Tropical Journal of Natural Product Research

and pharmacological activities are very rare. Owing to the potential medicinal value of plant exudates, we selected the resin of three plants for our current study; the oleo resin of A. excelsa (Salisb.), A. bidwillii Hook, and the kino resin of E. maculata


Introduction
Inflammation is a response, triggered by damage to living tissues, to protect them from infection and injury as well as the consequence of such injury i.e. the necrotic cells and tissues. 1 Inflammation is managed mainly with nonsteroidal anti-inflammatory drugs (NSAIDs). 2 Although, these anti-inflammatory drugs can treat many inflammatory conditions, they have numerous side effects, such as gastrointestinal injury, hepatotoxicity, sodium retention, obesity and osteoporosis, that might cause serious health problems. 3 As alternatives to synthetic medicines, natural products show good promise, few side effects and proved to be potential candidates to cure inflammatory conditions. 4 The adverse effects of oxidative stress on human health have become a serious issue. A lack of antioxidants in human diet, which can

Preparation of the extracts
The air-dried powdered resin (10 g each) of E. maculata exudate (EME), A. excelsa exudate (AEE) and A. bidwillii exudate (ABE) were extracted with methanol (3 x100 mL) using a sonicator for 15 min. Each extract was concentrated under reduced pressure to a constant weight to yield dry methanol residue; (EME, 9.5 g), (AEE, 9.4 g) and (ABE, 8.9 g), respectively. The three extracts were kept in a desiccator over anhydrous CaCl2.

Chemical analysis
The three methanol extracts were analyzed for their total phenolic content (TPC), total flavonoid content (TFC) and radical scavenging activity using DPPH assay. The most promising extract (E. maculata) was standardized using HPLC.
Determination of the total phenolic content Total phenolics were determined employing Folin-Ciocalteu method as previously described. 17,18 Briefly, 10 mL distilled H2O, 1.5 mL Folin-Ciocalteu reagent and 1 mL of extract (1 mg/mL) were mixed. After 3 min, 4 mL of 20% Na2CO3 were added then made up to 25 mL with distilled water and incubated for 30 min at room temperature, comparatively to gallic acid standard (80-280 µg/mL). Absorbance was measured at 765 nm. The assay was done in triplicate, the mean values were calculated and the results expressed as gallic acid equivalents (GAE)/mg extract from the pre-established calibration curve and calculated using the following equation: Where, Y = absorbance, X = corresponding concentration (µg/mL) and R 2 = linear regression coefficient.
Determination of the total flavonoid content Total flavonoid content was measured by the aluminum chloride colourimetric assay described by Saboo et al. 19 Briefly, an aliquot (1 mL) of extract (1 mg/mL) or standard solution of quercetin (5 -100 µg/mL) was added to 0.1 mL aluminum chloride (10%), 0.1 mL potassium acetate (1 M) solution and 2.8 mL distilled water were added and mixed well, the absorbance was read after 30 min at 415 nm. A blank was prepared in similar way using methanol instead of the sample. The assay was done in triplicate, and the mean values were calculated. Total Flavonoid content in the extract was expressed as quercetin equivalent (µg of quercetin/mg of sample) from the preestablished calibration curve and calculated according to the following equation: Where, Y = absorbance, X = corresponding concentration (µg/mL) and R 2 = linear regression coefficient Determination of radical scavenging activity using DPPH assay The in vitro antioxidant activity of the investigated extracts were determined using 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging assay as previously described. 20,21 This test provides information on the ability of a compound to donate a hydrogen atom, the number of electrons a given molecule can donate, and on the mechanism of antioxidant action. DPPH solution (0.2 mM) was prepared and 1.0 mL of this solution was added to 3.0 mL of extract solution in methanol at different concentrations (2.5 -25 µg/mL) of methanol extract. Thirty minutes later, the absorbance was measured at 517 nm. A blank was prepared without adding extract. Trolox (2.5 -25 µg/mL) was used as standard. Decreasing absorbance of the reaction mixture indicates higher free radical scavenging activity. The IC50 value of the radical scavenging activity is the effective concentration at which antioxidant activity is 50%. The IC50 of each methanol extract was expressed in µg/mL.

