ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS, ACUTE AND ...

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Cimanga et al. World Journal of Pharmacy and Pharmaceutical Sciences

ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS, ACUTE

AND SUBACUTE TOXICITY OF EXTRACTS FROM BRUCEA

SUMATRANA ROXB. (SIMAROUBACEAE) LEAVES COLLECTED IN

MAI-NDOMBE, DEMOCRATIC REPUBLIC OF CONGO

Tshodi Ehata M.1, Nsaka Lumpu S.

1, Kambu Kabangu O.

1, Lami Nzunzu J.

1, Cimanga

Kanyanga R.*1,2

, Vlientick A. J.2, Pieters L.

2

1Department of Medicinal Chemistry and Pharmacognosy, Laboratory of Pharmacognosy and

Phytochemistry, Faculty of Pharmaceutical Sciences, University of Kinshasa, P. O. Box 212,

Kinshasa XI, Democratic Republic of Congo. 2Department of Pharmaceutical Sciences, Natural Products & Food Research and Analysis

(Natura), University of Antwerp, Universiteitsplein1, B-2610, Antwerp, Belgium.

ABSTRACT

This study reported for the first time the antioxidant and anti-

inflammatory activities of extracts and fractions from B. sumatrana

leaves collected in Mai-Ndombe in Democratic Republic of Congo. In

the antioxidant test, results indicated that extracts and soluble fractions

from B. sumatrana leaves exhibited good and interesting antioxidant

activity against some selected reactive oxygen species (ROS) or

oxygen radical species like DPPH, ABTS, superoxide anion, hydrogen

peroxide and hydroxyl with IC 50 values ranging from 2.05±0.01 to

15.11±0.02 µ/ml. The most active sample were the lyophilized

aqueous and 80% methanol extracts, ethylactacete and residual

aqueous soluble fractions exerting this activity with IC50 < 10 µg/ml

against all ROS. When test for anti-inflammatory against carrageenan

induced increase of foot volume, the use of oral doses of 50 and 100

mg/kg body in animals with oedema induced significant reduction of

foot volume from 60 to 80% inhibition compared to negative control.

The high foot volume inhibition of oedema was obtained with the

administration of the highest oral dose of 100 mg/kg body weight. The inhibition of foot

volume showed by aqueous lyophilized, 80% methanol and total alkaloid extracts was more

than 85% and was dose-dependent. All soluble fractions including chloroform, ethyacetate, n-

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 2, 92-122 Research Article ISSN 2278 – 4357

Article Received on

27 Nov. 2019,

Revised on 17 Dec. 2019,

Accepted on 07 Jan. 2020

DOI: 10.20959/wjpps20202-15414

*Corresponding Author

Dr. Cimanga Kanyanga

Richard

Department of Medicinal

Chemistry and

Pharmacognosy, Laboratory

of Pharmacognosy and

Phytochemistry, Faculty of

Pharmaceutical Sciences,

University of Kinshasa, P.O.

Box 212, Kinshasa XI,

Democratic Republic of

Congo.


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butanol and residual aqueous phase showed more than 60% inhibition of foot volume at the

highest oral dose of 100 mg/kg body weight. These results showed that these extracts from B.

sumatrana possess good and appreciable anti-inflammatory activity and can thus, support and

justify the use of the plant part in traditional medicine for he treating of rheumatism and other

pains. In addition, the lyophilized did no induce mortality of animal at the highest oral dose

of 50000 mg/kg bodyweight. its LD50 was estimated to be greater than5000 mg/kg body

weight and the extract was considered practically non-toxic by oral route and safe.

KEYWORDS: Brucea sumatrana, leaves, antioxidant, anti-inflammatory, acute and

subacute toxicity.

INTRODUCTION

Medicinal plants used in many traditional medicine in the world were well known as

significant sources of natural antioxidant natural substances belonging to different

phytochemical groups such as phenolic compounds, alkaloids, tannins, essential oils, steroids,

terpenes, phenolic acids, etc (Maestri et al., 2006; Baiano et al., 2016; Dong-Ping et al.,

2017). They were in form of crude extract, fractions or isolated chemical constituents and

were very effective to block various processes of oxidation by neutralizing free radicals

species (Rezaeian et al., 2015).

On the other hand, reactive oxygen species (ROS) like DPPH, ABTS, superoxide anions,

hydroxyl and hydrogen peroxide played an important role in the development of some

illnesses such as inflammation, asthma, dementia, arthritis, cardiovascular disease and

Parkinson’s disease. They were generated in human body through aerobic respiration and

from exogenous sources, and reacted with various biological molecules mainly proteins,

lipids and nucleic acids resulting in the creation of imbalance between oxidants and

antioxidants (Narayanaswamy and Balakrishnan, 2011). They can contrecart with harmful

effects of excessive ROS by inducing endogenous defense system although synthetic

antioxidants played a major role in protecting biological systems against oxidative stress

associated with development of chronic diseases and neurodegenerative disorders (Bamorniri

eta la., 2010; Trigui et al., 2013).

Phenolic compounds and other phytochemicals in many medicinal plants extracts and

fractions exerted strong antioxidant effect against various ROS and may help to protect the

cells against the oxidative damage caused by these free radical species and finally terminate


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their action of free radical by protecting the body from various ailments (Narayanaswamy

and Balakrishnan, 2011; Narayanaswamy et al., 2011; Merghem et al., 2019). Hence, interest

had been increased for discovery antioxidants from plant source which are safe and suitable

for use in food and medicine (Rayasandra et al., 2013).

In the other hand, the cardinal signs of inflammation included: pain, heat, redness, swelling,

and loss of function. Some of these indicators can be seen here due to an allergic reaction.

Figure 1: Foot inflammation.

Inflammation (from Latin: inflammatio or Inflammare.) is a part of the complex biological

response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants,

and is a protective response involving immune cells, blood vessels, and molecular mediators.

The function of inflammation is to eliminate the initial cause of cell injury, to clear out

necrotic cells and tissues damaged from the original insult and the inflammatory process, and

to initiate tissue repair.

The five classical signs of inflammation are heat, pain, redness, swelling, and loss of function

(Latin calor, dolor, rubor, tumor, and functio laesa). Inflammation is a generic response, and

therefore it is considered as a mechanism of innate immunity, as compared to adaptive

immunity, which is specific for each pathogen (Abbas et al., 2009). Little inflammation could

lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise

the survival of the organism. In contrast, chronic inflammation is associated with various

diseases, such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis.

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial

response of the body to harmful stimuli and is achieved by the increased movement of plasma

and leukocytes (especially granulocytes) from the blood into the injured tissues. A series of

biochemical events propagates and matures the inflammatory response, involving the local

https://en.wikipedia.org/wiki/File:Allergy_to_Antibiotic_Cefaclor.JPG

https://en.wikipedia.org/wiki/Latin_language

https://en.wiktionary.org/wiki/en:inflammatio#Latin

https://en.wikipedia.org/wiki/Pathogen

https://en.wikipedia.org/wiki/Immune_cells

https://en.wikipedia.org/wiki/Blood_vessels

https://en.wikipedia.org/wiki/Cardinal_sign_(pathology)

https://en.wikipedia.org/wiki/Functio_laesa

https://en.wikipedia.org/wiki/Innate_immune_system

https://en.wikipedia.org/wiki/Adaptive_immune_system

https://en.wikipedia.org/wiki/Adaptive_immune_system

https://en.wikipedia.org/wiki/Hay_fever

https://en.wikipedia.org/wiki/Periodontal_disease

https://en.wikipedia.org/wiki/Atherosclerosis

https://en.wikipedia.org/wiki/Osteoarthritis

https://en.wikipedia.org/wiki/Blood_plasma

https://en.wikipedia.org/wiki/Leukocyte

https://en.wikipedia.org/wiki/Granulocyte


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vascular system, the immune system, and various cells within the injured tissues. While

prolonged inflammation, known as chronic inflammation, leaded to a progressive shift in the

type of cells present at the site of inflammation, such as mononuclear cells, and was

characterized by simultaneous destruction and healing of the tissue from the inflammatory

process.

Inflammation is not a synonym for infection. Infection describes the interaction between the

action of microbial invasion and the reaction of the body's inflammatory response-the two

components are considered together when discussing infection, and the word is used to imply

a microbial invasive cause for the observed inflammatory reaction. Inflammation on the other

hand described purely the body's immunovascular response, whatever the cause may be. But

because of how often the two are correlated, words ending in the suffix-itis (which refers to

inflammation) are sometimes informally described as referring to infection. (Abbas et al.,

2009; https://en.wikipedia.org/wiki/Inflammation, 2019).

It included a number of events which can be considered under three phase viz. acute

transient, delayed sub-acute and chronic proliferate phases. During inflammation, the

liberation of endogenous mediators like serotonin, bradykinin, histamine and prostaglandins

occurred and were substances that indicated and modulated cell and tissue responses involved

in inflammation. They are in small quantity eliciting pain response (Danya, 2017). Some

enzymes such as cyclooxygenase (COX) are involved in inflammation, pains and platelet

aggregation and were the key in the synthesis of prostaglandins and throxanes. In addition,

inflammation was a severe response by living tissues to any kind of injury and can be given

four primary indicators as pain, redness, heat, heat, warmness and swelling. It can also

happen in response to processes like injury, cell death, some diseases as cancer and ischemia,

etc. (Artis et al., 2015; Waisman et al., 2015; Azab et al, 2016).

