Analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of stem bark of Anogeissus latifolia Roxb

Stem bark of Anogeissus latifolia Roxb (family: Combretaceae) is used traditionally and ethnomedicinally to alleviate pain, inflammation and fever conditions. The current study investigates the analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of stem bark of Anogeissus latifolia Roxb. The HPLC studies were carried out to standardize the stem bark ethanolic extract of Anogeissus latifolia (ALEE) using ellagic acid as a marker. ALEE was screened for analgesic (formalin-induced pain and acetic acid induced writhing) and anti-inflammatory (formalin and carrageenan-induced paw oedema) activities in Wistar rats. Before 1 h of acetic acid or formalin or carrageenan injection, rats were orally fed with ALEE (100, 200 and 400 mg/kg), Aspirin (100 mg/kg) or Indomethacin (10 mg/kg). Antipyretic effect was studied in brewer’s yeast-induced pyrexia model in rats using Paracetamol (100 mg/kg) as a standard drug. HPLC analysis of ALEE revealed the presence of ellagic acid. ALEE treatment (200 and 400 mg/kg) significantly inhibited pain response in both models. ALEE treatments prevented the raise of paw volume in both in-vivo models with percent inhibition of 44.40 and 46.21, respectively at 5 h. ALEE also showed a significant reduction of yeast-induced pyrexia till 4 h of treatment. ALEE exhibited analgesic, anti-inflammatory and antipyretic property in experimental models and validates traditional use of ALEE in pain, inflammation and fever.


Background
The medicinal plants or their formulations are widely used in traditionally practices by different ethnic population worldwide for the prevention and/or treatments of several chronic diseases. In spite of development of newer technologies and advancement in modern medicine, a high proportion of world population still relies on traditional systems of medicine to fulfill their medical care [1]. The public interest in herbal is increasing exponentially day by day.
Inflammation is a defensive response protecting against the injured tissue caused by various stimuli such as heat, chemicals, and immunological reactions. Generally it is associated with dolorous condition of pain and hyperthermia and needs timely pharmacological treatment [2]. Though current treatment includes NSAIDs (Nonsteroidal anti-inflammatory drugs) for relieving painful conditions, inflammation as well as fever but their unrestricted use poses several side effects such as ulcers, hemorrhage [3], liver and kidney toxicities [4]. Hence, there is an inclination towards the herbal drugs for alleviation of pain and inflammation. In Ayurvedic system of medicine, plant based medicine are widely as Vedana shamak or shothhar [5]. Thus, there is increasing interest of peoples in the validation of traditional medicinal plants in India and other countries for treatment of inflammatory conditions.
In view of the traditional and ethnomedicinal use of stem bark of Anogeissus latifolia in inflammatory and dolorous conditions, it was thought worthwhile to study its analgesic, inflammatory and antipyretic effect in the different models of pain, inflammation and fever in Wistar rats, respectively.

Drugs and chemicals
Ellagic acid (percent purity> 95%) was purchased from Cayman Chemical Company Michigan, USA. Carrageenan was procured from Sigma-Aldrich, USA while Brewer's yeast was procured from M.P. Biomedicals, USA. Aspirin, indomethacin and paracetamol were obtained as gift sample from M/S Royal Research Center, Navsari, Gujarat, India. During the experimental study all analytical grade solvent, chemicals and reagents used in the research work.

Collection and authentication of plant material
Stem barks of Anogeissus latifolia Roxb (AL) were collected in the month of September, 2015 from Ghatigaon forest of Gwalior District, Madhya Pradesh, India and authenticated by the Chief Botanist (Mr. N. K. Pandey), Regional Ayurveda Research Institute for Drug Development, Gwalior, India. Voucher specimen submitted in the herbarium of the Institute for future (no. 5-4/ 10-11/NRIASHRD/Tech/Survey/1516).

Preparation of ALEE
Plant material was dried in dark at 25-32°C. The shade dried plant material was subsequently ground to powder (coarse) and passed through 60 no sieves. The dried plant material was treated with 95% ethanol for extraction in soxhlet assembly. The extract was concentrated in a rotary evaporator to obtain ALEE with 14.01% w/w percent yield.

Preliminary phytochemical screening
Phytochemical screening of ALEE was screened by the well establish standard methods [19]. The quantitative estimation of total phenolic and flavonoids were done by the previously described method [20].
High performance liquid chromatography (HPLC) analysis of ALEE HPLC analysis of ALEE was carried out at Royal Research Center, Navsari, Gujarat, India. The HPLC system (Shimadzu Corporation, Japan model no. LC-2010A) comprised of Enable C-18G HPLC column (particle size-250 × 4.6 mm size and 5 μm inner diameter). The photodiode array detector and class-VP software were available in the system. Samples (ellagic acid and ALEE at 0.1 and 50 mg/ml, respectively) were eluted in mobile phase  [21]. The percentage of ellagic acid was estimated by the using the areas obtained in the chromatograms of ALEE and standard.

