Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7

Research Article RJPP 2019 3:7

Research Journal of Pharmacology and Pharmacy (ISSN:2572-8377)

Anti-inflammatory Activity ofSaccharum Officinarum Linn (Poaceae) Juice in Animal Models

Agbaje Esther Oluwatoyin, Ayinla Adebola Christina, **Ujomo Tejumade Department Of Pharmacology, Therapeutics & Toxicology, College Of Medicine, University Of Lagos; **Department Of Pharmacology And Therapeutic, University Of Ibadan, Oyo State, Nigeria.

ABSTRACT

Saccharum officcinarum (SOC) juice has been used for treat- *Correspondence to Author: ment and management of several conditions including urinary Agbaje Esther Oluwatoyin tract infection, diabetes, constipation, tooth decay and bad breath Department Of Pharmacology, according to folklore. This study was carried out to explore the Therapeutics & Toxicology, College anti-inflammatory effects of the juice ofSaccharum officinarum in Of Medicine, University Of Lagos doses of 2.5, 5.0, 10.0 and 20.0 mL/kg using the following mod- els in rats - carrageenan, histamine, serotonin, formalin-induced paw oedema, and cotton-pellet granuloma. Also employing the How to cite this article: following models in mice - castor oil-induced diarrhea and xy- Agbaje Esther Oluwatoyin, Ayinla lene-induced ear oedema. Animals were pretreated with aspirin Adebola Christina, Ujomo Teju- (100 mg/kg; p.o), cyproheptadine (10 mg/kg; p.o.), atropine (1 mg/ made. Anti-inflammatory Activity kg p.o), dexamethasone (5 mg/kg; p.o.) and celecoxib (20 mg/ of Saccharum Officinarum Linn kg p.o.). Oral administration of SOC juice significantly (p<0.001) (Poaceae) Juice in Animal Models. reduced the right hind paw circumference induced by carrageen- Research Journal of Pharmacology an, histamine, serotonin and formalin compared to control group. and Pharmacy, 2019, 3:7 Furthermore, it inhibited xylene-induced ear oedema with peak effect at 20 mL/kg, as well as castor oil-induced diarrhea and cotton pellet granuloma with peak effect at 10 mL/kg. The an- ti-inflammatory activities of SOC were significant (p<0.05) when compared with aspirin, cyproheptadine and dexamethasone. The eSciPub LLC, Houston, TX USA. study revealed the potent anti-inflammatory activities of the juice Website: https://escipub.com/ of S. officinarum. Keywords: Saccharum officinarum, anti-inflammatory, carra- geenan, aspirin, histamine, rat

RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 1 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 INTRODUCTION Sugarcane has been used in various parts of the world for curing inflammatory diseases and other Inflammation is a generic body response, a part ailments. In the Ayurvedic system of medicine, of the protective mechanisms involving a sugarcane is used either as a single remedy or complex array of biochemical processes such as in combination with some other plant materials enzyme activation, mediator release and (7). Some natives and traditional healers of the extravasation of fluid, cell migration, and tissue world have recommended sugarcane juice for its damage and repair (1, 2) in response to harmful diuretic property (8). It is thought that regular use stimuli, such as pathogens, damaged cells, or of sugarcane juice will keep the urinary flow clear irritants. It is a mechanism of innate immunity and fast, which will further help the kidneys to involving immune cells, blood vessels, and perform their function properly. It is also used as molecular mediators, as compared to adaptive aphrodisiac, laxative, coolant, demulcent, immunity, which is specific for each pathogen. antiseptic, and as a tonic (9). According to the Too little inflammation could lead to progressive Unani system of medicine, sugarcane juice is tissue destruction by the harmful stimulus such considered good for patients with jaundice. Most as bacteria and compromise the survival of the of the ethnobotanical uses have not been organism. If not arrested or treated, acute scientifically evaluated. Efforts in this study were inflammation progresses to chronic, which may aimed at evaluating the acute toxicity profile, lead to a host of diseases, such as hay fever, phytochemical constituents and the anti- periodontitis, atherosclerosis, rheumatoid inflammatory activity of the juice of S. arthritis, and even cancer (gallbladder officinarum. The possible pathway of its anti- carcinoma). Inflammation therefore functions to inflammatory activity was also explored. eliminate the initial cause of cell injury, clears out necrotic cells and tissues damaged from the MATERIALS AND METHODS original insult and the inflammatory process, and Plant Materials initiate tissue repair (3). It is a process normally Collection and Preparation closely regulated by the body (4). The whole plant of Saccharum officinarum was Despite the availability of conventional drugs, obtained from the vegetable farm at Idi- araba, both old and new, the side effects of anti- Surulere LGA, Lagos State, Nigeria, for the inflammatory agents including gastrointestinal purposes of identification and authentication, upset, gastric ulcer, bleeding, which were carried out at the Herbarium of the immunosuppression and organo-toxicity are a Department of Botany and Microbiology, Faculty major concern in clinical use; which has of Science, University Of Lagos by Mr G.I. therefore called for a continuous search for safe Nodza. The plant specimen was deposited in the and effective newer agents. One of such herbarium and a voucher specimen number: research areas is the screening of medicinal LUH7935 was issued. plants with folkloric anti-inflammatory claims. A juice extractor was used to squeeze out the S. officinarum (Linn.) commonly known as juice from peeled fresh stems weighing 139.42 sugarcane is a large, strong-growing species of g. The juice was 84.39 g (80 mL) after extraction grass belonging to the family Poaceae. India is and always used fresh. the second largest producer of sugarcane, after Animals Brazil (5). It is a tropical, perennial grass that forms lateral shoots at the base to produce Eighty-eight (88) male Wistar albino rats multiple stems, typically 3-4m high and about weighing between 100-120 g and 52 albino mice 5cm in diameter. Sugarcane originated from weighing between 20-25 g were obtained from tropical South and Southeast Asia, but different the Laboratory Animal Center, College of species are found in different locations (6). Medicine, University of Lagos. The animals were RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 2 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 maintained under standard laboratory Ct – circumference of paw of rat treated with conditions, having free access to standard agent at time t rodent diet. All the animals were allowed to Co – circumference of paw of rat with initial paw acclimatize for 10 days before commencement circumference of experiment, and fasted 12 hours prior each Histamine-induced paw edema test experiment. All experimental procedures were carried out in compliance with the United States Experimental animals were divided into 3 groups National Institute of Health Guidelines for care of 4 animals each. Group 1 received distilled and use of Laboratory animals in biomedical water (10 mL/kg p.o), Group 2 SOC juice dose research (NIH) and recommendation of ISAP (20 mL/kg p.o) and Group 3- cyproheptadine (10 (10). mg/kg p.o). One hour post-treatment, edema was induced by injection of histamine into the Materials sub-plantar tissue of the right hind paw. The Atropine, carrageenan, serotonin, histamine, linear paw circumference was measured using a formalin, xylene and celecoxib were obtained digital vernier caliper. Measurements were from Sigma chemical company in USA. Castor made immediately before the injection of oil, aspirin, cyproheptadine and dexamethasone histamine and at a 30 minutes interval for 3 were purchased from a standard pharmacy shop hours and the percentage inhibition was at Idi-araba, Lagos, Nigeria. calculated (14, 15). Acute toxicity test [Ct−Co]control−[Ct−Co]test % inhibition = × 100 Fasted male mice were randomly selected into [Ct−Co]control groups (n =3), and each group dosed with 15, Ct – circumference of paw of rat treated with 20, 30 and 40 mL/kg of SOC juice agent at time t intraperitoneally, while 50 mL/kg was Co – circumference of paw of rat with initial paw administered orally. LD50 was scored circumference accordingly (11). Serotonin-induced paw edema test PHARMACOLOGICAL INVESTIGATIONS Experimental rats were divided into 3 groups of Carrageenan-induced paw edema test 4 animals each. Group 1 received distilled water Experimental animals were divided into 6 groups (10 mL/kg p.o), Group 2 SOC juice dose (20 of 4 animals each. Group 1- received distilled mL/kg p.o) and Group 3- cyproheptadine (10 water (10mL/kg p.o); Groups 2,3,4 and 5 SOC mg/kg p.o). One hour post-treatment, edema juice doses; 2.5, 5, 10, and 20 mL/kg per oral was induced by injection of serotonin into the and Group 6- aspirin (100 mg/kg p.o). One hour sub-plantar tissue of the right hind paw. The post-treatment, edema was induced by injection linear paw circumference was measured using a of carrageenan (0.1 mL, 1% w/v in saline) into digital Vernier caliper. Measurements were the sub-plantar tissue of the right hind paw (12, made immediately before the injection of 13). The linear paw circumference was serotonin and at a 30 minute interval for 3 hours measured using a digital Vernier caliper. and the percentage inhibition was calculated Measurements were made immediately before (14, 15). the injection and then at a 30 minutes interval for Castor-oil induced diarrhea in mice 6 hours after the injection, the mean increase in Experimental mice were randomly distributed paw swelling was measured and the percentage into 4 groups of 4 animals each. Group I inhibition was calculated. received distilled water (10 mL/kg), groups II and [ ] [ ] % inhibition = Ct−Co control− Ct−Co test× 100 III received SOC juice (10 mL/kg and 20 mL/kg [Ct−Co]control respectively), while group IV received atropine

