Artocarpus hirsutus Lam Leaf Extract-Evaluation of Analgesic and Anti-Inflammatory Activity

The study involved extraction, identification, and evaluation of pharmacological activities of the phytochemicals present. Artocarpus hirsutus Lam, commonly known as Wild jack is a greatly valued medicinal plant, which belongs to the plant family Moraceae. Preliminary phytochemical screening studies indicated the presence of flavonoids, saponins, tannins, glycosides, and alkaloids. This study estimated the analgesic and anti-inflammatory prospective of ethanolic leaf extract of Artocarpus hirsutus Lam. The findings showed that at various doses of 100, 200, and 400 mg/kg body weight when administered orally to rats, analgesic effects were produced, also the anti-inflammatory effect studied by carrageenan-induced rat paw edema test showed major anti-inflammatory action. The result indicates that the leaf extract of Artocarpus hirsutus Lam possesses major analgesic and anti-inflammatory activity and therefore requires further investigations to better understand the mechanism of action.

Artocarpus hirsutus lam is a tropical evergreen tree belonging to the genus Artocarpus and Moraceae family extensively known for its edible fruit and wood. Artocarpus hirsutus Lam is native to India, primarily in South India.
Artocarpus hirsutus Lam is an evergreen canopy tree that can grow to a height of 35 m and about 4.5 m in girth. Te various morphological parts of the plant are efective against skin diseases, intrinsic haemorrhage, and poisons; bark infusion is used to cure small pimples and cracks present on the skin. Te powdered bark is used to treat sores, dried leaves are applied to buboes and hydrocele .
Te present research work aimed to identify and isolate the phytoconstituents from the ethanolic leaf extract of Artocarpus hirsutus Lam and evaluate the analgesic and antiinfammatory activities.

Plant Leaf Collection, Authentication and Extraction.
Te leaves of the plant Artocarpus hirsutus Lam collected from Deralakatte, Mangaluru, Karnataka were authenticated by a botanist. Te fresh leaves were sun dried and subsequently pulverized using a mechanical grinder. Te dry powder was macerated in ethanol for a duration of 7 days with occasional stirring. Te total extracts of the leaves were distilled, fltered, concentrated using a rotary evaporator, and stored in a desiccator.

Preliminary Qualitative Phytochemical Investigations.
Presence of diverse phytochemicals in the total extracts of the leaves of Artocarpus hirsutus Lam was assessed by qualitative test for preliminary phytoconstituents.

Test for Alkaloids
(1) Dragendrof f's Test. In 5 mL of distilled water, 0.5 g of ethanolic leaf extract was dissolved, to which 2 M hydrochloric acid was added for the reaction to initiate. Subsequently, Dragendrof's reagent (1 mL) was added to the above solution. An orange red precipitate indicates the presence of alkaloids.
(2) Hager's Test. Hager's reagent was added to 1 mL of ethanolic extract of leaves. Yellowish colored precipitate formed confrms the presence of alkaloids.
(3) Wagner's Test. 1.5 mL of hydrochloric acid was added to 1 mL of ethanolic extract of the leaf. Subsequently few drops of Wagner's reagent were added. Buf colored precipitate formation indicates the presence of alkaloids.
(4) Mayer's Test. Mayer's reagent was added to 1 mL of ethanolic extract. Te presence of alkaloids was confrmed by observing the formation of pale yellow or white precipitate.

Test for Reducing Sugar
(1) Molisch Test. Freshly prepared 20% alcoholic α-naphthol was added to 2 mL of the leaf extract to which 2 mL of concentrated sulphuric acid was added dropwise along the walls of the test tube to facilitate the formation of a layer and to avoid mixing. Te formation of the purple ring at the layer formed is an indication for the presence of carbohydrates which vanishes on the addition of an excess of an alkaline reagent.
(2) Benedict's Test. Benedict's solution was added to the leaf ethanolic extract and heated. Te formation of a brick red color confrms the presence of carbohydrates.
(3) Fehling's Test. 10 mg of the ethanolic leaf extract was dissolved in 1 mL of water. 1 mL of Fehling's A and Fehling's B solution were added. Te presence of reducing sugar was confrmed by observing the formation of brick red color.
(4) Tollen's Test. In 1 mL of water 10 g of ethanolic leaf extract was dissolved. 1 mL of Tollen's solution was added and heated on a water bath. Te formation of a black precipitate or black silver mirror along the sides of the test tube confrms the existence of reducing sugars.

Test for Flavonoids
(1) Shinoda Test. 0.5 mL of ethanolic leaf extract was heated, and subsequently hydrochloric acid (10 drops) and magnesium powder were added. Te color change of the solution to reddish brown proves the presence of favonoids.

Test for
Saponins. 10 mg of ethanolic extract was dissolved in water and shaken vigorously. Te appearance of foam proves the presence of saponins.