HPLC analysis of the methanol extract of E. maculata Sample preparation
The methanol extract of the resin was subjected to fractionation and column chromatography following previously reported method. 22 Briefly, the total methanol extract of resin (120 g) was partitioned with CH2Cl2. The CH2Cl2 fraction (7 g) was chromatographed on a VLC column of Si gel 60 (270-400 mesh). Gradient elution was carried out starting with n-hexane, n-hexane /CH2Cl2, CH2Cl2, and finally CH2Cl2/EtOAc mixture, up to100% EtOAc. Fraction (1.2 g) eluted with CH2Cl2/EtOAc (4:1) was further purified on another Si gel column using CH2Cl2/MeOH (9:1 v/v) to give 300 mg of white microcrystalline powder (compound MA). The chemical structure of the compound was identified by comparing its 1 H-and 13 C-NMR spectra with those reported in literature. 22

Chromatographic conditions
Standardization of the methanol extract of E. maculata was achieved according to a previously reported method 23 with some modification using Agilent 1200 series (USA) HPLC equipped with quaternary pump, auto sampling injector, solvent degasser and UV detector.

Biological evaluation
The anti-inflammatory activity of the methanol extracts of resin exudate of E. maculata, A. excelsa and A. bidwillii were evaluated using carrageenan-induced foot paw oedema model following method of Winter et al. 24 with slight modification.

Animals
Sixty-six (11 x 6 in each group) Sprague-Dawley adult male rats, with average age 6-8 weeks and weighing 180 ± 20 g, were purchased from "Egyptian Organization for Biological Products and Vaccines", Giza, Egypt. All procedures were approved by the ""Research Ethics ISSN 2616-0692 (Electronic)

© 2020 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Committee"" for experimental and clinical studies at Faculty of Pharmacy, Cairo University, Egypt" under a serial number MP (2186).

Experimental protocol
Rats were divided into 11 groups (n = 6) of which nine groups were administered the methanol extract of the three plants at three doses (100, 200 and 400 mg/kg, p.o) and the remaining two groups of animals received carrageenan (0.1 mL of 1%, freshly prepared) and indomethacin as standard drug (5 mg/kg, i.p). 25 One hour after drug administration, inflammation was induced by sub planter injection of 0.1 mL of carrageenan (1% freshly prepared suspension) in saline into the paws of right hind limb of the rats of all groups.

Determination of the percentage oedema and percentage inhibition of oedema (I%)
After the injection of carrageenan, paw diameter of the right hind limb of the rats were measured once every hour for 4 h using digital caliper and the swelling rates of each group were calculated. The percentage oedema at the end of the experiment and percentage inhibition of the oedema (I%) of each group were calculated as follows: Where, PT0 and PTt are the paw diameter (mm) before and after carrageenan injection, respectively. Ed and Ec are the mean percentage oedema of the treated and carrageenan group, respectively. 26 Blood sampling Four hours after carrageenan injection, animals which received E. maculata resin extract at dose of 400 mg/kg (which showed the highest activity) were anesthetized using thiopental, and then blood samples were collected from the retro-orbital venous plexus. The serum samples were obtained by centrifugation (1000 × g for 15 min) and the serum was stored at -80°C until analysis.

Estimation of oxidative stress markers.
Malondialdehyde (MDA) is an index of lipid peroxidation, its content was quantified by reaction with thiobarbituric acid (TBA) and absorbance was measured spectrophotometrically at 532 nm according to the method reported previously. 27 Reduced glutathione (GSH) content was determined according to the method reported previously. 28 The absorbance of the product was measured spectrophotometrically at 412 nm. Superoxide dismutase (SOD) activity was determined by observing the SOD-inhibitable auto-oxidation of pyrogallol, as described previously. 29,30 Determination of inflammatory biomarkers Tumour necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) levels were assayed in serum using rat sandwich ELISA kits (Cusabio Biotech Co, USA). Additionally, Nuclear factor kappa B (NFκ-B) level was measured by sandwich ELISA kits (MyBiosource San Diego, CA, USA) according to the manufacturer"s instructions.