Experimental animals
Healthy adult male Wistar rats (200-250 g) were procured from Defense Research & Development Establishment (DRDE), DRDE Gwalior, India and housed in polypropylene cages with autoclaved husk bedding in a group of 2-3 animals/ cage. They were fed standard rodent chow (Ashirwad brand, Chandigarh, India) and water ad libitum and were housed at IPS College of Pharmacy, Gwalior, Madhya Pradesh (Registration: 1039PO/Re/S/07/CPCSEA) at housing temperature: 25 ± 2°C; humidity: 50 ± 5% and light-dark (1:1) cycle. All experimental protocols were performed during day time day (10 AM to 5 PM) after approved (proposal no. 1783/2015) by Institutional Animal Ethics Committee. The sample size (n) was determined by a power analysis (power > 0.80; a = 0.05, two tailed). The calculated sample size was 5 per group.

Acute toxicity study
Acute toxicity of ALEE was carried out by the following OECD 423 guidelines [22] and methods described previously [23,24]. The orally administration of ALEE with limit test dose of 2000 mg/kg was done in overnight fasted rats (sample size = 3) and observed for 14 days for any death or toxic signs.

Assessment of analgesic activity Writhing test
The analgesic effect of ALEE was screened using acetic acid-induced writhing test [25]. Intraperitoneal injection of acetic acid (10 ml/kg, 0.6%) in rats caused writhings (stretch, torsion to one side, retraction of the abdomen and opisthotonous so that the belly of the rodent touches to the floor) which are counted. Total 25 rats were randomly divided into 5 groups each containing 5 rats. Rats were pretreated with vehicle (4% gum acacia) or ALEE (100, 200 and 400 mg/kg) or aspirin (100 mg/ kg) 1 h before acetic acid administration and thereafter the observations were made. The number of writhing was counted for 30 min. Aspirin (100 mg/kg, orally) was as reference standard. The analgesic activity was estimated by the formula given below.
Where, Nc is mean number stretchings in control Nt is mean number stretchings in test.

Formalin-induced pain
ALEE was screened for inflammatory pain evaluated by formalin-induced pain model in rats [26] with some modifications. Total 25 rats were randomly divided into 5 groups each containing 5 rats. After 1 h orally pretreated rats with vehicle (4% gum acacia) or ALEE (100, 200 and 400 mg/kg) or indomethacin (10 mg/kg) intraperitoneal administration of 0.05 ml of 2.5% commercially available 37% formalin in dorsal surface of left hind paw in rats. Time of licking was recorded for 30 min followed by formalin administration. The early phase (10 min) and late phase (10 and 30 min) which represents neurogenic and inflammatory pain response, respectively. Analgesic activity (% Inhibition) at early and late phase was calculated by the following formula.
Where, Nc is average licking (sec) in control,

Assessment of anti-inflammatory activity Carrageenan-induced paw oedema
The anti-inflammatory activity of ALEE was assessed in rats using carageenan-induced paw oedema model as per the previously described method [27]. Carrageenan suspension (0.1 ml of 1% w/v in normal saline) was injected into the sub-plantar region of right hind paw. Total 25 rats were randomly divided into 5 groups each containing 5 rats. Rats were pretreated with vehicle or ALEE (100, 200 and 400 mg/kg, orally) or reference standard indomethacin (10/mg/kg) at 1 h earlier to carrageenan injection. The paw volume at 0, 1, 3, and 5 h after carageenan administration was measured in ml using Plethysmometer (Ugo Basile, Italy. The antiinflammatory activity (% Inhibition) was calculated by following formula.
Where, Pt = Paw volume after carrageenan administration P0 = Paw volume before carrageenan administration

Formalin-induced inflammation
The animals previously studied for pain were further assessed for measuring inflammatory response [28]. Total 25 rats were randomly divided into 5 groups each containing 5 rats. Similar treatments and evaluation parameters were opted in this model as above and the percent inhibition of anti-inflammatory activity was calculated.

Antipyretic activity
The antipyretic activity was assessed by using Brewer's yeast-induced pyrexia model in rats [29]. Before inducing pyrexia, initial rectal temperature was recorded using a digital thermometer. Pyrexia was induced by subcutaneously injecting 15% Brewer's yeast (10 ml/kg body weight) in 0.5% w/v in distilled water. After 18 h of yeast injection, the rats having increased temperature of more than 0.5°C were selected and further 25 rats were randomly divided into 5 groups each containing 5. Control group received 15% oral suspension of yeast in distilled water. Paracetamol (100 mg/kg, orally) was used as a reference standard drug in the study while ALEE was fed orally at 100, 200 and 400 mg/kg. The rectal temperature was noted at 0, 1, 2, 3, and 4 h for all groups.