Test for Tannins.
To 0.5 g of ethanolic leaf extract, 5 mL of chloroform was added. A reddish pink color formed by the addition of 1 mL of acetic anhydride and 2 drops of sulphuric acid to the solution confrms the presence of tannins.

Tests for Steroids
(1) Liebermann-Burchard's Test. 10 mg of the ethanolic extract was dissolved in acetic anhydride. Te solution was heated and cooled. Concentrated sulphuric acid (1 mL) was incorporated slowly, along the side of the test tube. Te development of a greenish color confrms the presence of steroids.
(2) Salkowski Reaction. To the ethanolic leaf extract, 1 mL of concentrated sulphuric acid was added along the side of a test tube. Te mixture was allowed to stand. Te red color formed in the chloroform layer confrms the presence of steroids.

Test for Triterpenoids.
Te leaf extract was dissolved in acetic anhydride. Te solution was heated and allowed to cool. Concentrated sulphuric acid (1 mL) was added along the sides of the test tube. Violet color formation confrms the presence of triterpenoids.

Test for Glycosides
(1) Molisch Test. 2 mg of ethanolic leaf extract was dissolved in 10 mL of water which was shaken and fltered. To the concentrated fltrate 2 to 3 drops of Molisch reagent was added, mixed, and subsequently concentrated sulphuric acid (2 mL) was added along the sides of the test tube. Reddishviolet ring proved the presence of glycosides.
(1) Evaluation. Te turbidity formed was measured spectrophotometrically at 660 nm. Te calculation for percentage inhibition of protein denaturation was done by the following formula: where V t � absorbance of test sample, V c � absorbance of control.

In Vivo Methods to Detect Analgesic and Anti-Infammatory Activity
(1) Analgesic Activity. Tail immersion method. Te tails of the albino rats (Table 1) were gently immersed in a thermostatic organ bath with a temperature maintained at 55 ± 10°C. Te animals which withdrew the tail from hot water within 3-5 seconds were selected for the test. Te reaction time of all animals towards thermal heat was noted. Group III, Group IV, and Group V animals were administered ethanolic extract in 2% w/v Gum acacia in normal saline orally. Group II was given the standard drug Pentazocine (10 mg/kg) intraperitoneally and Group I received 2% w/v of Gum acacia (2 mL/kg) with normal saline.
(2) Evaluation. Animals from all the groups were exposed to hot water subsequent to the administration of the test and reference compounds. Reaction time was noted at 30 and 90 mins interval, also the reaction time was noted as and when the animals withdrew their tail completely from the hot water bath. Te mean reaction time in each group was evaluated statistically.
Advances in Pharmacological and Pharmaceutical Sciences 3 RT AT and RT BT are the reaction time after treatment and before treatment, respectively. Acetic acid induced writhing method [56]. Te frst group of animals were administered acetic acid 06% v/v. i.p., which were considered as control. Te second group was administered the standard drug Diclofenac sodium (10 mg/kg body weight, i.p). Te third, fourth and ffth group animals were administered 100, 200, and 400 mg/kg body weight ethanolic extract which was suspended with 2% gum acacia 30 min prior ( Table 2).
(3) Evaluation. Te writhing action was indicated by the stretching of the abdomen along with the simultaneous stretching of at least one hind limb. Tis action was observed for 30 min. Percentage inhibition calculation was carried out by using the following formula: R t � Mean number of writhes in treated groups, R c � Mean number of writhes in control groups. Anti-infammatory screening method Carrageenan induced rat paw edema method [57].
Group I: control Group II: standard, Diclofenac sodium (10 mg/kg) Group III: test drug (100 mg/kg) Group IV: test drug (200 mg/kg) Group V: test drug (400 mg/kg) Albino rats weighing between 150-200 g were taken for the test. Prior to the day of the experiment the animals were starved overnight with water ad libitum. An identifcation mark was made on both the hind paws just below the tibiotarsal junction. Each time the paw was dipped up to the mark into the mercury column of the plethysmograph to get constant paw volume. Te second group was administered a standard drug, Diclofenac sodium 10 mg/kg body weight. Te third, fourth and ffth groups received ethanolic leaves extract at 100, 200, and 400, respectively. Acute infammation was induced after thirty minutes of administration of the drug by injecting into the subplantar region of the left hind paw 0.1 mL of 1% carrageenan (Table 3).
(4) Evaluation. Te paw volume was measured with the use of a plethysmograph at 0, 0.5, 1, 2, and 3 hours subsequent to the carrageenan injection. Te diference between 0 hour and consequent readings was considered as edema volume. Te calculation for percentage inhibition of edema was carried out by using the following formula: V t � volume of paw edema in drug treated group V c � volume of paw edema in control group.
(5) Statistical Analysis. Te data obtained were evaluated using one way analysis of variance (ANOVA), followed by posthoc test (Schefe's T-Test) using graph pad SPSS software version 16.0 where p value less than 0.05 was taken as statistically signifcant. Table 4).