Determination of total nitrite/nitrate (NOx) markers
The total nitrite/nitrate (NOx) products, an indicator of NO synthesis, were measured according to the methods described previously. 31,32 Statistical Analysis Data were expressed as mean  standard deviation (SD), where n= no. of rats. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons post hoc test. The level of significance was set at p 0.05. The GraphPad Prism V 6 (GraphPad Software Inc., San Diego, CA, USA) was used.

DPPH scavenging activity
The MEME kino resin reduced the stable radical DPPH to the yellow coloured diphenylpicrylhydrazine with IC50 value of 6.83 ± 0.77 μg/mL and was found to be more potent than the positive control; trolox (IC50 value of 21.18 ± 0.59). However, the methanol extracts of resin of AEE and ABE showed IC50 values of 617.86 ± 0.91 and 3796 ± 0.84 μg/mL.

Standardization of the most potent extract
Standardization of the MEME kino resin was made using HPLC utilizing 7-O-methyl aromadendrin (MA) as reference standard. The concentration of compound MA was determined from standard calibration curve to be 68.21 mg/g MEME.

Biological evaluation
Anti-inflammatory activity (carrageenan-induced paw oedema ( After 4 h of carrageenan administration, all treated groups that received methanol extract of the three plants at three doses (100, 200 and 400 mg/kg) significantly decreased the paw oedema at P < 0.05 and percentage oedema (Figure 2), when compared to the control group which received only carrageenan. The magnitude of the decrease was higher for the highest dose (400 mg/kg) among all tested groups, suggesting a dose-dependent effect. Interestingly, the MEME at 400 mg/kg showed E% of 35.5 ± 1.07, and a higher value of percentage inhibition of paw oedema (I%) of 50.91, while indomethacin group showed 66.1% ( Figure 3A-B). These results clearly showed that the methanol extract of E. maculata kino resin (400 mg/kg) had the highest anti-inflammatory activity.

Effect of MEME resin on antioxidant status
The role of E. maculata in enhancing the antioxidant status was investigated by measuring the enzyme levels of MDA ( Figure 4A), GSH ( Figure 4B) and SOD ( Figure 4C) in serum. Indomethacin administration attenuated the oxidative stress induced by carrageenan through decreasing MDA level by 47.5% from the control group (carrageenan only) and increasing the antioxidant enzyme GSH by 3.9-fold and SOD by 1.5-fold compared to the control group. The MEME resin (400 mg/kg) administration significantly attenuated the oxidative stress induced by carrageenan through decreasing MDA level by 42% from carrageenan group and increasing both antioxidant enzyme GSH and SOD levels by 2.6-, and 1.3-fold, respectively of carrageenan group. Moreover, MEME (400 mg/kg) showed no significant difference in the level of MDA and SOD when compared with indomethacin group.

