ANTI-INFLAMMATORY EFFECTS OF ROOT BARK METHANOLIC EXTRACT OF BOSWELLIA DALZIELII IN ALBINO MICE

Jeweldai Vedekoi1,4*image, Elizé Dadaya2image, Koubé Juliette3image, Sokeng Dongmo Sélestin4image

1Department of Biotchnologies and Pharmacognosy, Faculty of Science, University of Ebolowa, Ebolowa, Cameroon.

 2Department of Life Sciences, Higher Teachers Training College, University of Bertoua, Bertoua, Cameroon. 

3Department of pharmaceutical sciences, Faculty of Medicine and Pharmaceutical Sciences, University of Douala, Cameroon. 4Department of Biological Sciences, Faculty of Science, University of Ngaoundere, Ngaoundere, Cameroon.

 

 

 

Abstract

Background and objective: Boswellia dalzielii has been traditionally used in the Far North region (Cameroon) to treat fever, rheumatism, asthma and various inflammatory conditions. This investigation was designed to determine the anti-edematous and antioxidant properties to confirm its storied claims.

Methods: The DPPH radical scavenging and reduced Fe3+to the Fe2+ assays were two methods used to evaluate the antioxidant activities. The swelling caused by carrageenan paw and xylene ear edema to cause inflammation were applied to evaluate the anti-inflammatory activities of the extract. The mechanisms of action of the methanol extract were evaluated in the serotonin test. Fever induced by yeast was exploited to value antipyretic activity in albinos animals. Diclofenac, ascorbic acid, and paracetamol were utilized as standard medicaments.

Results: Results published the existence of cardiac glycosides, flavonoid, simple phenols, flavonoids, tannins, coumarins and alkaloids. The results indicated also no acute toxicity and the LD50 was above 3000 mg/kg. The methanolic extract exhibited the best activity IC50 (178.12 µg/mL) against DPPH radical with, which was comparable with ascorbic acid (179.12 µg/mL) and a strong reducing power with an IC50 of about 147.85 µg /mL. In addition, the roots extract of B. dalzielii significantly inhibited (p<0.001) the volume of paw edema, ear edema and the fever.

Conclusion: Finally, the present study published clearly that the B. dalzielii methanolic extract (BDME) possess important antioxidant and anti-edematous effects. The phytoconstituents found in this methanol extract were responsible for the anti-inflammatory and antioxidant effects.

Keywords: Anti-inflammatory, B. dalzielii, Carrageenan, DPPH, FRAP, IC50, LD50.

 

 

 

INTRODUCTION

 

 The reactive oxygen species (ERO) are unstable, very reactive elements and their reaction rate constants are very high, ranging from 10-9 to 10-6 second1. By their particular structure, free radicals tend to attract electrons from other atoms and molecules to gain stability2. In fact, free radicals, capable of independent existence (hence the term free), are chemical, atomic or molecular species, characterized by instability and/or strong oxidizing power. In the biotic life, these unpaired electrons (the superoxide anion (O2.˙-), the hydroxyl radical (.OH), nitrogen monoxide (NO.), the peroxide radical (ROO.) and the alkoxyl radical (RO.)) are produced continuously by the respiratory chain in order to destroy bacteria within phagocytic cells, to regulate lethal cellular functions, to participate in the functioning of certain enzymes, in the transduction of cellular signals, in the regulation of capillary dilation3. However, most of the damage to cellular components and tissue structures results in an expression called oxidative stress caused either by a shortage of available antioxidants or by an overproduction of free radicals4. Oxidative stress corresponds to effect to a disturbance of the intracellular oxidative status. In terms of counter-effect, an arsenal of (antioxidants) is being put in place to prevent these harmful oxidizing products5. But, the use of synthetic antioxidants provokes the appearance of side effects of uncontrolled, inadequate and abusive use and a potential risk of toxicity to human health such as tetragene, mutagenic and carcinogenic effects. Today, antioxidants from natural sources are causing renewed interest because of their therapeutic values via the chelation of metals and the scavenger effect, the inhibition of enzymes generating free radicals6. The search for natural substances as sources for the production of new anti-inflammatory and antioxidant potion has emerged as an alternative to chemical synthesis molecules. There is assuredly that Africa is fortunate with extensive greenery whose medicinal effects are to be rappe7. B. dalzielii belongs to Burseraceae family is grows up to 13 meters in height. It is native to the Northern Ivory Coast, Northern Nigeria, Cameroun and Central African Republic and is also plenty in the tropical zones of Africa and Asia. This vegetal is very utilized in traditional medicine: the stem bark extract is employed in North-Central zone of Nigeria to treat brainsickness, to show antiulcer activity and reduced gastrointestinal motility8.The fresh bark was caused vomiting and cured of vertiginousness and pulses9.The stem bark of B. dalzielii was used in the Far North region (Cameroon) folk medicine to manage pyrexia, rheumatism, asthma and arthritis. Moreover, this plant is used as an anti-seizure medication. The pharmacological study was approved that the plant methanol extract has been antioxidant, anti-inflammatory and antipyretics properties using classical experimental models. A phytochemical screening was too appraised.