Acute Toxicity Studies. Te ethanolic extract of leaves of
Artocarpus hirsutus Lam up to 2000 mg/kg body weight by the oral route of administration were found to be safe. Even after 24 hours the animals were found to be well tolerated.
Tere was no indication of toxicity and death. Tree doses, 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight were selected for the experiment. Tables 5 and 6).

Anti-Infammatory Activity.
Efect of ethanolic extract of Artocarpus hirsutus Lam on carrageenan induced rat paw edema method Table 7).

Discussion
Te present study was designed to evaluate the analgesic and anti-infammatory activity of ethanolic leaf extract of Artocarpus hirsutus Lam. Te peripheral analgesic activity was carried out using the chemical induced writhing method ( Table 6) and central analgesic activity by tail fick using immersion of the tail (Table 5). Te carrageenan induced paw edema model was carried out to identify the acute infammatory activity. Preliminary phytochemical screening of ethanolic leaves extract of Artocarpus hirsutus Lam signifes the presence of saponins, tannins, favonoids, triterpenoids, glycosides, alkaloids, and resins (Table 4). When the ethanolic extract of the leaves were administered orally at the doses of 200 and 400 mg/kg, signifcant analgesic activity was exhibited (p < 0.05) by inhibition of writhing induced by acetic acid in the mice. Te writhing is associated to the increase in levels of PGE2 and PGF2 in the peritoneal fuid and elevation in levels of lipoxygenase products by the peritoneal injection of acetic acid [60]. Acetic acid induced writhing methods also release endogenous substances like bradykinin, histamine, and serotonin, which activates the sensory nerve endings [61]. Antinociceptive action of ethanolic extract of Artocarpus hirsutus Lam leaves could be due to its action on visceral receptors which are reactive to acetic acid thereby preventing prostaglandin synthesis.
In the tail immersion test the pain produced by the thermal stimulus is characteristic for central-mediated activity [62]. Opioid agents like morphine bring about analgesic action via supra spinal and spinal receptors [63]. Tis activity could be due to the stimulation of the periaqueductal gray matter area to discharge endogenous peptides [64].
Tese endogenous peptides crash the spinal cord and inhibit pain impulse transmission at the synapse in the dorsal horn. Te possible actions of ethanolic extract of Artocarpus hirsutus Lam leaves may be due to its action on the central receptors or promoted discharge of endogenous opioid peptides. In this study, the action of the drug extract at a dose of 100 mg/kg was not signifcant. But it showed a signifcant increase (p < 0.05) at a dose of 200 mg/kg body weight and tail fick latency was highest (p < 0.05) at a dose of 400 mg/kg body weight at 90 min. Hence an expressive increase in reaction time and decrease in writhing are generally taken as signifcant parameters of analgesic activity in the heat conduction method and acetic acid induced writhing test respectively.
Te carrageenan technique was chosen due to its sensitivity in identifying anti-infammatory agents which are orally active, mainly in the acute stage of infammation. Carrageenan is used to produce acute infammation and is biphasic. Te initial phase (0-2.5 hours after carrageenan injection) mainly occurs due to the concordant release of mediators such histamine, kinins, and serotonin. Te next phase is related with an increase in the production of prostaglandin, oxygen derived free radicals, and inducible cyclooxygenase production [65,66]. Te carrageenan induced paw edema method in rats is avowed to be reactive to cyclooxygenase (COX) inhibitors as well as lipoxygenase inhibitors thereby restricting the enzyme COX involved in the synthesis of prostaglandins. When compared to the control, the dose of 400 mg/kg body weight at 30, 60, 120,  . Based on the result it can be concluded that the inhibitory action of ethanolic leaves extract on Carrageenan induced infammation in rat paw may be because of the restriction of the enzyme cyclooxygenase which further leads to restriction in the synthesis of prostaglandin (Tables 8  and 9). Te denaturation of protein method was selected for the evaluation of in vitro anti-infammatory activity of the ethanolic extract of Artocarpus hirsutus Lam. Tissue protein denaturation is the main causes of infammatory and arthritic disease. Substances that restrict the denaturation of protein are meritorious in the anti-infammatory drug development [67]. From the results, it can be deduced that the ethanolic extract of Artocarpus hirsutus Lam is more effective in less concentration than that of diclofenac sodium and thus it has anti-infammatory activity.

Conclusion
Artocarpus hirsutus Lam has been used in folklore, Ayurvedic and traditional medicines for the treatment of numerous health concerns. Trough this study, the phytoconstituents present in ethanolic leaf extracts of Artocarpus hirsutus Lam were identifed. Using animal models, it was concluded to have good analgesic and antiinfammatory potential. Tis study should enable researchers to identify and isolate new constituents from Artocarpus hirsutus Lam, understand the mechanism of action of the phytoconstituents and formulation of drugs for various diseases.

Data Availability
Te data used to support the study are included in the paper.

Conflicts of Interest
Te authors declare that they have no conficts of interest.