For the treatment if inflammation, nowadays, steroids, nonsteroidal anti-inflammatory drugs

and immunosuppressants were usually used for the relief of inflammatory disease and

required long-term treatment, and their use is often associated of the occurring of serious side

effects such as bleeding gastrointestinal and pectic ulcers (Oukacha et al., 2018).

https://en.wikipedia.org/wiki/Vascular_system

https://en.wikipedia.org/wiki/Immune_system

https://en.wikipedia.org/wiki/Mononuclear_cell_infiltration

https://en.wikipedia.org/wiki/Healing

https://en.wikipedia.org/wiki/Infection

https://en.wikipedia.org/wiki/Correlation

https://en.wiktionary.org/wiki/-itis

https://en.wikipedia.org/wiki/Inflammation


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MATERIALS AND METHODS

2.1. Vegetal material

Plant material (leaves of B. sumatrana Roxb. (Simaroubaceae) were collected In Mai-

Ndombe in DR-Congo. It was identified at the National Institute of Studies and Researchs in

Agronomy (NISRR), Departement of Biology, Faculty of Sciences, University of Kinshasa.

A voucher specimen of the plant No BSL2209014NL had been deposited in the herbarium of

this institute. Leaves were dried at room temperature and reduced to powder using an

electronic blender were kept in brown bottles hermetically closed.

Figure 2: Brucea sumatrana leaves and fruits.

2.2. Preparation of extracts and fractionation of lyophilized aqueous extract

50 g of powdered leaves were mixed with 300 ml distilled water and boiled on hotplate for

15 minutes. The mixture was cooled and filtered on a filter paper F001 grade (CHLAB

GROUP, 08205, Barcelone, Spain and the filtrate was evaporate in vacuum to give a died

extract with was further lyophilized to give a dried lyophilized aqueous extract BSLAE-1

(32.54 g). 20 g of BSLAE-1 were dissolved in 200 ml distiller water, filtered as described

above and successively and exhaustively extracted with solvents of different polarities as

chloroform, ethylacetate, n-butanol together with the resulting residual aqueous extracts

treated as described above to yield corresponding dried extracts denoted as BSLAE-1.1 (4.51

g0, BSLAE-1.2 (4.65 g), BSLAE- 1.3 94.15 g0 and BSLAE-1.4 96.05 g 9Harborne, 1998;

Trease and Evans, 2000).


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BSLAE-1 (20 g)

Dissolved in 300 ml distilled water

Filtration

Successive and exhautive extractionwith solvents of different polarities

CHCl3 (BSLAE-1.1) EtAOC (BSLAE-1.2) n-BuOH (BSLAE-1.3) Res.aq phae (BSLAE-1.4)

Figure 3: Fractionation of lyophilized aqueous extract BSLAE-1.

On other hand the same amount of plant material was macerated with 80% methanol for 24 h

and after filtration, the marc was exhaustively percolated with the same solvent. The

macerate et percolate were combined and evaporate in vacuum to give a dried extract denoted

s BSME (38.02g).

2.3. Extraction of polysaccharides

20 g of lyophilized aqueous extract ws dissolved in 30 ml distillled water and filtered on

paper Whaman No

1. In the filtrate, 150 ml of alcohol 95oC was added and the mixture was

left in fridge at 4oC for 24 h. After this period, un white precipitate was formed, filtered and

dried to give a dried extract (12.03 g). This extract gave a positive test with phenol/H2SO4

conc. (red-violet colour) for polysaccharides (Soniamol et al. 2010). Next, 10 g of this crude

polysaccharide was submitted to coloumn chromatography on Sephadex elutated with

MeOH/H2O 1:1 resulting in the obtaining 3 fractions, which analysed by CCM indicated the

presence of 1 spot in each fraction, suggesting thus the isolation of 3 pure polysaccharide

2.3. Qualitative phytochemical screening

The major phytochemical groups in lyophilized aqueous extract BSLAE-1 of B. sumatrana

leaves was carried out tubes solution in tubed and by TCL using different chemical reagents

and mobile phases described by Harborne (1998)et Evans and Teases, (2000).

2.4. Evaluation of acute and subacute toxicity

2.4.1. Toxic effects and determination of lethal dose 50 (DL50)

The acute toxicity of lyophilized aqueous extract of B. sumatrana leaves was evaluated on

Wistar rats (143-150 g body weight (bw)) using the procedure described by the Organisation

of Economic Co-operation Development (OECD) for chemical products, TG425. Briefly, for

this, sixteen rats were divided in four groups as followed:

- Group I (2 rats) orally received 5 ml distilled water as negative control group.


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- Groups II, III and IV (4 rats for each oral dose) were orally administered a single dose of

5000 mg/kg bw once. While the subacute toxicity of the same extract was determined

according OECD for chemical products, TG407.The same groups of rats were maintained

and groups II, III and IV daily received 500, 100 and 5000 mg/kg bw of lyophilized aqueous

extract BSLAE-1. Animals were placed in individual plastic cage and fed with water and

pellets. They were continually observed for toxic effects during the first 4 hours and daily

weighed. All animals were observed for 28 days. All abnormal suspect movements and death

of animals were recorded. Blood of animals having received 5000 mg/kg bw of lyophilized

aqueous extract was collected for haematological and biochemical analysis.

2.4.2. Evaluation of effects of lyophilized aqueous extract of B. sumatrana leaves of

haematological and biochemical parameters of Wistar rats.

The haematological parameters analysis of animals was carried out on a haematological

analyser (Couleter STK, Beckam) using respective appropriate kits. For biochemical

parameters, blood of animals having received 50000 mg/kg bw of lyophilized aqueous extract

of the studied plant part, was collected and centrifuged at 4000 g to obtain plasma. This last

was kept at 20oC: glucose, creatinine, aspartate aminotransferase (ASAT), alanine

aminotransferase (ALAT), serum glutaminopyruvate transferase 9SGPT), serum

glutamooxalate transferase (SGOT), total cholesterol high density lipoprotein (HDL), low

density lipoprotein (LDL) were quantified on an automatic apparatus Architect (AbootR)

using respective appropriate kits. Proteins were measured by Biuret method.

2.5. Evaluation of antioxidant activity

2.5.1. Effects of lyophilized aqueous extract BSLAE-1 and fractions on of DPPH (1,1-

diphenyl-2-picryzyl-hydrazyl)

The ability of free radical extracts and fractions from B. sumatrana leaves against the radical

DDPH was evaluated using the methods previously described by Selvakumar et al. (2011)

and Manthal et al. (2019). Briefly, 1 ml of test sample dissolved in methanol was mixed with

1 ml methanol solution DDPH 0.4 M and the mixture was left in obscurity for 30 minutes

before the measure of absorbance on a spectrometer Perkin-ElmerLamdda at 517 nm. DPPH

0.4 M MeOH solution was used as negative control. A series of test concentrations of test

samples from 0.1 to 500 µg/ml was used. The effect of tests sample on DPPH was calculated

using the following formula:


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AbsNc- AbsTs

AbsNc

x 100% inhibition DPPH =

Where AbsNc is the absorbance of the negative control and AbsTs is the absorbance of the

tested sample. The inhibition concentration 50 (IC50) of each tested sample was derived from

linear courbes concentrations-responses (n=3).

2.5.3. Effects of lyophilized aqueous extract and fractions of B. sumatrana leaves on

ABTS (2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonate)

Methods previously reported by Ilhami et al. (2010) and Vargas et al. (2016) based on the

oxidation of ABTS were used. The oxidated ABTS solution is prepared by reaction of 2 mM

ABTS in deionized water with potassium persulfate (K2S2O8). Before used, ABTS solution is

diluted with phosphate sodic tampon (0.1 M, pH 7.4) to have an absorbance of 0.750 at 734

nm. After, 1 ml of ABTS solution is added to 3 ml of test sample dissolved in ETOH-

H2O:1:1 (1-500 µg/ml), well mixed and kept in obscurity for 4 h before to measure the

absorbance. ABTS solution was taken as negative control. Absorbances were taken on the

same apparatus at 734 nm and inhibition percentages was calculated using the same formula

described also above. The concentration inhibitory 50 (IC50) of each tested sample was

derived in the same manner (n=3).

2.5.4. Effects of lyophilized aqueous extract and fractions of B. sumatrana leaves on

superoxide anion (O 2.-

)

For this test, 5 mg of each testes samples including extracts and fractions we dissolved in 5

ml ETOH to have respective stock solution of 1 ml/ml. They were diluted in two fold dilution

to have a series of test concentrations from 500 to 0.1 µg/ml. Test was carried out in

microtiter plates with 6 holes. Each hole contained a known concentration of test sample

mixed with 250 mM nitobleuterazolium (NBT, 100 µL) and 390 µM NAD (100 µL).

Absorbances were recorded at the same apparatus and the same formula was used to

calculated the inhibition of production of superoxide anions (de Vargas et al. (2016).