 

MATERIALS AND METHODS

 

Authors are thankful for the University of Ebolowa, Ebolowa, and University of Ngaoundere, Ngaoundere, Cameroon to provide necessary facilities for this work. 

Planting material and identification

The B. dalzielii root barks belong to Burseraceae family was selected for the study. The herbarium of vegetable material was collected in December, 2021 at Mandaka Village, Far North Region, Cameroon. The fresh material plant was collected from original source after kindly identification and authentication from plant taxonomist, Professor MAPONGMETSEM Pierre-Marie. The voucher specimen was kept in the Cameroon National Herbarium, Yaoundé, Cameroon, (Voucher number 20532/SRF-CAM) was assigned. The collected root barks were cut into small pieces, cleaned up and dry in the shade with regular turning to avoid decaying, until crispy.

Experimental animals 

The white animals of both sexes (25-30 grams) wereplaced in the clean polypropylene cages of classic size. The experimental albinos were acclimatized at ambient weather (natural, 22±2oC temperature, 35–60 % humidity), fed with eat selectively, had free access to drinking fresh water ad libitum, and purchased from LANAVET (National Veterinary Laboratory), Garoua, Cameroon. Male and female albinos’ mice 2 to 2.5 months old are healthy and not pregnant (female). The mice were acclimated for 2 weeks prior they were subjected to the investigation. This living being deprived of water only during the experiment and fasting for six hours before to commencement of the experiments.

Preparation of the roots bark of B. dalzielii methanolic extract (BDME)

For preparing extract, the fresh roots of B. dalzelii subjected for air dried, were chopped into small pieces and was finely powdered with pestle and mortar and was mixed with absolute methanol. In brief, 20 g of plant powder were extracted with 200 mL at room temperature under agitation. After the extraction, the mixture filtered with Whatman No. 1 filter paper (Maidstone, UK) and was condense by evaporation at 45°C in a Rotavap (UV-2100 Spectrophotometer) to get solid residue. This viscous mass was then stored in a specimen bottle at room temperature for use throughout the study. 

Acute oral toxicity study 

The toxic effect was studied in healthy female living being (30-35 g) following the OECD guidelines No 42310. The mice were abstained from food for 12 h foregoing to the experiments. These mice were (n=6) were divided into 2 groups of 3 animals each. Mice were administered with single dose of BDME, 3 g/kg, orally; while the negative grouping was obtained normal saline (10mL/kg) andthe food may be withheld for a further 2–3 hours. The mice were seen perpetually for first seventy-two hours and fifteen consecutive hours of sunlight for their extensive physiological manifestations: signs of toxicity, other parameters (weight gain, food and water consumption) and mortality after which the LD50 was calculated. 

Qualitative phytochemical determination

Qualitative phytochemical determination of the plant material was tested employing the next chemical compounds and interactant. It was verified the presence or absence of saponin (frothing test), tannins (FeCl3), cardiac glycosides (Salkowski test), flavonoid (NaCl and HCl), phenol (FeCl3 and K3Fe(CN)6), terpenes/sterols (H2SO4), coumarins and alkaloids (Dragendorff). These different groups of chemical compounds have been selected for their importance in the therapy of inflammatory pathologies.