(1 mg/kg, s.c), 30 min prior to the oral RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 3 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 administration of castor oil (0.2 mL) (16). All cotton pellets were implanted subcutaneously, other treatments were orally administered. one on each side of the abdomen of the animal Following castor oil treatment, the animals were under light ether anaesthesia. Test drugs; placed on a filter paper covered with a funnel in distilled water (10 mL/kg), Saccharum order to restrict movement. Defecation was officinarum juice (10 and 20 mL/kg), and observed for 5 hours. celecoxib (20 mg/kg) were administered once Formalin-induced rat Paw edema daily throughout the experimental period of 7 days. On the 8th day after implantation, rats were Experimental rats were distributed into 6 groups anaesthetized with pentobarbital sodium. The of 4 animals each. Group 1- distilled water (10 pellets were dissected and dried at 60º for 18 h, mL/kg), Groups 2 and 3 SOC juice doses at 10 weighed after cooling. The mean weight of the mL/kg and 20 mL/kg, Group 4- aspirin (100 cotton pellets of the control group as well as of mg/kg), Group 5- dexamethasone (5 mg/kg) and the test groups was calculated. The wet and dry Group 6- aspirin + SOC juice dose 20 mL/kg. weights, granuloma formation and percent One-hour post-treatment, edema was induced granuloma inhibition of the test compound were by formalin (0.02 mL, 2%) in the right hind paw calculated (17). of the rats. The paw thickness was measured ( ) using a vernier caliper, 1 hour after formalin % inhibition = 퐺푟푎푛푢푙표푚푎 푐표푛푡푟표푙 −퐺푟푎푛푢푙표푚푎 (푡푒푠푡) × 퐺푟푎푛푢푙표푚푎 (푐표푛푡푟표푙) injection. The drug treatment was continued for 100 7 days and formalin was also induced on the 3rd Phytochemical screening day. The increase in paw thickness and percentage inhibition were calculated (15). The juice of SOC was subjected to preliminary phytochemical screening following standard Xylene-induced ear edema test procedures (18 – 20). Experimental mice were allotted to 4 groups of 4 Anti-oxidant assay of aqueous extract of animals each. Group 1- distilled water (10 Saccharum officinarum juice mL/kg), Groups 2 and 3 received SOC juice doses at 10 mL/kg and 20 mL/kg and Group 4- Nitric oxide radical scavenging assay dexamethasone (5 mg/kg). One hour post- Nitric oxide (NO) generated from sodium treatment, edema was induced in each mouse nitroprusside (SNP) was measured (21). Briefly, by applying 0.1 mL of xylene to the inner surface the reaction mixture (5.0 mL) containing SNP (5 of the right ear. The animals were then sacrificed mM) in phosphate buffered saline (pH 7.3), with by cervical dislocation after 30 minutes, both S. officinarum juice at different concentrations, ears were then cut off to approximately equal was incubated at 25˚C for 180 min in front of a size and weighed. The mean differences visible polychromatic light source (25W tungsten between the right and left ears were determined lamp). The NO radical thus generated interacted for each group and percentage inhibition was with oxygen to produce the nitrite ion (NO) which calculated respectively (13). was assayed at 30 min intervals by mixing 1.0 % inhibition = mL of incubation mixture with an equal amount [Right ear−left ear]control−[Right ear−left ear]test of Griess reagent (1% sulfanilamide in 5 % × 100 [Right ear−left ear]control phosphoric acid and 0.1% Cotton pellets granuloma in rats naphthyethylethelene-diamine-dichloride). Experimental rats were divided into four groups The absorbance of the chromophore (purple azo of four rats each. Adsorbent cotton wool was cut dye) formed during the diazotisation of nitrite into pieces weighing 20±1 mg and made into ions with sulphanilamide and subsequent pellets. The abdomen of the rats was shaved, coupling with naphthylethylenediaminedi- swabbed with 70 % ethanol and two sterilized hydrochloride was measured at 546 nm. The

RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 4 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 nitrite generated in the presence or absence of (23). A mixture containing 2.5 mL of 0.2 M the plant extract was estimated using a standard phosphate buffer pH 6.6 and 2.5 mL 1% curve based on sodium nitrite solutions of known potassium hexacyanoferrate was added to 1.0 concentrations. Each experiment was carried mL of the different solvent extracts and out two times and the data presented as an standards (25 μg/mL to 400 μg/mL). The average of two independent determinations. resulting mixture was incubated for 20 min at DPPH (2,2-Diphenyl-1-picrylhydrazyl) 50°C. After incubation, 2.5 mL of 10% TCA (w/v) was added to terminate the reaction and The free radical scavenging activity of S. centrifuged for 10 min at 3000 rpm. 2.5 mL of the officinarum was measured with stable DPPH in supernatant was removed and mixed with 2.5 terms of hydrogen donating or radical mL of distilled water and 0.5 mL of 0.1% freshly scavenging activity. 100μL of DPPH solution prepared FeCl , FeCl , was added. The mixture (0.36 mm DPPH in methanol) was added to 1 mL 2 3 was left to stand for 10 min, and the absorbance extract (100-1000 μg/mL in methanol), vortexed was read at 700 nm. A mixture of the buffer thoroughly and kept in the dark at room instead of the sample served as control. temperature for 30 min, after which the decrease Increased absorbance of the reaction mixture in absorbance was measured at 517 nm using indicated higher reducing power of the plant ultraviolet-visible (UV-VIS) spectrometer. fractions. The results were reported as µg of Ascorbic acid was used as the positive control ascorbic acid equivalents (AAE) per ml. (22). RESULTS The scavenging effect was calculated using: Phytochemical screening Percent inhibition (%) = [A0 - A1] × 100, The results of the phytochemical tests revealed A0 the presence of alkaloids, saponins, flavonoids, where A0 is the absorbance of the control steroids, quinone, coumarin, tannins, phenols, solution and A1 is the absorbance in the glycosides and terpenoids. presence of aqueous leaf extract of S. Acute Toxicity officinarum and standard. Intraperitoneal administration of 15, 30 and 40 Ferric reducing power assay mL/kg showed 0 %, 66.67 % and 100 % mortality The ferric reducing antioxidant power (FRAP) of respectively. The juice was found to elicit these the different solvent fractions was assessed effects; itching, grooming and tachypnea.

TABLE 1: Acute toxicity test (Intraperitoneal route) Dose (mL/kg) Log dose Mortality % mortality Probit 15 1.176 0/3 0 0 30 1.477 2/3 66.67 5.52 40 1.602 3/3 100 8.09

LD50 was calculated to be 25 mL/kg of juice dose.

PHARMACOLOGICAL INVESTIGATIONS and 20 mL/kg after 30 minutes showed Carragenan-induced paw oedema in rats significant inhibition (p<0.001) of edema similar to the standard drug (aspirin). S. officinarum For the control group, carrageenan produced a juice dose at 20 mL/kg produced the highest progressive swelling of the rat’s paw 7.74±0.05 percentage inhibition of 83.20 %, which mm at 30 minutes, which gradually increased to compared effectively with the standard drug the peak at 240 minutes (4th hour) 8.30±0.03mm (82.40 %). (Table 2). Administration of the juice 2.5, 5, 10 RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 5 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 TABLE 2: EFFECT OF S. OFFICINARUM ON CARRAGEENAN-INDUCED PAW OEDEMA IN RATS

RAT PAW EDEMA (mm) MEASURED AT VARIOUS TIMES IN MINUTES

Treatme Dose 0 30 60 90 120 150 180 210 240 270 300 330 360 1440 nt mL/kg

Control 10 6.13±0 7.74± 7.79± 7.93± 7.95± 7.97± 8.23± 8.27± 8.30 ± 8.20± 8.20± 8.16± 8.01 ± 7.09± .09 0.03 0.06 0.11 0.03 (DW) 0.05 0.04 0.04 0.13 0.03 0.02 0.02 0.05 0.072

SO 2.5 5.38±0 6.37± 6.64± 6.73± 6.99± 6.76± 6.58± 6.49± 6.46± 6.45± 6.44± 6.44± 6.44± 6.17± .06 0.06* 0.05* 0.06* 0.01* 0.01* 0.16* 0.18* 0.09* 0.05* 0.03* 0.05* 0.05* 0.055* (42.8) (50.2) (48.3) (17.7) (38.5) (24.1) (14.0) (11.5) (25.0) (48.1) (48.8) (47.8) (43.9)