Statistical analysis
All data were analyzed by one-way ANOVA followed Dunnet's multiple comparision post hoc tests or two way ANOVA followed by Bonferroni post hoc test, wherever applicable using prism pads software. A value of P < 0.05 was taken significance in all cases.

Preliminary phytochemical screening
Phytochemical tests revealed the occurrence of carbohydrates, phenols, saponins, alkaloids, steroids, flavonoids, quinones, furanoids and triterpenoids. The estimated total phenolic content was and flavonoid content were 62.86 mg gallic acid equivalent and 40.64 mg quercetin equivalent per gram ALEE, respectively.

HPLC analysis
HPLC analysis estimated ellagic acid in ALEE. The peaks represented by standard ellagic acid and ALEE were represented in Fig. 1 (A and B, respectively). The percentage of ellagic acid in ALEE was estimated as 0.38% w/w. There was a linear relation observed between the peak area verses concentration of ellagic acid over the concentration range of 5-15 μg/ml with the regression coefficient (r 2 ) of 0.999. The recovery rates were higher than 99% indicating high accuracy. The intra-day and interday RSD were 1.17 and 1.20, respectively indicating high precision or repeatability of the method. The calculated LOD and LOQ of ellagic acid were 0.38 and 0.5 μg/ml, respectively. The validation parameters are mentioned in Table S1-S4 (Supplementary material).

Acute toxicity of ALEE
Acute toxicity study at limit oral dose 2000 mg/kg revealed normal behavioral and exhibited no death or lethargy and no signs of toxic effects in any behavioral patterns up to 14 days. The LD 50 of ALEE is greater than 2000 mg/kg, orally, and it seems safe and non-toxic.

Analgesic activity
Effect on acetic acid-induced writhing ANOVA (One way) followed showed that ALEE exhibited a significant change on writhing test (Table 1).
Post hoc test showed that ALEE caused significant inhibition of number of writhing at the dose of at 200 and 400 mg/kg (P<0.05-P < 0.001). The maximum inhibition exhibited at 400 mg/kg (66.83%). The effects were comparable to that of standard drug aspirin that also showed significant inhibition (71.34%) of writhing (P < 0.01) ( Table 1).

Effects on formalin induced pain response
Effect on early and late phase licking One-way ANOVA showed that ALEE showed significant influence on formalin-induced licking of animals. Post hoc test indicated that pretreatment with ALEE (200 and 400 mg/ kg) showed significant (P < 0.05-P<0.001) inhibition of the early phase and late phase licking when compared to vehicle control (Table 2). Indomethacin at 10 mg/kg also also showed similar effect on early phase licking (P < 0.001) as well as the late phase licking (P < 0.01) when compared to vehicle control.

Antipyretic activity
In antipyretic study, two-way ANOVA showed significant influence of ALEE on yeast-induced pyrexia ( Table 5). ALEE (100, 200 and 400 mg/kg) as well as paracetamol significantly reduced hyperthermia in rats in time dependent manner. ALEE (200 and 400 mg/kg) exhibited a significant change (P < 0.05 to P < 0.001) in hyperthermia from 3 h onwards (Table 5) when comparision made with control. The standard drug paracetamol also showed inhibition (P < 0.01 to P < 0.001) of fever from 2 h as compared to control.