DPPH radical scavenging properties

The antioxidant properties of the BDME and the vitamin C was estimed on the base of the DPPH (1,1-diphenyl-2-picryl-hydrazyl) as previously described with minor modifications. 11In this method, the different extract solution (100, 200, 400, 600, 800 et 1000 µg/ml) of vegetable methanol extract and vitamin C were added to 2 mL of 10 mg/L. The solution was shuddered and was incubed for 30 min to perform complete reaction. Vitamin C was utilized as reference drug. Finally, the absorbance (Abs) of plant material extract was measured at 517 nm by U.V. spectrophotometer.

Ferric reducing antioxidant power (FRAP) activity

The protocol used in the laboratory is founded on the methodology described previously. 12In brief, 200 µl of BDME at 100, 200, 400, 600, 800 and 1000 µg / ml was added in 0.5 ml of a 0.2 M phosphate buffer solution (pH 6.6) and 0.5 ml of a 1 % solution potassium ferricyanide K3Fe(CN)6. The mixture was cover in a bain-marie at 50°C for twenty minutes, and then 0.5 ml of 10% trichloroacetic acid was put in to the reaction medium. The solutions were eccentric at 3000 rpm for 10 min. The fractional share (0.5 ml) of floating was mixed with 1 ml of a liquid of FeCl3 (0.1% ferric chloride) newly formed in distilled water. The optical density of the solution was measured at 700 nm against a white prepared under the same conditions but by substitute the extract with methanol which makes it possible to measure the device.

Carrageenan induced mice paw edema

The hoof of mice was conducted based on a previously described13.Thirty adult mice of both sexes (25-30 g) were arbitrarily divided into the following 5 groupings of six mice each. Group 1, the negative grouping, was obtained normal saline (10 mL/kg); group 2 was obtained diclofenac sodium (10 mg/kg) and Groups 3, 4 and 5 were given the BDME of 100, 200 and 400 mg/kg, respectively. After 60 min, the edema was caused by injecting 0.1 ml of sterile saline stay of 1% carrageenan generate the beneath the sole of the foot of right hind paw of each mouse and the left hind paw served as control. The hind paw diameter was measured immediately the nat 60, 120, 180, 240 and 300min after carrageenan administration trough vernier caliper.

Xylene induced ear edema

Mice lughole swelling was generated by xylene solvent as previously described15. Thirty adults’ mice of any sex were arbitrary allocated into 5 lots of six albinos each. At the end of the abstain from food moment, animals were received the BDME or standard medicament. Group 1 received physiological solutionat the dose of 10 mL/kg and served as negative group. Group 2 received diclofenac solution (10 mg/kg) and served as reference group. The grouping 3, 4 and 5 were pretreated with 100, 200, and 400 mg/kg of the BDME, respectively. At one hour following orally, edema was instantly persuaded on either surfaces of the right hearing of any mouse by employment of 0.05 mL of xylene utilizing a runner pipette. The left ear is preserved natural. Thirty minutes after xylene treatment by performing cervical dislocation, the mice were loss of consciousness under ether anesthesia then; 6 mm of both treated and natural ears were pierced and circular sections were taken utilizing a laboratory cork borer and massed.

Serotonin-produced edema test

The paw swelling caused by the serotonin solution (0.05 ml) into the right hoof of albinos’ mice was accorded on that described previously with slight modifications14. A total of thirty experimental animals were equally grouped into five (1-5) of 6 per group. Mice were impoverished of water only during the experiment to guarantee uniform hydration and to reduce variableness in edematous reaction. The mice were handled as follows: Group 1, the negative control, took physiological solution (10 mg/kg); Group 2, the reference group, administrated diclofenac sodium solution (10 mg/kg) and the remaining groups received the BDME of 100, 200 and 400 mg/kg, orally. The standard drug and plant material extract were carry out 1 h prior to induce inflammation. The right hoof perimeter was determined through a Vernier caliper at 0, 60, 120, 240 and 360 min following the oral administrations of the conventional medicament and the plant extract. Edema was aimed as the variation in paw largeness between the negative group and the group tests.