5 5.48±0 6.18± 6.50± 6.77± 6.98± 6.83± 6.73± 6.66± 6.58± 6.54± 6.54± 6.50± 6.47± 6.01± .11 0.11* 0.17* 0.09* 0.09* 0.16* 0.15* 0.11* 0.12* 0.12* 0.12* 0.14* 0.11* 0.10* (56.5) (38.5) (28.7) (17.6) (26.6) (40.5) (44.8) (49.2) (49.3) (48.8) (49.8) (47.6) (44.8)

10 5.98±0 6.63± 6.81± 6.94± 6.95± 7.00± 6.83± 6.80± 6.69± 6.68± 6.61± 6.60± 6.49± 6.31± .06 0.05* 0.13* 0.15* 0.14* 0.15* 0.26* 0.15* 0.15* 0.18* 0.18* 0.19* 0.18* 0.15* (65.6) (59.6) (50.0) (46.9) (46.1) (44.5) (59.5) (61.6) (67.2) (74.0) (68.9) (69.4) (73.0)

20 5.68±0 5.99± 6.09± 6.32± 6.43± 6.68± 6.78± 6.66± 6.59± 6.39± 6.31± 6.21± 6.07± 5.75± .21 0.17* 0.16* 0.20* 0.09* 0.04* 0.09* 0.14* 0.16* 0.13* 0.04* 0.02* 0.03* 0.08* (78.0) (83.2) (65.6) (59.1) (52.2) (36.3) (40.9) (49.0) (55.8) (66.2) (70.0) (72.9) (76.7)

Aspirin 100 5.53±0 5.88± 5.89± 5.92± 5.91± 5.87± 5.97± 5.96± 5.94± 5.92± 5.93± 5.89± 5.86± 5.79± .03 mg/kg 0.02* 0.02* 0.02* 0.04* 0.03* 0.03* 0.03* 0.03* 0.04* 0.03* 0.04* 0.02* 0.05* (78.2) (78.3) (79.5) (82.4) (81.5) (79.0) (79.9) (81.1) (81.1) (80.6) (82.3) (82.5) (72.9)

Results are expressed as mean± SEM (n=4); SOC Saccharum officinarum; DW: Distilled Water;*p<0.001 statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post-test). Percentage inhibition of oedema in parenthesis.

Histamine- induced paw oedema in rats while the standard drug recorded 85.71% The extract at 20 mL/kg dose produced a high percentage inhibition at that same time (Fig. 1). percentage inhibition of 60.71% at 180 minutes,

7.5 Control DW 10 mL/kg 7.0 S. officinarum 20 mL/kg Cyproheptadine 10 mg/kg 6.5

6.0

Paw size (mm) size Paw 5.5

5.0

Basal 30 mins 60 mins 90 mins 120 mins150 mins180 mins Time Figure 1: Paw size of rats in histamine-induced paw oedema. Results are expressed as mean± SEM (n=4); SOC Saccharum officinarum; DW: Distilled Water; bp<0.001, a p<0.01 and *p<0.05statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post- test).

Serotonin- induced paw oedema in rats comparatively effective inhibition 70.1 % of paw The dose 20 mL/kg of SOC juice after 60 edema in rats with aspirin recording 77.6 % minutes produced a significant (p<0.05) and a (Fig.2). RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 6 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7

7.5 Control DW 10 mL/kg 7.0 S. officinarum 20 mL/kg cycproheptadine 10 mg/kg 6.5

6.0

Paw size (mm) size Paw 5.5

5.0

Basal 30 mins 60 mins 90 mins 120 mins150 mins180 mins Time

Figure 2: Paw size of rats in serotonin-induced paw oedema. Results are expressed as mean± SEM (n=4); SOC Saccharum officinarum; DW: Distilled Water; *p<0.05 statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post-test).

Castor oil-induced diarrhea in mice dependent decrease in the frequency of purging Pre-treatment of mice with the different doses of (reduction of number of wet stool) and, weight of SOC juice caused a significant and a dose wet stools (Table 3).