2.6. Estimation of total phenolic compounds

The quantity of total phenolic compounds in lyophilized aqueous extract of B. sumatrana

leaves was determined using Folin-Ciocalteu’s (FC) reagent using the method described by

Sheila and Mahmmod (2010) and Narayanaswamy and Balakrisham (2011). Gallic acid was

used as a standard (5-25 µg/ml). Different concentrations of standard and extract (50-250

µg/ml) were introduced separately in different tubes mixed with 1 ml of FC (1:1 dilution). 5


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minutes after, 2 ml sodium carbonate sodic 20% were added, mixed and left in obscurity for

30 minutes. After this period, absorbances were recorded at 765 nm using the same

spectrometer apparatus. The quantity of total phenolic compounds was expressed in term of

gallic acid equivalent per g of dried extract (n=3). 2.7.

2.7. Estimation of total flavonoids

The content of total flavonoids in lyophilized aqueous extract of B. sumatrana leaves was

determined quantitatively using ACl3 % MeOH solution using methods described by Talla et

al. (2016) and Manthal et al. (2019). 5 mg of lyophilized aqueous extract BSLAE-1 of B.

sumatrana leaves ere dissolved in5 ml MeOH to have stock solution of 1 mg/ml. It was

further diluted in two fold dilution of have a series of test concentrations from 500 to 0.1

µg/ml. To 1 ml of test sample with a known concentration, 1 ml of ACl3 5% in MeOH was

added, carefully mixed and incubated at room temperature for 60 minutes. After, absorbances

were measured at 430 nm on a spectrophotometer Perkin-ElmertLambda. Quercetin was used

as a reference and the quantity of total flavonoids was expressed in term of quercetin (mg/g

dried extract) (n=3).

2.8. Evaluation of anti-inflammatory activity

The method Ouedrago et al. (2015) was used to evaluate the anti-eremitical effect of

lyophilized aqueous extract of B. sumatrana leaves and its soluble fractions, 80% methanol

and total alkaloids extracts in animal model. Wistar rats with 115-165 g body weight were

used and divided in groups as followed: - Group I : (3 rats) received orally distilled water as

negative control,

- Group II (3 rats) received by the same way 5 mg/kg bw of diclofenac as positive control,

- Group II and III received in the way 50 and 100 mg/kg bw 993 rats for each oral dose) of

lyophilized aqueous extract BSLAE-1,

- Groups IV to VII received orally 50 and 100 mg/kg bw of chloroform, ethylacetate, n-

butanol and residual aqueous soluble fractions respectively (3 rats for each oral dose).

On hour after administration of test sample separately, inflammation was induced by the

administration of 50 µL of carrageenan 1% in NaCl 5% in cousin-plantar of right foot of each

treated animal. Volumes of treated foot of each animal as measured 5h after administration of

carrageenan (n=3). The anti-inflammatory activity of each test sample was evaluated using

the following formula:

%Inhibition of carrageenan = (Vt-Vo)nc – (Vt-Vo)ts / (Vt-Vo)nc x 100


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Where Vt is the foot volume at time t after injection of carrageenan and Vo the volume of

foot before the injection of carrageenan in negative and treated animals.

3. RESULTS AND DISCUSSION

3.1. Qualitative phytochemical screening

Results from the qualitative phytochemical screening revealed the presence of alkaloids,

flavonoids, steroids, terpenoids, catechic and gallic tannins, proanthocyanidins, reductor

sugar, polysaccharides, aminated compounds and saponins. Anthocyanins, anthraquinones,

coumarins and cardiotonic heterosides were not detected in the studied extract in our

experimental conditions.

Table 1: Qualitative phytochemical screening.

Chemical groups Results Chemical groups Results

Alkaloids ++ Saponins +

Flavonoids ++ Steroids +

Coumarins - terpenoids +

Anthocyanins - Reductor sugars +

Cardiotonic heterosides - Polysaccharides ++

Aminated compounds + Proanthocyanidins ++

The chemical composition of the lyophilized aqueous extract of B. Sumatran leaves was not

yet reported in the literature. This is the first report of these results.

3.2. Antioxidant activities against some radical species

3.2. Effects of lyophilized aqueous extract and its fractions against DPPH

The radical DPPH id a stable free radical showing a maximum absorption at 571 nm an is

currently used for the assessment of antioxidant of many plant extracts and isolated natural

products and other (Smith and Adanlovo, 2014, Mancarz et al., 2016; Nwokolo et al, 2019).

The used protocol is often simple, realisable without many difficults and is less expensive

(Nur et al. 2013). Mensor et al. (2001) and Veerapur et al. (2009) had considered also this

method easy and fast for the evaluation of potentials antioxidant properties of natural

products with a big advantage that the test is prepared and carried out in room temperature to

eliminate all risk of thermic degradation of substances under study. In this test, antioxidants

are capable to reduce the stable radical DPPH in violet colour to yellow coloured compound

diphenylpicrylhydrazin:


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N N N N

NO2

NO2

NO2

NO2

Diphenylpycryzylhydrazyl

(Free radical: violet

Diphenylpiicrylhydrazin

(non-radical: yellow)

Antioxidant +

Figure 4: Oxidation of DDPH radical to DPPH-H non radical by antioxidant agents.

The method was based on the reduction of DPPH alcoholic solution in the presence of

hydrogen as a donor of an antioxidant due the formation of the non-radical DPPH-H. Thus,

the antioxidant activity of the test sample on this radical is expressed in term of its ability to

reduce it, its effect is due to the presence of hydrogen donor present in these test sample

antioxidants (Conforti et al., 2005, Kusuma et al., 2014).

For good interpretation of results from the antioxidant testing, following criteria were

adopted: IC50 ≤ 10 µg/ml: pronounced activity, 10 < IC50 ≤ 20 µg/ml; good activity, 20 < IC50

≤ 30 µg/ml; moderate activity, 30 < IC50 ≤ 40 µg/ml; weak activity, IC50 > 40 µg/ml:

inactive.

In the present study, results revealed that lyophilized aqueous extract BSLAE-1, 80%

methanol extract BSME, as well as ethylacetate and residual aqueous soluble fractions rich in

flavonoids and phenolic compounds other than flavonoids respectively, exhibited pronounced

antioxidant activity against this radical with IC50 values < 10 µg/ml (Table 2). Soluble

fractions chloroform BSLAE-1.1 and n-butanol BSLAE-1.3 rich in steroids and terpenes, an

saponins respectively also showed good antioxidant activity against DPPH with IC50 values

9.22±0..01 and 11.03±0.04 µg/ml respectively. The activity of the samples in increase order

ca be established as BSMSE > BSALAE-1 > BSLAE-1.2 > BSLAE >BSLAE-1.3.

Table 2: Antioxidant of lyophilized aqueous extract a from B. sumatrana and its

fractions, 80% methanol and total alkaloids extracts against some oxygene radical

species.

Samples DPPH ABTS O 2. H2O2 HO

BSLAE-1 3.15±0.02 5.21±0.04 4.08±0.03 8.04±001 2.38±0.01

BSLAE-1.1 9.22±0.01 11.45±0.04 15.08±0.03 17.35±0.03 8.74±0.04

BSLAE-1.2 4.08±0.02 6.12±0.01 5.45±0.03 6.24±0.01 3.52±0.02

BSLAE-1.3 11.03±0.04 10.25±0.03 13.68±0.02 15.11±0.02 10.25±0.03


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BSLAE-1.4 6.04±0.01 8.64±0.03 7.75±0.02 10.21±0.01 5.68±0.02

BSAT - - - -

BSME 2.89±0.01 3.95±0.03 2.11±0.03 3.47±0.02 2.05±0.01

Vitamin C 2.54±0.03 3.85±0.05 3.15±0.02 10.4±0.03 3.01±0.01

Trolox 3.25±0.04 4.62±0.03 - - 6.24±0.02

Gallic acid 1.54±0.02 1.25±0.01 7.50±0.01 5.07±0.01 5.02±0.03

α-Tocopherol 10.30±0.02 9.65±0.04 14.36±0.03 7.56±0.02 6.35±0.04

BSLAE-: lyophilized aqueous extract, BSLAE-1.1 to 1.4: chloroform, ethyacetate, n-butanol

and residual aqueous soluble fractions from the partition of BSLAE-1 extract, BSAT: total

alkaloids extract, BSME: 80% methanol extract.

The radical ABTS (2,2’-azino-bis (3ethylbenthiazoline-6-sulphonate) radical scavenging

method for the evaluation of effects of antioxidants against this radical, was firstly reported

by Rice-Evans and Miller and was then modified by Rey et al., (1990). The modification

introduced was based on the activation of methaemoglobin with hydrogen peroxide in the

presence of ABTS+ to produce a radical cation. The improved method generates a blue/green

ABTS+

chromophore via the reaction of ABTS and potassium persulfate. Along with DPPH

method, the ABTS radical scavenging method is one of the most extensively used antioxidant

assay for plant samples. It is currently used to assess antioxidant activity of some lipohilic

and hydrophilic compounds like flavonoids and carotenoids as well as medicinal plant

extracts and their resulting different fractions (Cai et al. 2004). This assay is based on the

inhibition of radical cation ABTS+ with the non participation of other intermediate radical.

