% was observed at 200 μg/ml. Diclofenac, a standard antiinflammation drug showed the maximum inhibition 93.7% at the concentration of 20 μg/ml compared with control (Table 6).

Another method of invitro antiinflammatory study is the HRBC membrane stabilization method because the erythrocyte membrane is analogous to the lysosomal membrane [53,54] and its stabilization indicates that the Ethanolic, Ethyl acetate and Hexane extracts of Ethanolic extract/Organic Soluble Fractions of Origanum majorana may well stabilize lysosomal membranes. Lysosomal membrane Stabilization is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil, such as bacterial enzymes and proteases, that causes tissue inflammation and damage upon extra cellular release causes various disorder. and are said to be related to acute or chronic inflammation. The NSAIDS act either by inhibiting these lysosomal enzymes or by stabilizing the lysosomal [55]. Ethanolic extract/Organic Soluble Fractions of Origanum majorana were effective in stabilising the membrane. Maximum stabilisation of 69.87% was observed at 200 μg/ml. Diclofenac, a standard antiinflammation drug showed the maximum stabilisation 73.7% at the concentration of 20 μg/ml compared with control (Table 7).

Commonly used experimental animal model for screening acute inflammation is Carrageenan induced oedema and is believed to be biphasic. The early phase (1-2 h) in the damaged tissue surroundings is mediated by histamine, serotonin and increased synthesis of prostaglandins. The late phase is characteriused by release of prostaglandins and mediated by bradykinin, leukotrienes, polymorphonuclear cells [56-57]. Since the extract/fractions significantly reduced paw edema induced by carrageenan in the second phase and this may be attributed due to inhibition of cyclooxygenase synthesis by the extract/fractions particularly ethylacetate fraction and this effect is similar to that produced by non-steroidal anti-inflammatory drugs such as diclofenac, whose mechanism of action is inhibition of the cyclooxygenase enzyme.

In addition, the several ROS release [58] and excessive Nitric Oxide (NO) due to the stimulation of neutrophils during tissue damage and inflammation [59] causes a variety of disease like atherosclerosis, cancers, migraine, Parkinsonism [60-62] demonstrated that polyphenols are more potent inhibitors of NO synthase activity and NO production.

As Ethylacetate fraction of Origanum majorana exhibited significant free radical as well as NO scavenging activity, so this can be responsible for the reduction of inflammation in the carrageenan induced paw edema in rats. Furthermore, [63,64] it has been observed that polyphenols (including phenolic compounds and flavonoids) rich Origanum majorana possess antioxidant as well as anti-inflammatory activity. So polyphenols including flavonoids present in the Origanum majorana may induce the antioxidant properties as well as may also plays an important role in mediating the anti-inflammatory effects.

Fever occurs as a result of infection or one of the sequelae of tissue damage, inflammation, graft rejection or other disease state [65]. Body temperature regulation requires a delicate balance between the production and loss of heat. The hypothalamus is the one that regulates the set point at which body temperature is maintained. Antipyretic potential of the Origanum majorana test extract/fractions exhibited significant reduction in the brewer’s yeast provoke elevated body temperature in the animals. In suppressing TNF-α, flavonoids have been used as an antipyretic agent by [66]. The antipyretic properties of the Origanum majorana test extract/fractions could possibly be associated with the presence of these Phytochemicals.

Phytochemical analysis revealed that the extract/fractions contain alkaloids, glycosides, phenolic compounds, tannins, steroids and flavonoids. Flavonoids, phenolic compounds and glycosides were associated with anti-inflammatory, antipyretic and antioxidant activities [67]. Phenolic Compounds and flavonoids are known to inhibit both the cyclooxygenase and 5-lipoxygenase pathways [68]. Apart from this flavonoids also have anti-inflammatory properties due to their inhibitory effects on enzymes involved in the production of the chemical mediator of inflammation [69].

Conclusion

The present research study indicated that the analgesic, anti-inflammatory, antipyretic and antioxidant effects observed in this research study are perhaps due to the activity of one or more of the identified classes of compounds. This information should be considered for future purification of anti-inflammatory and antioxidant active compounds from this natural source. Our results indicate that Origanum majorana ethanolic extract/organic soluble fractions has both effective pronounced analgesic, antipyretic, anti-inflammatory and strong antioxidant activity.

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