TABLE 3: CASTOR OIL INDUCED DIARRHEA IN MICE

Treatment Dose Onset of No of wet Weight of No of semi- Weight of semi- No of solid Weight of diarrhea (min) stool wet stool (g) solid stool solid stool (g) stool solid stool (g)

Control DW 10 mL/kg 11.50±5.68 7.50±2.06 0.22±0.05 6.50±1.56 0.23±0.05 3.50±1.44 0.09±0.04

S.O 10 mL/kg 53.25±16.62a 6.00±2.35 0.22±0.04 4.25±1.49 0.10±0.04 3.25±1.25 0.06±0.02

S.O 20 mL/kg 26.25±13.93 4.50±1.56 0.22±0.06 6.25±2.00 0.20±0.07 4.50±1.44 0.06±0.03

Atropine 1 mg/kg 42.50±22.46b 11.25±1.80 0.37±0.13 8.25±2.25 0.24±0.10 4.25±1.65 0.12±0.05

Results are expressed as mean± SEM (n=4); S.O: Saccharum officinarum; DW: Distilled Water; ap<0.001 and bp<0.01 statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post-test).

Formalin-induced paw oedema in rats mL/kg juice dose showed a significant inhibition st nd The SOC juice at a dose of 10 mL/kg produced (p<0.01) on the 1 and 2 day. Dexamethasone a significant inhibition of paw edema (p<0.05 showed a significant inhibition (p<0.05) on the th th and 0.01 respectively) on the 5th and 6th day. 4 day. On the 7 day, the juice at a dose of 10 Also, the standard drug, aspirin produced a mL/kg produced a percentage inhibition significant inhibition of paw edema (p<0.05) on (72.09%) that was higher than that of the the 2nd, 3rd, 4th and 6th days. Aspirin and 20 standard drugs (68.60% and 69.77% for aspirin and dexamethasone respectively) (fig 4). RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 7 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7

8 Control DW 10 mL/kg S. officinarum 10 mL/kg 6 S. officinarum 20 mL/kg Aspirin 100 mg/kg 4 Aspirin(100 mg/kg)+S.o20 mL/kg Dexamethasone 5 mg/kg

Paw size (mm) size Paw 2

0

Basal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Time

Figure 4: Paw size of rats in formalin induced paw oedema. Results are expressed as mean± SEM (n=4); SOC Saccharum officinarum; DW: Distilled Water;a p<0.01 and * p<0.05 statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post-test).

Xylene-induced ear edema in mice revealed the difference between both ears in the In this experiment; xylene produced an control group. (Fig 5). immediate swelling of the right ear which

0.15 Control DW 10 mL/kg S. officinarum 10 mL/kg 0.10 S. officinarum 20 mL/kg Dexamethasone 5 mg/kg

ear weight ear 0.05

0.00

RE Wt LE Wt

(RE-LE) Wt Treatments

FIGURE 5: Xylene-induced ear oedema

Cotton pellet-induced granuloma reduction with 36.56 % inhibition when The oral administration of SOC juice produced compared to the control group. Celecoxib 20 significant (p< 0.05 and p<0.01)) reduction in mg/kg showed the highest inhibition of 37.63 % inflammation when compared to control group. which was significant (p<.0.01) when compared Figure 6 shows the results obtained in the cotton to control after 7 days drug treatment. The anti- pellet-induced granuloma model in which the inflammatory effect produced by the SOC juice juice 10 mL/kg produced a significant (p<0.001) was low dose-dependent with the highest inhibition produced by a dose of 10 mL/kg. RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 8 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7

0.15 Control DW 10 mL/kg S. officinarum 10 mL/kg 0.10 S. officinarum 20 mL/kg Celecoxib 20 mg/kg

Weight (g) 0.05

0.00

weight of dry cottonweight pellet of(g) initial cotto pellet (g)

difference in weight of cotton pellet (g)

Treatments

Figure 6: Cotton pellet granuloma in rats. Results are expressed as mean± SEM (n=4); S.O: Saccharum officinarum; DW: Distilled Water; a p<0.01 and * p<0.05 statistical significance compared to control (Two-Way ANOVA followed by Bonferonni post-test).