The ABTS radical cation is generated by oxidation of ABTS with potassium persulfate. It is

a decolouration reaction leading to the formation of a radical cation by addition of an

antioxidant agent taking continually in the presence of an antioxidant (Rajamurugan et al.,

2013.).

Results reported in the present study indicated that 80% methanol extract BSME was the

most active simple against this radical with IC50 value of 3.95±0.03 µg/ml. It was followed

by lyophilized aqueous extract BSLAE-1, ethylacatate and residual aqueous soluble fractions

presenting IC50 values of 5.21±0.04, 6.21±0.01 and 6.84±0.03 µg/ml respectively as also a

sign of their pronounced activity. Chloroform and n-butanol soluble fraction also showed

good activity against this radical with IC50 values of 11.45±0.04 and 10.25±0.03 µg/ml

respectively .


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The hydrogen peroxide or oxygenated water (H2O2) possessed strong oxidative property and

commonly found in biological substances. It is an important reactive oxygen species due to

its ability to penetrate biological membranes and it is regarded as one of the most reactive

free radical, which can induce damage of biomolecules (Han et al., 2013). It is less reactive

than OH. having a half live from second nanoseconds. It can be formed in vivo by many

oxidative reactions such as superoxide dismutase (Alves et al., 2010). This radical is a pro-

oxidant capable to go through membranes to oxide a number of biological compounds. In

can give an increase to hydroxyl radical by intercalation with cellular constituents and cause

thus the damage of issues which can eventually lead to cell necrosis. As other antioxidant

assays, it also widely employed in many recent studies to evaluate antioxidant effects of plant

extracts (Krishnaiah et al. 2011).

Results from the present study revealed that B. sumatrana leaves extracts and its soluble

fractions exerted inhibitory effect against H2O2 activity in dose-dependent manner as

illustrated in figures 3 and 4. At the highest tested concentration of 100 µg/ml, lyophilized

aqueous extract BSLAE-1 and 80 % MeOH extracts BSME produced more than 80%

inhibition of hydrogen peroxide activity with BSME as the most active (86% inhibition)

against the lyophilized aqueous extract with 84% inhibition of the activity this oxygen radical

species.

Figure 5: % Inhibition of H2O2 activity by lyophilized aqueous and80% MeOH extract

from B. sumatrana leaves.

Soluble fractions also showed good inhibition of the activity of H2O2 in dose-dependent

manner (Fig 4) with the high activity presented by ethylacetate soluble fraction BSLAE-1.2


105


(81%) followed by residual aqueous phase BSLAE-1.4 (78%), n-butanol BSLAE-1.3 (75%)

and chloroform BSLAE-1.1 (71%) at the highest tested concentration of 100µg/ml.

Figure 6: % Inhibition of H2O2 activity by soluble fractions from the partition of

lyophilized aqueous extract BSLAE-1 extract from B. sumatrana.

Regarding their IC50 values, , It was observed that lyophilized aqueous BSLAE-1 and 80%

methanol BSME extracts, and ethylacetate BSLAE-1.2 and residual aqueous BSLAE-1.4

soluble fraction exhibited pronounced inhibitory effect against this oxygen radical species

with IC50 values < 10 µg/ml (Table 2). Chloroform BSLAE-1.1 and n-butanol BSLAE_1.3

soluble fractions displayed good inhibitory effect with IC50 values of 17.35± 0.03 and

15.11±0.02 µg/ml respectively. The activity all samples from B. sumatrana leaves was

weaker compared to all reference products, but the activity of BSLAE-1.4 seemed to be

comparable to vitamin C (Table 2).

The superoxide anion O2.-

is another nocive oxygen radical species causing dedomagement of

cellular compounds in various biological systems. The couplage reaction PMS-NADH

(methylphenzinne methosulphate-nicotinamide adenine dinucleotide) accelerated the yield of

superoxide anion radicals from dissolved oxygen (Smith and Adanlovo, 2014). It is

considered as a weak oxidant agent, but it produced strong and dangerous radical as well as

oxygen alone, both contributed to stress oxidative (Nur et all. 2013). It is also constantly

produced in organism by divers cellular processes like electron transport in mitochondrial

chain, in microsomes towards some enzymes such as xanthine oxidase, NADPH oxidase and

can increase as a part or caused some pathologies. However, xanthine oxidase is an enzyme

responsible for conversion of xanthine to uric acid causing gout, resulting in the production

of hydrogen peroxide and superoxide anions. It is considered as major biological source of


106


reactive oxygen species. Il is also possible to considere the inhibition of enzymatic process by

substances with antioxidant properties having therapeutic use (Alves et al., 20100. It is a

large reagent with an important number of biologically molecules including lipid barriers

towards initiated peroxidation processes (Oh et al., 2006).

Results from the present study revealed that lyophilized aqueous BSLAE-1 and MeOH

BSME extracts, as well as ethylacetate BSLAE-1.2 and residual aqueous BSLAE-1.4 soluble

fractions significantly (p < 0.05) inhibited the production of superoxide anions with IC

values < 10 µg/ml (Table 2). BSME being the most active sample (IC50 = 2.11±0.03 µg/ml)

followed by lyophilized aqueous extract BSLAE-1 (IC50 = 4.08±0.03 µ/ml, ethylacetate

BSLAE-1.2 and residual aqueous soluble fractions with IC50 values of 5.45±0.03 and 7.75.

±0.02µg/ml respectively. Chloroform BSLAE-1.1 and n-butanol BSLAE-1.3 soluble

fractions also showed good inhibitory effect on the production of superoxide anions with IC50

values of 15.33.08±0.03 and 13.68±0.02 µg/ml respectively and their effects was considered

as good. MeOH extract BSME showed high activity while the activity of remaining sample

was weaker compared to all reference products, but the activity of BSLAE-1.4 soluble

fraction seemed to be comparable to gallic acid (Table 2).

Against hydroxyl radical (HO.), I was observed that lyophilized aqueous BSALAE-1 and

MeOH BSME extracts, chloroform BSLAE-1.1 and ethylacetate BSLAE-1.2 and residual

aqueous BSLAE-1.4 soluble fractions exhibited pronounced antioxidant activity against this

oxygen radical species with IC50 values < 10 µ/ml with BSME extract as the most active (IC50

= 2.05±0.01) followed by lyophilized aqueous extract BSLAE-1 (IC50 = 2.38±0.01 µ/ml),

ethylacetate BSLAE-1.2 (IC50 = 3.52±0.02 µg/ml), residual aqueous soluble fraction (IC50 =

5.68±0.02 µg/ml, chloroform BSLAE-1.1 (IC50 =8.74±0.04 µg/ml). n-butanol BSLAE-1.3

(IC50 = 10.21±0.01 µg/ml) exhibited also good antioxidant activity against this selected

radical. In general the activity of lyophilized aqueous BSLAE-1 and MeOH BSME extracts is

higher while the activity of remaining samples was weaker compared to all used reference

product. (Table 2). The total alkaloid BSAT was devoid with antioxidant activity against all

selected oxygen radical species suggesting the absence of alkaloids with phenolic group in

this extract.

From these results of B. sumatrana samples against some selected oxygen radical species, It

can be suggest that their activity is due to presence of some secondary metabolites or

phytochemical groups such as flavonoids, phenolic compounds, phenol acids, tannins,


107


steroids and terpenes containing phenolic groups in their structure since the antioxidant

activity of these chemicals were already previously reported in other studies ( Cimanga,

1997; Smith and Adanlowo, 2014; Talla et al., 2016, Vargas et al., 2016) and it also known

that temperature had some effects on the antioxidant properties of tested samples (Ogueke et

al., 2018).

As already mentioned above, in comparising antioxidant activity of test samples to reference

products, it was observed that Vitamin C, Trolox and gallic acid exhibited higher activity

against DPPH compared to B. sumatrana samples with IC50 values ranging from 1.54±0.02 to

3.25±0.04 µg/ml. This activity of α-tocopherol (10.30±0.02 seemed to be comparable to

soluble fraction BSLAE-1.3 (IC50 = 11.03±0.04 µg/ml), but weaker compared to remaining

samples (Table 2). Against ABTS radical, the activity of MeOH BSME extract from B.

sumatrana leaves was higher compared to Trolox and α-tocopherol, but weaker compared to

Vitamin C and gallic acid (Table 2). The remaining samples chloroform BSLAE1.1 (IC50

11.45±0.04 µg/ml) and n-butanol BSLAE-1.3 (IC50 = 10.25±0.03 µg/ml) showed weaker

activity compared to all reference products with IC50 values between 1.25±0.01 and

4.62±0.03 µg/ml while the activity of aqueous residual phase BSLAE-1.4 was higher

compared to α-tocopherol and weaker compared to Vitamin C and gallic acid.

When tested against superoxide anions O2.-

lyophilized aqueous BSLAE- and MeOH BSME

extracts, and ethylacetate BSLAE-1.2 and residual aqueous soluble fractions exhibited higher

activity with IC50 values ranging from 2.11±0.03 t 5.45±0.03 µg/ml compared to gallic acid

and α-tocopherol, but their activity was weaker compared to Vitamin C (Table 2). Moreover,

against hydroxide (HO.) radical, lyophilized aqueous BSLAE-and MeOH BSME extracts

showed higher activity with IC50 values of 2.38±0.01 and 2.05±0.01 µg/ml respectively while

the activity of the remaining samples against this radical species was weaker compared to

Vitamin C, Trolox, α-tocopherol and gallic acid with IC50 values from 3.01±0.01 µg/ml and

6.35±0.04 µg/ml. Eltyacetate soluble faction BSLAE-1.2 exhibited higher activity compared

to gallic acid and α-tocopherol, but its activity was weaker compared to Vitamin C (Table 2).