Discussion
The current study clearly indicates the analgesic, antiinflammatory and antipyretic potential of ALEE in animal models. HPLC study of ALEE reveals the occurrence of ellagic acid, an important phytoconstituent of Anogeissus latifolia ( Fig. 1a and b) in the concentration range of 0.38% w/w. Acute toxicity suggests that ALEE was safe when administered orally at limit test dose of 2000 mg/kg. The results of the analgesic activity suggest that ALEE exhibited significant inhibition of pain response in both chemically-induced pain models. The effect of ALLE was comparable to aspirin in amelioration of acetic acidinduced pain in rats which suggest the role of ALEE in inhibition of cyclooxygenase or lipoxygenase pathway Results are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.001 compared to Control. ALEE: Ethanolic extract of stem bark of A. latifolia Results are expressed as mean ± SEM(n = 5). *P<0.05, **P < 0.01, ***P<0.001 compared to control ALEE: Ethanolic extract of stem bark of A. latifolia which is the general pathway of common peripherally acting analgesic drugs [30]. Formalin paw licking test produces a distinct biphasic response. Early phase is characterized by neurogenic pain (non-inflammatory) which which is generated by the activation of nociceptors [31] and involves substance P and bradykinin. The formalin-induced pain model also describes the inflammatory pain in the later phase [32,33]. The peripheral inflammation may be induced due to the production of prostaglandins, serotonin and histamine [34]. Hence, this biphasic model is used extensively used to differentiate the exact mechanism of analgesics [35]. In present study, ALEE inhibited both early and late phase paw licking due to formalin. This suggests that ALEE has inhibitory influence on both neurogenic and inflammatory pain.
ALEE showed significant inhibition of carrageenan and formalin induced inflammation in dose dependent manner. In carrageenan-induced paw oedema model the early phase is not influenced of inhibited by NSAIDs and occurs due to the release of inflammatory mediators like histamine, serotonin and bradykinin while the late phase is marked by elevated production prostaglandins and induction of cyclooxygenase [36]. Late phase is accelerating phase of swelling due to increased vascular permeability and oedema caused by prostaglandins and can be inhibited by NSAIDs [37]. In the present study, treatment with ALEE and indomethacin showed significant inhibition of increase in paw volume at 3 and 5 h of carrageenan administration without influencing paw volume at 1 h. This further confirms the anti-inflammatory action of ALEE. The exact mechanism of anti-inflammatory action of ALEE is difficult to interoperate, however, it can be contemplated that ALEE has influence on arachidonic acid pathway and inflammatory mediators like prostaglandins mainly by inhibiting COX enzyme as like indomethacin.
Formalin-induced induction of pain is known to be caused due to the inflammation of peripheral tissue [35]. Acute inflammatory response is mediated primarily by blood leukocytes such as neutrophils and macrophages [38]. Neutrophils stimulation also causes increased vascular permeability and produce oedema [39]. In formalin induced paw oedema model, results showed that subplantar injection of formalin caused significant increase in paw volume as compared to normal rat paw. Administration of ALEE significantly prevented the raise of paw oedema after formalin injection. Indomethacin also inhibited the paw volume increment at 3 and 5 h after formalin injection. Thus, it indicated the anti-inflammatory nature of the extract. Recently, Agnihotri et al. [40] demonstrated antioxidant and anti-inflammatory potential of Anogeissus latifolia stem bark on vascular and capillary permeability and inflammatory mediators and supports the present findings.
The etiology of yeast-induced fever which is considered as pathogenic fever suggests the role of prostaglandins in the thermoregulation of body temperature. The prostaglandin (PGE 2 ) during the arachidonic acid is Results are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001 compared to Control. ALEE: Ethanolic extract of stem bark of A. latifolia considered an important factor for induction of fever [41,42]. The results of the current study yeast-induced fever model indicates the antipyretic activity of ALEE (Table 5) and its effect was comparable to paracetamol. Hence, it can be contemplated that the possible mechanism of antipyretic action of ALEE may be due to the inhibition of prostaglandin synthesis [35]. Most of NSAIDs suppress inflammation-associated hyperthermia [43] through inhibition prostaglandins synthesis [44]. Since, ALEE manifested antipyretic activity which may be related to its anti-inflammatory action. It is quite difficult to conclude the exact constituent which may be accountable for the observed analgesic, antiinflammatory and antipyretic action of ALEE. ALEE contains ellagic acid as main polyphenolic chief constituent. Ellagic acid possesses good anti-inflammatory property in carrageenan-induced paw oedema model [45]. HPLC analysis has also quantified the fair amount of ellagic acid in ALEE which may be responsible for amelioration of inflammation. The quantitative estimation of phytoconstituents revealed the high content of phenolic and flavonoids in ALEE. The stem bark is also reported to have phytoconstituents like quercetin, rutin [46], tannin, (+) leucocyanidin [16], glucoside ester [17], β-sistosterol, hydroxy acetytaraxaren and gallotannin and β-penta-O-galloylated glucose [18]. The phytoconstituents like quercetin [47], rutin [48] and βsistosterol [49] were reported to have anti-inflammatory activity and might be responsible for the biological activities of ALEE in the present study. The results were in concordance with the previous study [50]. Thus, the analgesic and anti-inflammatory may be exhibited by the presence of phenolics and flavonoids in ALEE. The study needs further confirmation using estimation of markers of inflammation like COX and interleukins.

Conclusion
In conclusion, the ALEE exhibited analgesic, antiinflammatory and antipyretic effects. The presence of phenolic and flavonoid compounds which may be responsible for the effects. The study validates the use of stem bark of Anogeissus latifolia for treatment of painful inflammatory conditions.