Yeast-induced pyrexia in mice

The fever induced pyrexa tests in miceas previously described with minors’ modifications16. Briefly, hyperthermia was inducted in albino’s animals by intraperitoneal vaccination of 20 mL/kg of 20% aqueous stay of sterilized pyrexia stimulus compound. A well lubricated clinical thermometer (Panamedic, Cheonan Choongnam, Korea) was inserted about 2cm into the rectum of the animals. Eighteen hours’ post-injection, animals showing an augment of rectal pyrexia of at smallest 0.5°C were chose for the study. The anal porehyperthermia was determinated on 30, 60, 120 and 240 minutes’ time intervals through the thermal-probe. The experimental animals were completely randomized into 5 groupings containing 6 mice in anylot and were qualified to continue private in a bridle stockade. Physiological solution was administered to negative grouping (group 1); the second lot of animals received pharmaceutical drug, paracetamol (150 mg/kg), orally. While experimental group received doses of 100, 200 and 400 mg/kg of the BDME (groups 3, 4 and 5 respectively).

Ethics approval

The experimental protocol was accepted by the Animal Ethical Committee, Cameroon (number 075/16/L/RA/ DREPIA) and in agreement with NIH-Care and Use of Laboratory Animals (8th Edition).

Statistical analysis

Results of this study were used three replicates values of antioxidant potentials of specimen for statistical inquiries. The data were evaluated with ANOVA (SPSS, version 20.0) and the Dunnett’s post-hoc test to estimate variations between vehicle groups and treated groups. p=0.05 or less were observed statistically significant.

 

RESULTS

 

Authors are thankful for the University of Ebolowa, Ebolowa, and University of Ngaoundere, Ngaoundere, Cameroon to provide necessary facilities for this work. 

Acute toxicity study

In this investigation during seventy-four hours of observation, test animals were found normal. Following the fifteen days, the mice were no significant change in characters such as decreased sensitivity to disconfort, noisy breathing, runs, muscular contractions, expectoration, fraily, movement, hypnosis, fight, discharge from eyes and ears, offence, refuse to take food or water, slim, fatigue, paralysis. 

Phytochemical screening

The phytochemical profile from the BDME was detected the presence of cardiac glycosides, flavonoid, simple phenols, flavonoids, tannins, coumarins and alkaloids. The saponin and terpenes/sterols were not found.

Presence of Radical scavenging activity by DPPH

The capability of the methanol extract of B. dalzielii to reduce DPPH radicals was presented an decrease in the percentage inhibition with increasing concentrations (Figure 1). The DPPH unpaired electrons scavenging effects results of BDME and ascorbic acid (standard antioxidant) were presented in Figure 1. The maximum scavenging capacity of methanolic extract was 51.12 % at 0 mg/ml concentration as compared to conventional drug, vitamin C (60.12%) at the same concentration. 

The IC50 being inversely linked to the antioxidant capacity of the plant extract, it expressed the quantity of antioxidants needed to decline the concentration of the free radical to fifty per cent.  The IC50 was determined from the logarithmic regression equations of plotted curves illustrated in Figures 1. The BDME was presented an average IC50 of the order of 0.17 mg/ mL compared to an IC50=2.47 mg/mL of that of vitamin C.

Ferric-reducing antioxidant power (FRAP) assay

The decreasing power effect results of BDME and ascorbic acid are shown in Figure 2. According to the results obtained, the methanolic extract has an important reducing power; their IC50 is 0.03 mg/ml, a value slightly lower than that of ascorbic acid whose IC50 value is around 0.14 mg/ml.

Carrageenan produced mice hoof edema

The results of the carrageenan-induced paw swelling and percentages of inhibition in mice were presented in Table 1. The results were showed that in the first hour, the extract strongly inhibited edema (p<0.001) at all the doses (100, 200 and 400 mg/kg comparable to negative groups. The great reduction 72.22% was watched with 400 mg/kg of methanol extract at 5 h, while the 200 mg/kg presented 59.52% at four hours next the treatment. Diclofenac sodium (10 mg/kg) was showed 55.56%, the maximum inhibition, at 5 h of oral administration. Data are reported as mean ± SEM, n = 5. Each value in parentheses designate the proportion of reduction. ** Highly significant (p<0.01), *** Very highly significant (p<0.001) compared with the normal saline at the same time.