In addition the antioxidant activity displayed by chloroform BSLAE1.1, n-butanol BSAE-1.3

and aqueous residual soluble fractions was weaker compared to all reference antioxidant

products (Table2).


108


3.3. Content of total phenolic compounds and flavonoids

Total phenolic compounds were determined using Folin Ciocalteu’s reagent. This assay also

served to detected phenol acids, flavonoids, tannins, proanthocyanidins and other phenolic

compounds (Talla et al., 2016). In lyophilized aqueous extract of B. sumatrana leaves, total

phenolic compounds were determined and expressed in mg equivalent to gallic acid/g dried

extract while total flavonoids were estimated spectrophotometrically using Al3 % MeOH

solution and expressed in quercetin/g dried extract. Results indicated that lyophilized aqueous

extract of B. sumatrana leaves contained 32.56±0.05 mg of total phenolic compounds

equivalent of gallic acid in mg/g dried extract and total flavonoids of 4.56±0.02 mg expressed

in quercetin in mg/g died extract. These results suggested that phenolic compounds are in

high amount compared to flavonoids since they contained other phenolic compounds extra

flavonoids such as particularly tannins and its antioxidant activity may due to its abundance

in these chemicals acting in synergistic manner.

In general manner, antioxidant activity of medicinal plant extracts is due the presence of

different secondary metabolites or phytochemical groups such as alkaloids, flavonoids,

essential oils, steroids, triterpenes, polyphenol compounds, saponins, tannins ( Cimanga et al,

1997; Cai et al., 2003; Bourkhiss et al., 2010; Soheila et al., 2010; Bi et al, 2012; Akinpelu et

al., 2014;Pande et al., 2014; Smith et al., 2014; Tapondjou et al., 2015; Talla et al, 2016).

3.4. Anti-inflammatory activity of lyophilized aqueous extract BSLAE and its fractions,

methanol and total alkaloids extracts

The inflammation is a common phenomenon. It is a reaction occurring in different tissues

towards the dedomagements. Anti-inflammatory agents destroy and possibly induced the

redistribution of blood peripheric lymphocytes (Ejebe et al., 2010; Saleem et al., 2015).

Acute inflammation can be treated in spontaneous manner or by treating with known steroid

glucocorticoids and non-steroid anti-inflammatory agents (NSA). These substances although

effective are associated with iatrogenic effects such as digestive damage 9gastro-duodenal

ulcer, stenose and perforation and kidney toxicities (acute kidney insufficiency and

hydrosodated retention) (Boukhiss et al., 2010).

The induction of oedema by injection of carrageenan in foot of rats is animal model well

established. The induction for the evaluation of anti-inflammatory activity of medicinal plant

extracts, isolated natural and synthetic product is currently used. This treatment in animal


109


foot provoked a biphasic anti-inflammatory response for which the initial phase last about

1h30 minutes after carrageenan injection, showed by the release of serotonin, histamine and

bradykinin or again by development of oedema in foot after carrageenan injection due to the

release of biochemical mediators (Georgewill et al., 2010a, b; Saleem et al. 2015). The

second which takes place after is due to the biosynthesis of prostaglandins (Sonialmol et al.,

2011, Livia de Paulo et al., 2012, Ouedraogo et al., 2015.

Results obtained in this pharmacological model, showed that samples of B. sumatrana leaves

were found to cause significantly (p < 0.05) decrease of treated foot oedema induces by

carrageenan in dose-dependent manner.

Table 3: Anti-inflammatory activity of extracts, fractions and polysaccharides from B.

sumatrana leaves in Wistar rats.

Samples Treatment

(mg/kg bw) Foot volume (ml)

% inhibition of

oedema after 5h

1h 3h 5h

NC 5 ml distilled water 0.27 0.31 0.35 -

BSLAE-1 50 0.20 0.11 0.09 74.28±0.01

100 0.12 0.09 0.06 82.85±0.03

BSLAE-1.1 50 0.20 0.18 0.16 54.28±0.02

100 0.16 0.18 0.12 65.71±0.02

BSLAE-1.2 50 0.17 0.5 0.10 71.42±0.01

100 0.15 0.12 0.08 77.42±0.04

BSLAE-1.3 50 0.14 0.21 0.18 48.57±0.01

100 0.22 0.17 0.14 60.00±0.02

BSLAE-1.4 50 0.19 0.16 0.13 62.85±0.01

100 0.14 0.12 0.10 71.42±0.03

BSLAE-1’ 50 0.21 0.19 0.17 51.42±0.02

100 0.17 0.15 0.13 62.85±.04

BSAT 50 0.16 0.10 0.04 88.57±0.03

100 0.14 0.09 0.03 91.42±0.02

BSME 50 0.16 0.09 0.05 85.71±0.04

100 0.13 0.10 0.04 88.57±0.02

PBSLAEb 50 0.11 0.07 0.05 85.57±0.03

100 0.09 0.06 0.03 91.42±0.01

PBSLAE-1 50 0.13 0.10 0.06 82.85±0.04

100 0.12 0.08 0.05 85.74±0.02

PBSLAE-2 50 0.14 0.11 0.09 74.28±0.01

100 0.12 0.09 0.07 80.00±0.03

PBSLAE-3 50 0.15 0.13 0.10 71.42±0.01

100 0.13 0.11 0.08 77.14±0.02


110


They thus exerted anti-inflammatory activity (Table 3). The lyophilized aqueous extract

BSLAE-1 at dose of 50 and 100 mg/kg bw leaded to significant reduction (p <0.05) of foot

volume (oedema from 0.09 to 0.06 ml of foot volume after 5h corresponding to the inhibition

of 74.28±0.01 and 82.85±0.03% compared to untreated animal showing 0.35 ml of foot

volume.

At the highest dose of 100 mg/kg bw, its soluble fractions had also the same effect producing

significant reduction of foot volume (oedema) from 0.08 to 0.23 ml after 5h corresponding to

percentage inhibition of oedema from 34.28±0.03 to 77.14±0.04% with ethylacetate soluble

fraction as the most active (7.14±0.04) followed by residual aqueous soluble fraction

(68.57±0.02%), chloroform BSLAE-11 soluble fraction (65.71±0.02%) and n-butanol

BSLAE-13 soluble fraction (60.00±0.02%). The detannified extract BSLAE-1’extract leaded

to 0.18 ml of foot volume corresponding to 48.57±0.01% inhibition of oedema. At the same

high dose of 100 mg/kg bw, total alkaloids BSAT and MeOH BSME caused 0.03 and 0.04 ml

foot volume corresponding to 91.42±0.02 and 88.57±0.02% inhibition of oedema. BSAT

showed higher ant-inflammatory activity compared to lyophilized aqueous BSLAE-1 and

MeOH BSME extract (Table 3).

The investigation of natural phytochemical products from various medicinal plant extracts

know empirically to treat rheumatism in traditional medicine can lead to the discovery of lead

compounds with interesting anti-inflammatory effects in animal model without significant

side effects as alternative to known corticoids and non-steroid anti-inflammatory agents

having many sides effects (Saprong et al., 2016).

Figures 4 and 5 revealed the decrease of foot volume in treated food of rats induced by

carrageenan by lyophilized aqueous extract BSLAE-31 and its fraction BSLAE-1.1 to

BSLAE-1.4, MeOH BSME and total alkaloids extracts. Extracts BSLAE-1, BSME and

BSAT produced 74.28. 85.71 and 85.71 % inhibition of foot volume respectively at the

administered oral dose of 100 mg/kg bw after 5h (Fig. 3). This liquid volume began to

decrease from 1 h and continued to decrease gradually with time compared to negative

control and was important after 5h. Soluble fractions BSLAE-1.1 to SLAE-1.4 produced also

60.00 to 78.14% inhibition of foot volume with ethylacetate soluble fraction as the most

active (Fig. 5) at the same highest oral dose.


111


Figure 7: % inhibition of foot volume by lyophilized aqueous BSLAE-1, MeOH BSME

and total alkaloids BSAT extract at oral dose of 100 mg/kg bw.

Figure 8: % Inhibition of foot volume by soluble fractions BSLAE-1.1 to BSLAE-1.4

from the partition the lyophilized aqueous extract BSLAE-1.

Diclofenac uses as reference anti-inflammatory product produced 97.14 % inhibition of foot

volume and exhibited thus high anti-inflammatory activity compared to B. sumatrana

samples.