Effect of MEME resin on TNF-α, NFκ-B, COX-2 and NO serum levels
To investigate the role of MEME resin as an anti-inflammatory, the NFκ-B ( Figure 5A), TNF-α ( Figure 5B), COX-2 ( Figure 5C) and NO ( Figure 5D), the key regulators in inflammation were measured in serum. Treatment with indomethacin significantly reduced the serum levels of inflammatory biomarkers by 60%, 59%, 65% and 43%, respectively, from carrageenan group. Treatment with the methanol extract of MEME resin (400 mg/kg) significantly reduced the serum levels of inflammatory biomarkers NFκ-B, TNF-α, COX-2, and NO by 42.7, 40.5, 42 and 23%, respectively compared to the control group. Carrageenan is widely used to induce paw oedema in rodents to investigate anti-inflammatory effect of drugs or herbal extracts. 33 Carrageenan-induced severe oedema in rats includes two different phases: the initial phase (0-1 h) of oedema is attributed to the release of histamine, bradykinin and serotonin which mediates the increased synthesis of prostaglandins from surrounding tissues of the injured area. 34 The second phase (3-5 h) is mainly mediated by prostaglandin, cytokines, cyclooxygenase, nitric oxide, and neutrophil derived free radicals. 35 With the aim of finding new natural remedies for the management of inflammation, three exudate resin extracts were screened for their potential anti-inflammatory activity using carrageenan-induced rat paw oedema model. The results showed that oral administration of methanol extracts of E. maculata kino resin, A. bidwillii and A. excelsa oleo-resins (100, 200 and 400 mg/kg) pretreatment produced a significant improvement in the paw inflammation. The MEME at a dose of 400 mg/kg demonstrated the most potent oedema inhibition (50.9%) compared to control group. In addition, EME resin possessed higher antioxidant potential evaluated by DPPH assay, which can be attributed to its higher phenolic and flavonoid contents than the other tested resins. To get a deeper insight about the mechanism involved in alleviating inflammation, attenuation in oxidant status and inflammatory biomarkers in serum were further studied. Cyclooxygenase-2 (COX-2) represents a vital key enzyme in inflammation and is considered as a rate-limiting enzyme, which catalyzes prostaglandins production, which is responsible for the formation of inflammatory mediators. 36 NO is also responsible for vasodilatation, the increase in vascular permeability and oedema formation at the site of inflammation. 37 NF-κB is recognized to be the main transcription factor regulating the expression of proinflammatory enzymes and cytokines, such as iNOS, COX-2, TNF-α and interleukin-1 (IL-1). 38 TNF-α, a mediator of carrageenan-induced inflammation, is able to induce the further release of kinins and leukotrienes, which is suggested to have an important role in the maintenance of long-lasting inflammatory response. 39 Hence, inhibition of these markers leads to reduction of inflammatory conditions. Pretreatment with MEME resin significantly decreased these biomarkers in serum after carrageenan injection. In a number of pathophysiological conditions associated with inflammation or oxidative stress, reactive oxygen species (ROS) have been reported to mediate cell damage via a number of mechanisms including the initiation of lipid peroxidation, the inactivation of a variety of antioxidant enzymes such as GSH and SOD. MDA production after carrageenan injection is due to free radical attacking plasma membrane. Thus, inflammation would result in the accumulation of MDA and reduction of anti-oxidative status by reduction of GSH and SOD activity. 40 Additionally, the level of MDA was decreased significantly, although the level of GSH and SOD enzyme were significantly increased from control group by treatment with the MEME resin (400 mg/kg), confirming its anti-oxidative effect. These results are greatly supported by the previously reported antioxidant activity of MEME kino resin. 16 The potent anti-inflammatory and antioxidant activities of the MEME kino resin might be attributed to its high phenolic and flavonoid contents. 41 Therefore, it may be used as a potential agent in the management of diseases in which free radical formation is a pathogenic factor. Data are expressed as mean ± SD, n= 6, P< 0.05 (* compared to carrageenan group, # compared to indomethacin group) using regular one-way ANOVA followed by Tukey's multiple comparisons test. Data is expressed as mean ± SD, n = 6, P < 0.05 (* compared to carrageenan group, # compared to indomethacin group) using regular one-way ANOVA followed by Tukey's multiple comparisons test. Data is expressed as mean ± SD, n= 6, P< 0.05 (* compared to carrageenan group, # compared to indomethacin group) using regular one-way ANOVA followed by Tukey's multiple comparisons test. Data is expressed as mean ± SD, n = 6, P < 0.05 (* compared to carrageenan group, # compared to indomethacin group) using regular one-way ANOVA followed by Tukey's multiple comparisons test.

Conclusion
In the current study, we evaluated the antioxidant and antiinflammatory activities of E. maculata kino resin, A. bidwillii and A. excelsa resin exudates. Methanol extract of E. maculata (400 mg/kg) showed the highest antioxidant activity. The results confirmed that the methanol extract of E. maculata kino resin, A. bidwillii and A. excelsa oleo-resin (100, 200 and 400 mg/kg) reduced carrageenan-induced paw oedema in rats. The methanol extract of E. maculata kino resin (400 mg/kg) was the most potent through its anti-inflammatory and antioxidant activity in a TNF-α, NFκ-B and COX-2 dependent manner and this can be attributed to its high content in phenolics.

MA Retention time (minutes)
Intensity