Antioxidant capacity of SOC juice Figure 9 and showed potency. Values are The juice of S. officinarum exhibited good nitric expressed as Mean ± SEM. oxide scavenging activity leading to the DISCUSSION reduction of the nitrite concentration in the assay Anti-inflammatory models employed in this medium. The nitric oxide scavenging capacity investigation included xylene induced ear was concentration dependent with 100 µg/mL edema in mice, carrageenan-induced rat paw scavenging most efficiently. The juice of S. edema, formalin induced rat paw oedema (sub- officinarum significantly inhibited (p < 0.05) the acute), cotton pellet-induced granuloma and in accumulation of nitrite, a stable oxidation the process of elucidating mechanism of action product of nitrc oxide liberated compared to the of extract, the following models were used: standard ascorbic acid (Figure 7). The result of serotonin and histamine induced rat paw effects of S. officinarum on DPPH radical oedema, and castor oil induced diarrhoea for the scavenging activity is presented in Fig 8 which biosynthesis of prostaglandins. Anti- indicated potent radical scavenging activities. inflammatory activity is determined by analyzing The reducing power of S.officinarum juice as the reduction in oedema size and calculating % well as the standard Ascorbic acid increased inhibition of oedema. A mean reduction in progressively in a concentration-dependent oedema when compared with control and manner. The reducing power of Ascorbic acid increase % inhibition in the treated groups is an observed was greater than S.officinarum juice. indication of anti-inflammatory activity. The reduction capability of S.officinarum juice The carrageenan induced is based on the when compared to Ascorbic acid is shown in principle of release of various inflammatory RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 9 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 mediators. Oedema formation due to induced paw edema in rats, with peak inhibition carrageenan is a biphasic event. The initial of 83.23 %. The inhibition of induced paw edema phase is attributed to the release of histamine, produced by SOC was dose dependent with serotonin and kinins while the second phase is peak inhibition of 83.23% obtained at a dose of due to the release of prostaglandin, protease 20 mL/kg. Interestingly, this effect was and lysosome (24 - 25; 15). The juice of SOC comparable to that produced by 100 mg/kg of (2.5, 5, 10, and 10 mL/kg) in a dose-dependent aspirin (82.40 %), a non-steroidal anti- manner significantly inhibited the carrageean inflammatory (NSAIDs) drugs.

100 SO 80 Ascorbic acid

60

40 % Inhibition % 20

0

20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml Concentration

Figure 7: Nitric oxide scavenging activity of S. officinarum juice in comparison with ascorbic acid.

100 SO 80 Ascorbic acid

60

40 % Inhibition % 20

0

20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml Concentration

Figure 8: DPPH scavenging activity of S. officinarum juice in comparison with ascorbic acid.

RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 10 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7

Figure 9: Reducing power activity of S. officinarum juice in comparison with ascorbic acid.