Polysaccharide were also evaluated for their potential anti-inflammatory activity against

carrageen induced paw oedema in rat foots. Results indicated that crude polysaccharide

exhibited anti-inflammatory activity by producing 91.42±0.01% inhibition of foot volume of

treated rats at oral dose of 100 mg/kg bw of the extract. The 3 chromatographically pure

isolated polysaccharides had the same effect by causing significant reduction of foot volume

of treated animals by 80.00 to 85.74% inhibition at the same oral dose. The activity of pure


112


polysaccharide was weaker compared to crude polysaccharide and this finding suggested that

the chemical cam react in synergistic manner. They can partly considered as active anti-

inflammatory principles of B. sumatrana leaves extract. Our results are in good agreement

with other studies concerning the reported anti-inflammatory activity of polysaccharides

isolated in other medicinal species (Soniamol et al., 2011; Hur et al., 2012; Wang et al., 2013,

Ibrahim et al., 2014; Zhou et al;., 2014).

The literature had reported some natural metabolites and phytochemical groups with anti-

inflammatory activity in animal model. They included polysaccharides (Soniamol et al, 2011;

Livia de Paulo et al., 2012), phytosterols like lupeol, β-carophyllene 8R,9R-oxide,

tsoonglanolides A, B and D, ligularenolide, β- stostero-glucopyranoside, stigmaterol,

sistosterol, α and –amyrin (battachara et al., 1980; Garcia et al., 1990, Magina et al., 2009,

Arccinega et al., 2015, biflanonoids like kolaviron (Braide , 1993), ametoflavone (victor,

19960, terpenoids like ginkolides A et B, ginketine, sciadopitysine et bilobalide (Della Logia

et al., 19960, triterpenoids (Atta-ur-Rhaman, 2000), essential oils (Menezes et al., 1990),

alkaloids (Barbosa-Filho et al., 2006) and saponins (Sur et al., 2001) and others (Maestri,

2006). The presence of some Phychemical groups identified in B. sumatrana leaves may

account for the observed anti-inflammatory activity in extracts and fractions of the studied

plant part.

In general, the anti-inflammatory effects showed by B. sumatrana leaves samples is

interesting and obtained results can support and justify the use of the plant part in traditional

medicine to treat rheumatism other pains without significant side effects in humans.

3.5. Acute and subacute toxicity of lyophilized aqueous extract of B. sumatrana leaves

3.5.1 Observation of toxic effects and determination of DL50

In acute and subacute toxicity after administration once the highest oral dose of 5000mg/kg

and every day 500, 1000 and 5000 mg/kg bw respectively of the lyophilized aqueous extract

of B. Sumatran leaves, animals were observed for toxic effects foe the four first 4 hour and a

total of 28 days. During this period of observation, no sign of toxicity no change in mobility,

tiredness convulsions, gastrointestinal disorder, behaviour and necrosis, consumption of food

and water, major physiological activities was observed. Nevertheless, treated animal of this

extract gained body weight compared to untreated (Fig 6). This effect may be due the

stimulation f appetite induced by the administration of the extract as also reported for other

medicinal extracts in other studies (Ogbannia et al., 2010; Tripa et al., 2011; Naka et al, 2012.


113


Cimanga et al. 2015, Oloro et al,. The administrated at this highest oral dose had no adverse

effect on the bevioural responses of the treated animals up 28 days.

Figure 9: Evolution of bodyweight of Wistar rats after administration of lyophilized

aqueous extract in treated animals. NC; negative control TR: treated rats.

of observation. Physical observations indicated no sign of changes in skin, fur, eyes, mucous

membrane, behaviour patterns, tremors, salivation, vomiting and other gastro-intestinal

disorders. All treated animal were healthy. In addition no mortality of animals was recorded

at the highest oral dose of 5000 mg/kg bw of the administered extract. Thus, its lethal dose 50

(DL500 was estimated to be greater than 5000 mg/kg bw. According to Kennedy et al. 91960

substance presenting a DL50 > 5000 mg/kg bw is considered as no-toxic and consequently

lyophilized aqueous extract of B. sumatrana was considered practically an non-toxic by oral

route.

In general, results from that analysis of haematological parameters of treated animal indicated

that the administration of lyophilized aqueous extract BSLAE-1 at the highest oral dose of

5000 mg/kg bw induced a significant increase of level parameters of treated animals

compared to untreated. It was not a sign of the presence of any pathology. But all values were

comparable to other reported in other studies and remained in acceptable physiological limits

(table 4) (Ogbonnia et al., 2010, Tripa eta l.,2011; Nsaka et al., 2012; Cimanga et al., 2015;

Oloro et al. 2016).


114


Table 4: Effects of lyophilized aqueous extract of haematological parameter of Wistra

rats.

Parameters Negative

control

Treated animals

with B. sumatrans:

5000mg/kg bw

References

(Nsaka et al.,

2012

White corpuscules 9x 103 µL 8.1±0.2 9.8±0.3 6.6 – 10.5

Red corpuscules x 103 µL 7.5±0.3 8.7±0.1 7.6 -10.29

Haemoglobin (g/dl) 15.8±0.1 17.1±0.2 15 -18.2

Hematocrit (%) 48.2±0.2 46.3±0.1 40.7 -50

Plauetels 9x 103 µl-1

128.6±0.2 1356.8±0.1 995 -1713

Basophiles 0.0±0.0 0.0±0.0 0.0±0.0

Eosinophiles 0.8.±01 1.2±0.3 1.2-2

Lymphocytes 78.6±0.2 76.5±1 75-90

Segmented lymphocy3tes 14.3±0. 17.2±.3 12 -25

3.5.3. Effects of lyophilized aqueous extract of B. sumatrana on biochemical parameters

Results revealed that the oral administration of lyophilized aqueous extract of B. sumtrana at

the highest oral dose of 5000 mg/kg bw caused significant reduction of glucose level in

treated animals compared to untreated. This effect would be due probably to the

hypoglycemic property of the extract or to an increase of hormone T and T4 (Crook,2006 as

also previously reported by Ogbonnia et al, 2010, Nsaka et al, 2021, Cimanga et al, 2015).

The level of creatinine in treated animals did not how significant difference compared to

untreated group (p > 0.050. It was also observed slight increase of the level of ASAT

(153.1±0.3) in treated animals compared to untreated (147.3±0.3) with significant difference

( p < 0.05), but it did not affect the function of kidney and liver. In contrary slight decrease

of levels of ALAT (53.2±0.70 and SGOT (20.4±0.1) was observed in treated animals

compared to untreated groups (54.8±0.50 and 121.0±0.) respectively and no significant

difference was observed (p < 0.05).The level of SGOT in treated animal 933.7±0.20 was

comparable to that of untreated group (33.8±0.2) without no significant difference (P < 0.05)

and this finding suggested that the administered extract at this high oral dose did not possess

adverse effects against kidney and liver for which the function was well maintained in good

state in treated animals. Our results are in good agreement with other studies reporting the

effect of some plant extracts on kidney and liver function (Nsaka et al, 2012; Cimanga et,

2015).


115


Table 5: Effects of lyophilized aqueous extract of B. sumatrana on biochemical

parameters.

Parameters Negative

control

Treated animals with B.

sumatrana: 5000 mg/kg bw

Glucose (mg/dl) 85.7±0.4 80.1±0.2

Creatinine 0.87±0.3 0.84±0.1

ASAT (UI/L) 153. 1±0.3 147.6±0.3

ALAT (UI/L) 53.2±0.2 54.80.3

SGOT (UI/L) 120.4±0.1 121.0±0.3

SGPT (UI/L) 33.7±0.2 33.8±0.2

Total cholesterol (mg/dL) 53.8±0.2 50.7±0.2

LDL (mg/dL) 43.3±0.1 40.2±0.3

HDL (mg/dL) 34.2±0.2 37.8±0.2

Triglycerides (mg/dL) 44.5±0.1 41.5±0.3

Proteins (mg/dL) 8.2±0.2 8.6±0.4

Moreover, significant decrease of total cholesterol, low density lipoproteins (LDL) and

triglycerides in treated animals compared to untreated group and significant difference was

deduced (p < 0.05) (Table 5). A significant increase of high density lipoproteins (HDL)

(43.3±0.1) was also observed in treated group compared to untreated (40.2±0.3). The

decrease of LDL and increase of HDL levels as well as total cholesterol levels is beneficial

for human health since it allowed to prevent cardiovascular disease which can be mortal for

diabetic patients as already reported in other studies (Nsaka et al. 2012, Cimanga eta la.

2015).

In general, the evaluated biochemical parameters levels of in the present study were in the

same ranks with those reported in other studies and were in acceptable physiological limits

(Crook et al. 2006). From these results, it was concluded that lyophilized aqueous extract

BSAE-1 of B. sumarantra leaves was non-toxic and safe or harmless, and can not be

considered as source of any pathology.

CONCLUSION

The present study reported for the first time the antioxidant and anti-inflammatory activity of

extracts and fraction of B. sumatrana leaves collected in Mai-Ndombe in Democratic

Republic of Congo. Results indicated that extracts and fractions exhibited good and

interesting antioxidant activity against some selected oxygen radical species such as DDPH,

superoxide anions, ABTS, hydrogen peroxide and hydroxyl. They also showed good and

interesting anti-inflammatory effect demonstrated by the reduction of increasing foot volume

of treated animals by the administration of carrageenan. Thus the traditional use of B.


116


sumatrana leaves to treat rheumatism and other pains in popular medicine can be supported

and justified by these reported results in the present studies. Further studies are planified

aiming the isolation and structure elucidation of active constistuents.