Xylene induced animal’s ear edema

The results of the mice ear swelling induced by xylene were presented in Table 2. Compared to the normal saline, methanol extract of B. dalzielii at 100 and 200 mg/kg presented a highly significant of the ear inflammation. The doses tested of methanol extract (100 and 200 mg/kg) was presented highly significant (p<0.001) inhibition ear edema by 45.71% and 41.22% respectively. The vegetable extract at a dose of 400 mg /kg produced 56.33% sixty minutes following the induction of inflammation and used a highly significant anti-edematous property (p<0.001) in comparison with the vehicle grouping, considered to be 100% swelling. Diclofenac at the quantity of 10 mg/kg had had a very highly significant (p<0.001) decreasing effect on the ear edema in mice induced by application of 0.05 mL of xylene (56.73%).

Mice paw inflammation induced by serotonin

The BDME and diclofenac sodium effects on mice hoof swelling induced by serotonin was showed in Table 3. In untreated mice (vehicle grouping) paw edema was grown progressively and was reached its best point 4 hours following the serotonin solution injection. Oral administration of BDME at a dose 400 mg/kg was presented very highly significant (p<0.001) percentage inhibition compared to control grouping. The maximum reduction percentages caused at the three respective test doses(100, 200 and 400 mg/kg) were 72.97 %, 62.16% and 81.08% paw edema, next 1, 4 and 4 hours respectively of serotonin injection. The diclofenac showed the maximum inhibition percentages of paw swelling (51.35%) afterward 3 h of serotonin injection (Table 4).

Antipyretic effect on yeast induced pyrexia

The results of yeast induced pyrexia test was shown in Table 4. All the mice in different groups were received DBME (100, 200 and 400 mg/kg, respectively) and diclofenac sodium solution (10 mg/kg). The results were showed highly significant percentage inhibition of fevers (***p<0.001). The highest antipyretic effect was remarked at 400 mg/kg (81.08%) at 5 hrs, an activity higher than that of the pharmaceutical medicament. The antipyretic properties of the conventional medicament, paracetamol, were 51.35% at 5 hrs.

 

DISCUSSION

 

Qualitative chemical contents screening of BDME (Table 1) was revealed its richness of cardiac glycosides, flavonoid, simple phenols, tannins and coumarins. These chemical components were generally known for their effects against oxidative stress that had been implicated in inflammation pathologies and the mechanism of action of a methanol extract used in this work17. 

For the toxic effect study, the results indicated that albinos, mice do not presented signs of acute toxicity at the tested dose (3000 mg/kg) of BDME. During the fifteen days, the acute toxicity assessment observation period, there were presented no signs of toxicity. Accordingly, the finding indicates that this methanol extract was been presented non-toxic and was considered safe for mice testing. The LD50 was considered superior at 3000 mg/kg body weight.

The Vitamin C used as the standard antioxidant compound. DPPH radical scavenging capacity has been extensively utilized for studying antioxidant activities of different natural drugs. It is when the value of IC50 is very low that the antioxidant effect of the extract is strong, so the methanolic extract has a very powerful capacity to trap DPPH ROS. The DPPH (proton sensor) is an easy, acceptable and great extent used method to study antioxidant effects of pure molecules as well as plant extract18. It is fast, sufficiently and duplicable method. Noting that the powerful vertue of these substances in stopping or neutralizing unpaired electrons is basically due to their phenolic contents with the presence of hydroxyl groups acting as an electron donor, which makes them important antioxidant agents19. There is a power full relationship between phenolic compounds and antioxidant effects20.

Reducing power ability of a substance is the indicator of the potential antioxidant properties of a compound. Under not pathological conditions, natural antioxidants kept for happening oxidative stress21. Indeed, the antioxidant properties of a compound depend not only on its chemical structure, the nature, the concentration, and the power of this molecule but also according to the type of the generated radical which it can neutralize. These substances, thanks to their redox capacities, act as reducing agents, donors of hydrogen and singular oxygen22. Therefore, the inhibiting power of a substance can serve as anessential indicator of its antioxidant capability. The presence of great oxidant molecules and unpaired electrons-reducing abilities of plants justifies the importance of plants for medical purposes23.