Xylene induced ear oedema model is partially at doses of 100 mg/kg and 5 mg/kg which associated with substance P which is an produced inhibition of 68.60 % and 69.77 % undecapeptide that is widely distributed in the respectively on the same day 7. Co- central and peripheral nervous system and it administration of SOJ and aspirin produced functions as a neurotransmitter or 31.40 % inhibition on day 7. neuromodulator in a variety of physiological The effect of formalin is biphasic, an early processes (26). Release of substance P from the neurogenic component followed by a later sensory neurons causes vasodilation and tissue-mediated response. In the first phase, plasma extravasations suggesting its role in there is a release of histamine, 5HT, and kinin, neurogenous inflammation. Thus, it causes the while the second phase is related to the release swelling of ear in mice. This test distinguishes of prostaglandin (27). The peak inhibition of NSAIDs and steroidal anti-inflammatory drugs. formalin on day 7 by the juice suggests the The juice of SOC (20 mL/kg) showed a inhibition of both phase 1 (histamine production) significant dose dependent inhibition of and phase 2 (prostaglandin production) by the inflammation when compared with distilled water extract, an effect which was dose dependent. control. Peak inhibitory effect was obtained at a In order to elucidate the mechanism of action of dose of 20 mL/kg of the juice (50 %) and has a the juice of SOC, selected anti-inflammatory better effect to that produced by 5 mg/kg mediators were used, especially, those dexamethasone (40 %). This showed that SOC produced by carrageenan (serotonin, histamine, also possesses mild steroidal anti-inflammatory and prostaglandin). The juice of SOC (20 mL/kg) property. showed a significant inhibition of serotonin when The effect of the juice on formalin-induced paw compared with the control. The peak inhibition oedema in mice suggests its anti-arthritic effect. (70.15 %) was obtained at 60 minutes post The juice of SOC showed a significant inhibition induction compared to that of 10 mg/kg of inflammation induced by formalin compared to cyproheptadine (77.61%). This showed that control. The extract at a dose of 10 mL/kg juice of S. officinarum has a good anti- produced 77.70 % inhibition of formalin on day serotonergic effect as a result of inhibition of 7, and this was however comparable to serotonin. On the other hand, the same dose of acetylsalicylic acid (aspirin) and dexamethasone RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 11 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 the extract (20 mL/kg) showed a significant of the present investigation has elucidated as inhibition of histamine when compared with the part of its mechanism of action, its inhibitory control (10 mL/kg distilled water). The peak involvement in prostaglandins synthesis as well inhibition (60.71%) was obtained at 180 minutes as on serotonin and histamine, which are compared to 10 mg/kg cyproheptadine inflammatory mediators. (85.71%). This showed that the juice is equally a Reactive oxygen species and free radicals, mild inhibitor of histamine. which are products of normal cellular To further investigate the mechanism that might metabolism are thought to act indirectly as account for the anti-inflammatory action of SOC cellular messengers and elicit an inflammatory juice, castor oil-induced diarrhoea, which results response. in biosynthesis of prostaglandin in rat was used The juice was evaluated to determine its (16). The juice (10 mL/kg), produced a possible radical scavenging activity, a known significant delayed onset of diarrhoea (53 mechanism by which antioxidants inhibit lipid minutes) when compared to the control (11 oxidation. A balance between free radicals and minutes). Moreover, there was a marked antioxidants is necessary for proper reduction in the production of wet stools with physiological function in the body (31). juice dose 20 mL/kg. This showed an inhibition 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) is of prostaglandin synthesis, which accounted for scavenged by antioxidants through the donation the characteristic diarrhoea seen in control of hydrogen forming a reduced DPPH, which can group with its rapid onset (11 minutes) and an be quantified by its decrease of absorbance. increased number of wet stools with a decrease The DPPH radical scavenging of the juice in the number of solid stool. The delayed onset indicated potent radical scavenging that of diarrhea (53 minutes) observed in SOC suggested antioxidant property, which treated animals is comparable to that of aspirin compared effectively with ascorbic acid. The (43 minutes). ferric ion reducing power of SOC was also The results of phytochemical analysis revealed evaluated. This measures the electron donating the presence of flavonoids, glycosides and activity of the extract. Reducing agents would quinine in high amounts. Kim et al., (2004) (28) cause a reduction of Fe3+ to Fe2+. The ferric ion reported that flavonoids possess anti- reducing power was concentration dependent inflammatory property. Ahmadiani et al, (2000) when compared to ascorbic acid. Nitric oxide (29) also reported that flavonoids were found to (NO) is generated from amino acid L-arginine by have anti-inflammatory and anti-nociceptive vascular endothelial cells, phagocytes, and activity, therefore flavonoid found in SOC could certain cells of the brain. Nitric oxide is classified have contributed to its observed anti- as a free radical because of its unpaired electron inflammatory property. Flavonoids are known to and displays important reactivity with certain target prostaglandins which are involved in the types of proteins and other free radicals. The late phase of acute inflammation and pain toxicity of NO becomes adverse when it reacts perception (15). Glycosides, the active with superoxide radical, forming a highly reactive component in willow bark have also been found peroxynitrite anion (ONOO−). The nitric oxide to exhibit anti-inflammatory effects (30, 15). radical scavenging of the juice indicates a potent From the foregoing, SOC has been established radical scavenging that was concentration to possess anti-inflammatory property, which dependent suggesting antioxidant property. compared effectively with both standard CONCLUSION steroidal and non-steroidal anti-inflammatory In conclusion, the investigation carried out drugs in both the acute and sub-acute models demonstrated that the juice of SOC possesses employed in this study. Furthermore, the scope RJPP: https://escipub.com/research-journal-of-pharmacology-and-pharmacy/ 12 Agbaje Esther Oluwatoyin et al., RJPP, 2019; 3:7 anti-inflammatory activities. Further studies are 10. Zimmerman M. Ethical guidelines for needed to elucidate the mechanism of action investigations of experimental pain in conscious animals. Pain, 1983; 16(2): 109-110. and therapeutic value in the treatment of 11. Lorke, D. A new approach to practical acute inflammation. Also, it can be concluded that the toxicity testing. Arch Toxicol, 1983; 54: 275–87. use of SOC in the treatment of chronic 12. Winter CA, Risley EA, Nuss GW. Carrageenin- inflammation is not as effective as its use in induced edema in hind paw of the rat as an assay acute and sub-acute conditions, howbeit, the for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med, 1962; 111: 544 – 547. juice at 10 and 20 mL/kg doses, compared well 13. Agbaje E.O, Fageyinbo M.S. Evaluating anti- with the standard drugs aspirin, inflammatory activity of aqueous root extract of dexamethasone, and celecoxib, and it is Strophanthus hispidus (DC.) 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