REFERENCES

1. Abbas A.B.; Lichtman A.H.. "Ch.2 Innate Immunity". In Saunders (Elsevier) (ed.). Basic

Immunology. Functions and disorders of the immune system (3rd ed.). ISBN 978-1-4160-

4688-2. 2009.

2. Akinpelu BA, Ignghu OA, Awotunde AL, Iwalewa EO, Oyedapo OO. Antioxidant and

antibacterial activities of saponin fractions of Eurthophleum suavelons (Guill. and Perri.)

stem bark extract. Scient Res Essays, 2014; 9(18): 826-833.

3. Aktre S, Iman MZ, Hassan SMR, Hossain Md, Mazumber MDEH, Rana MdS.

Antioxidant, antidiarrhoeal and cytotoxic properties of aerial parts of Trichosanthes

dioica Roxb. Am J Food Nutr, 2011; 1(1): 95-101.

4. Ali KZ, Hazilawath H, Hutheyfa S, Mohd RS, Shammugavelu S, Hemn HO. Acute and

sub-acute dermal toxicity of Morinda citrifolia L. fruit extract in Sprague Daweleys rats.

Asian J Pharm Clin Res, 2015; 8(2): 400-408.

5. Alves CQ, David JM, Bahia MV, Aguiar RM. Methods for determination of in vitro

antioxidant activity of extracts and organic compounds. Quimica Nova, 2010; 33(10):

2202-2210.

6. Arciniega A, Pérez-Camacho A, Maldonado JL, Villasenor JL, de Vivar AR Chemical

composition and anti-inflammatory activity of Roldana platanifolia. Quimica Nova,

2015; 38(9): 1-7.

7. Atta-ur Rhaman HEJ. Bioactive natural products. Studies in natural products chemistry.

Elsvier, Amsterdam. 2000.

8. Azab A, Nassar A, Azab AN. Anti-inflammatory activity of natural products. Molecules,

2016; 21(10): 3-19.

9. Azeem AK, Dilip C, Prasnth SS, Junisse V, Hanan S. Anti-inflammatory activity of the

glandular extracts of Thumus alalunga. Asian Pac J Trop Med, 2010; 3(2): 412-420.

10. Baiano, A.; del Nobile, M.A. Antioxidant compounds from vegetable matrices:

Biosynthesis, occurrence, and extraction systems. Crit Rev Food Sci Nutr 2015, 56(12):

2053–206.

https://en.wikipedia.org/wiki/International_Standard_Book_Number

https://en.wikipedia.org/wiki/Special:BookSources/978-1-4160-4688-2

https://en.wikipedia.org/wiki/Special:BookSources/978-1-4160-4688-2


117


11. Barbosa-Filho JM, Piuvezam MR, Moura MD, Sila Ms, Lima KVB, da-Cunha EVL,

Fechine IM, Takemua OS. Anti-inflammatory activity of alkaloids; a twenty century

review. Revista Brasil Farmacogn, 2006; 16(1): 1-23.

12. Bhattacharya TK, Gosh MN, Sakara Subamanian S. A note on anti-inflammatory activity

of carpesterol. Fitoterapia, 1980; 51(5): 265-268.

13. Bi L, tian X, Dou F, Hong L, Thang H, Wang S. New antioxidant and antiglycation active

triterpenoid saponin from the root bark of Aralia taibaiensis. Fitoterapia, 2012; 83(1):

234-240.

14. Bourkhiss M, Hnach M, Polini J. Propriétés antioxidante and anti-inflammatory des

huiles essemtielles des différentes parties de Teraclinis articulata Vahl.) Masters du

Maroc. Bull Soc Roy Sci Lg, 2010; 79(1): 141-154.

15. Braide V. Antiinflammatory effect of kolaviron, a biflavonoid extract of Garcinia kola.

Fitoterapia, 1993; 5(3): 433-436.

16. Cai Y, Luo Q, Sum M, Corke H. Antioxidant and phenolic compounds of 112 traditional

Chines. medicinal plants associated with anticancer. Life Sci, 2004; 74(17): 2157-2184.

17. Cimanga K. The biological active constituents of two African medicinal plants:

Cryptolepis sanguinolenta (Lindl.) Schlechter (Periplocaeae) and Morinda morindoides

(Baker) Milne-Redhead (Rubiaceae) Doctorat Thesis, Universiteit Instelling Antwerpen,

Antwerpen, Belgium. 1997.

18. Cimanga KR, Kikueta MC, Mbamu MB, Kambu KO, Apers S, Vlietinck AJ, Pieters L.

Assessment of acute and subacute toxicological profiles of the aqueous extract

(decoction) from the leaves of Triclisia gilletii (De ilD.) Staner (Menispermaceae) in

Wistar rats. Schol Acad J Pharm, 2015; 4(7): 351-357.

19. Conforti F, Loizzo MR, Statti GA, Menichini F. Comparative radical scavenging and

antidiabetic activities of methanolic extract and fractions from Achillea ligustica. Biol

Pharm Bull, 2005; 28(9): 1791-1794.

20. Crook MA. Clinical chemistry and metabolic medicine. 7th

Edition. Hoker Arnold,

London. 2006.

21. de Vargas FS, Amleida PDO, de Boletii AP, Pereira MM, de Souza TP, de Vascncellos

MC, Numez CV, Pohlit AM, Lima ES. Antioxidant activity and peroxidase inhibition of

Amazonian plant extracts traditionally used as anti-inflammatory. Compl Alternat Med,

2016; 16(83): 119-130.


118


22. Danya U. In vivo anti-inflammatory activity of the endemic medicinal plant Caralluma

sarkatiiae R.BR. using carrageenan induced paw oedema in swiss albino mice. J Medl

Plants Studies, 2017; 5(2): 133-135.

23. Della Logia R, Sosa S, Tubaro P, Morazzoni E, Bombaedelli A, Griffini A. Anti-

inflammatory activity of some Gingko biloba constituents and their phospholipid-

complexes. Fitoterapia, 1996; 67(3): 257-264.

24. Dong-Ping Xu 1, Ya Li 1, Xiao Meng 1, Tong Zhou 1, Yue Zhou 1, Jie Zheng 1, Jiao-

Jiao Zhang 1 and Hua-Bin Li 1,2,*, Natural Antioxidants in Foods and Medicinal Plants:

Extraction, Assessment and Resources. Int J Mol Sci, 2017; 18(96): 1-32.

25. Ejebe DE, Siminialayi IM, Emudainowho JOT. Analgesic and anti-inflammatory

activities of the ethanol extract of the leaves of Helianthus annus in Wistar rats. Asian

Pac J Trop Med, 2010; 3(5): 341-347.

26. Georgewill OA, Georgewill UO. Evaluation of anti-inflammatory effects of Moringa

oleifera Lam. extract in rats. Asian Pac J Trop Med, 2010; 3(2): 133-135.

27. Geogwill OA, Geogewill UO. Evaluation of anti-inflammatory activity of Vernonia

amygdalina. Asian Pac J Trop Med, 2010; 3(2): 150-151.

28. Garcia MD, Sáenz MT, Gόmez MA, Fernández MA. Tropical anti-inflammatory activity

of phytosterols isolated from Eryngium foetidum on chronic and acute inflammation

models. Phytother Res, 1999; 13(1): 78-80.

29. Han L, Li L, Li B, Zhao D, Li Y, Xu Z, Liu G. Hydroxyl radical induced lipid in Maillard

reaction model system promotes died –derived N- (ꜫ)-carboxymethyllysine formation.

Food Chem Toxicol, 2013; 60: 536-541.

30. Harborne JB. Phytochemical methods. A guide to modern techniques for plants analysis.

Chapaman & Hall. London. 1998.

31. Hur SJ, Choi SY, Lim BO.. In vitro anti-inflammatory activity of Russula virescens in the

macrophage like cell line RAW 647.7 activated by lipopolysaccharide. J Nutr Food Sci,

2012; 2(4): 1-4.

32. https://en.wikipedia.org/wiki/Inflammation, 2019

33. Ibrahim AY, Mahmoud MG, Asker MMS. Anti-inflammatory and antioxidant activities

of polysaccharides from Adansonia digitata: an intro study. Int J Pharm Sci Rev Res,

2014; 25(2): 174-182.

34. Ilhami G, Zϋbeyr H, Mahfuz E, Haassan YAE. Radical scavenging and antioxidant

activity of tannic acid. Arabian J Chem, 2010; 3(2): 43-53.

https://en.wikipedia.org/wiki/Inflammation


119


35. Kennedy GL, Frenz BL, Burgers BA. Estimation of acute oral toxicity in rats by

determination of the approximative lethal dose rather the LD50. J Apll Toxicol, 1986;

6(3): 145-148.

36. Kripa KG, Chamubdeeswari, Thanka J. Acute and sub-acute toxicity evaluation of

ethanol extract of Leucas aspera (Lamiaceaae) in experimental rats. Int J Drugs Devel

Res, 2011; 3(2): 339-347.

37. Krishnaiah S, Sarbatly R, Nithanandam R. A review of the antioxidant potential of

medicinal plant species. Food Bioprod Processing, 2011; 89(3): 217-233.