Paw edema induced by carrageenan was usually studied in detecting orally active anti-inflammatory compounds and to predict the vertue of anti-inflammatory drugs acting by reducing the mediators of acute and local inflammation24,25. This work is an acute study using carrageenan as a phlogistic agent. The mice paw swelling induced by carrageenan is triphasic: the first phase (0-1 h) directly to release of 5-Hydroxytryptamine, histamine, bradykinin from mast cells, after which grown vascular permeability is maintained by the release of (2-4 h) maintained by kinins (second phase), third phase (4-5 h) produced by prostaglandins, protease and lysosomes26. These mediators augmented quantity of prostanoids generally such as prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) also lipoxygenase secretein the region and reduce arteriolar resistance27. As a result, the evacuate break out from the bloodstream to the interstitial space. This exudate is responsible of localized swelling, which, in turn, contracts the nerve endings and thus determines a feeling of sorrow, the greatest dreadful sign of inflammation28. Thus, this suggests that the inhibitory action of BDME could have an antagonistic action on histamine, bradykinin, serotonin and the biosynthesis of prostaglandins29,30.

The test of mice ear edema induced by xylene was associated to neurogenous edema released of inflammatory molecules such as histamine, kinins, prostaglandin, and bradykinins31,32. These mediators induce ear edema the formation of edema leading to an increase in vascular permeability and plasma leakage. Edema, characteristic of acute inflammation, gradually increases in volume and its thickness reaches its maximum half hour next employment of xylene. The results (Table 2) can be claimed that the BDME was presented pathological alterations occurred through reducing free radicals and attenuation of cytokines as well as causing edematous conditions. The phytoconstituents observed in this methanol extract is known to possess these properties23.

Sserotonin is the first inflammation mediator, a powerful vasodilator which increases vascular permeability33. Since the methanol extract effectively suppressed the oedema caused by serotonin, it is evident that methanolic extract has an acute effect on inflammation. The same is true for pharmaceutical medicament (non-steroidal anti-inflammatory drugs, corticosteroids and glucocorticoids) that have been widely employed to effectively inhibit inflammation34.

This indicates an activity of the BDME which is equivalent to that of aspirin (NSAID) which is a pure molecule. Pyrexia manifests itself clinically by an increase in body temperature above normal limits. Pyrexia is the rise of body temperature above normal range cause of PGE2 synthesis in hypothalamus, which occurs owing to produce ILs, TNF-α and interferon35. Intradermal injection of the yeast solution provoked fever by increasing prostaglandin secretion. These chemical mediators synthetize this substrate called arachidonic acid by the action of cyclooxygenase enzyme. Their production increases in the inflamed zone and may function either in the production and reduction of swelling36.  The hyperthermia induced by yeast solution is mentioned to as pyrexia, and its origin call for prostaglandin production. Blocking of prostaglandin synthesis is attain by inhibiting cyclo-oxygenase (COX) enzyme activity37.

The antipyretic effect of the methanol extract could be due to the reduction of cytokine release and prostaglandin biosynthesis. The fever induced by the injection of yeast is associate to the release of cytokines (TNFα, IL-1β, IL-6) which, having reached the blood vessels, stimulate the biosynthesis of prostaglandins (PGE2) around the center hypothalamic thermoregulatory38.

 

CONCLUSIONS

 

This study constitutes a scientific basis which validates the folkloric medicine of the methanolic extract used in the northern part of Cameroon in the treatment of inflammatory pathologies. The healing power of plants comes from the effects of their secondary metabolites including cardiac glycosides, flavonoid, phenols (simple phenols, flavonoids, tannins and coumarins), and alkaloids. For the rest, it is urgent if not necessary to isolate and analysis the active compounds of the extract in order to have a better comprehension of the mechanism’s action.

 

ACKNOWLEDGEMENTS

 

Authors are thankful for the University of Ebolowa, Ebolowa, and University of Ngaoundere, Ngaoundere, Cameroon to provide necessary facilities for this work. 

 

AUTHORS' CONTRIBUTIONS

 

Vedekoi J: investigation, data analysis and wrote the draft paper. Dadaya E: editing and formal the idea. Juliette K: methodology, data analysisand reviewed the manuscript. Sélestin SD: statistical work, calculations to manuscript proofing and carried out discussion. Final manuscript was checked and approved by all authors.

 

CONFLICT OF INTEREST

                

None to declare.

 

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