38. Kumar RBS, Kar B, Dolai N, Bal A, Haldar PK. Evaluation of antihyperglycemic and

antioxidant properties of Streblus asper Louragainst in streptozocin-induced diabetes rats.

Asian Pac J Trop Disease, 2012; 1(1): 139-143.

39. Kusuma IW Mudiyano, Arung ET, Syafrizal, Yong-ung K. Antimicrobial and antioxidant

properties of medicinal plants seed by Benian tribes from Indonesia. Food Sci and Hum

Well, 2014; 3(1): 191-196.

40. Livia de Paulo P, Kaira ES da S, racquel O, Anan MSA, Maria GP. Anti-inflammatory

polysaccharides of Azadiractha indica seed tegument. Rev Brasil Farmacogn, 2012;

22(1): 1-8.

41. Maestri DM, Nepote V, Lamarque AL, Zygallo JA. Natural antioxidants. Phytochemisy,

2006; 2: 105-135.

42. Magina MDA, Pietrovski EF, Comig FDB, Falkenker DBm Cabrini DA, Pizzolllati MG,

Brighente IMC. Tropical antiinflammatory activity and chemical composition of the

epicutilar was from the leaves of Eugenia beaurepaireana (Myrtaceae). Brazilian J

Pharm Sci, 2009; 45: 171-176.

43. Manthal KEI, Lairini S, Boudkhili M, Bouslamti R, Errbii M, Guiss S. Assessment of

ultrasound extraction for the recovery of phenolic compounds from Opuntia tricta peel:

evaluation of antioxidant and antibacterial activities. Europ J Biochem Pharm Sci, 2019;

6: 293-301.

44. Mancarz GFF, Lobo, ACP, Baril MB, Franco FA, Nakashimaa T. Antimicrobial and

antioxidant activity of leaves, bark and stem of Liquidambar styracifua L. (Altingiaceae).

Int J Curr Microbiol Apll Sci, 2016; 5: 306-317.

45. Menezes, AMS Almeida FRC, Rao VSN, Matoa MEO. Anti-inflammatory activity of the

essential oil of Vanillosmopsis arbórea. Fitoterapia, 1990; 3(2) : 252-254.


120


46. Mensor LL, Menezes FS, Leităo GG, Reis As, Santos TC, Coube CS el al.. Screening of

Brazilian plant extracts for antioxidant activity by use DPPH free radical method.

Phytother Res, 2001; 15(1): 127-130.

47. Narayanaswamy N, Balkrishman KP. Evaluation of some medicinal plants for their

antioxidant properties. Int J Pharm Technol Res, 2011; 3(1): 381-385.

48. Nigam V, Sodhi JS. Some medicinal plants with antioxidant activity. A review. Int J

Pharm Biol Sci, 2014; 4(41): 173-178.

49. Nsaka LS, Tshodi EM, Tona, LG, Kambu KO, Cimanga KR, Apers S, Pieters L,

Vlientick AJ. Assessment of the antidiahhroeal properties of the aqueous extract, the 80

methanol extract and its fractions of the leaves of Alstonia congensis Engl.

(Apocynaceae) in Wistar rats. J Ethnopharmacol, 2012; 142(3): 620-626.

50. Nur MDA, Nustrat JB, Rafiquzzaman Md. Review om in vivo and in vitro methods

evaluation of antioxidant activity Saudi Pharmaceutical Journal, 2013; 21: 143-152.

51. Nwokolo LN, Onyekwelu KC, Ene MC, Adilieje CM, Ezechukwu IN, Ezeh RC. In vitro

antioxidant scavenging potential of methanol extracts of Uvaria chamae leaves and roots.

International journal of Pharmacy and Pharmaceutical sciences, 2019; 11: 67-71.

52. Ogbonnia SO, Mbaka GO, Anyika EEN, Osegbo OM, Igbkwe NH. Evaluation of acute

toxicity in mice and subchronic toxicicty of hydorethanol extract of Chromalaenna

odorata (L.) King and Robinson (Fam. Asteraceae) in rats. Agriculture and biology

Journal of North America, 2010; 1: 859-865.

53. Ogueke CC, Azeke, Ehije A, Owuamanam, Cifford I, Ojuku, Moses, Agunwah IM,

Baeber L, Bede EEN. Comparison of antimicrobial properties spices commonly

consumed in Nigeria and effect of temperature of their antioxidant properties. African

journal of Biotechnology, 2018; 17: 1473-1483.

54. Oh YS, Jang ES, Bock JK, Yoon SH, Jung AY. Singlet oxygen quenching activies of

various fruit and vegetable juices and protective effects of apple and pear juices against

hematolysis and protein oxidation induced by methylene blue photosensitization. Journal

of Food Sciences, 2006; 7(1): 206-208.

55. Oloro J, Kiguli JM, Nabirumbi R, Tanayen JK, Imanirampa K, Bajunirwe F, Ganafa AA.

Acute and sub-acute toxicity evaluation in rats of PPOJ5 and ADOJ herbal remedies used

traditionally in the management of HIV infection. European Journal of Medicinal Plants,

2016; 16: 1-6.


121


56. Ouedrago N, Gnoula C, Lompo M, Sawadogo WR. Activités anti-inflammatoire,

analgésique et antioxidante de l’ extrait aqueux des tiges de feuilles de Saba senegalensis.

Phytohérapie, 2015; 10.1007/s10298-015-0992-5.

57. Oukacha A, Abderrahmane Z, Abdellah B, Saida T, Abdelhakim H. Anti-inflammatory

activity of emthanolic extract from Pistacia atlatica Desf. leaves. Pharmacognosy

Journal, 2018; 10(1): 71-76.

58. Pande PS, Mana VD, Mishra MN. Evaluation of antioxidant activity of saponin and

tannin fractions isolated from the leaves of Tridax procumbens. International Journal of

Pharma and Boi Sciences, 2014; 5: 396-400.

59. Subba B, Sharma A, Budhathoki A. Assessment of phytochemical content, antioxidant

and antibacterial activies of three medicinal plants of Nepal Journal of Medicinal Plants

Research, 2014; 10: 829-837.

60. Sur P, Chauhri T, Vedasiromoni JR, Gomes A, Ganguly DK. Aniinflammatory and

antioxidant property of saponins of tea Camelia sinensis (L.) O. Kuntze root extract.

Phytotherapy Research, 2001; 15: 174-176.

61. Talla E, Nyem JN, Tiabou AT, Djoou SGZ, Biyanzi P, Sophien L, Elst LV, Tanyi JM.

Antioxidant activity of and new-triterpene from Vitellara pardoxa (Sapotaceae) stem

barks. European Journal of Medicinal Plants, 2016; 16: 1-10.

62. Tapondjou LA, Nyaa LB, Tane P, Ricciutelli M, Quassinti L, Bramuci M, Lupidi G,

Ponou BK, Barboni L. Cytotoxic and antioxidant triterpene saponins from

Butyrospermum markii (sapotaceae). Carbohydrate Research, 346: 2699-2704.

63. The Organization of Economic Co-operation Development (OCED). Test No 425 Acute

oral Toxicity. Up-and-down procedure, OECD guidelines for testing of chemicals, 2008;

10: 03.) [2017-11.htts://ntp.nieths.nh.gov/ccvam/suppdocsfeddocs/oecd/oecdtg425.pdf.

64. The Organization of Economic Co-operation Development (OCED). Test No 407

repeated dose 28-day oral toxicity study in rodents. OECD guidelines for testing of

chemicals, 2008; 10(03): 20217-11.

Http://ntp.nieths.nh.gov/ccvam/suppdocsfeddocs/oecd/oecdtg407-2008.pdf.

65. Trease GE, Evans. Pharmacognosy. WB Saunders, Philadelphia, USA, 2000.

66. Tshodi EM, Mbenza PA, Nsaka LS, Kikweta MC, Bakana PD, Tona LG, Kambu KO,

Cimanga KR, Matheussen A. Cos P, Apers S, Pieters L, Vlietinck AJ. In vitro

antiprotozoal and cytotoxic activity of aqueous extract, the % methanol and its fractions

from the seeds of Brucea sumatrana Roxb. (Simaroubaceae) growing in Democratic

Republic of Congo Chinse Medicine, 2012; 3: 65-71.


122


67. Veerapur VP, Prabhakar KR, Parihar VK, Landadi MR, Ramkitishana S, Mishra B, Satish

Rao SB, Srinivasan KK, Priyadarsini KI, Unnikriishanan MK. Ficus racemosa stem bark

extract: a potent antioxidant and a probable natural radioprotector. Evidence based

Complement and Alternative Medicine, 2009; 6: 317-324.

68. Venkatachalam D, Saraswat R, Sampath KKP. Screening of Euphorbia hirta extracts for

antioxidant activity. European of Biomedical and Pharmaceutical Sciences, 2019; 6: 618-

628.

69. Waisman A, Liblau RS, Becher B. Innate and adaptive immune responses in the CNS.

Lancet Neurology, 2015; 14 (9): 945-955.

70. Wang P, Li XT, sun L, Shen l. Anti-inflammatory activity of water-soluble

polysaccharide of Agaricus blazei Murill onovariectomized osteopenic rats. Evidence-

based Complementary and Alternative Medicine, 2013.

http://dx.doi.org/10.1155/2013/164817: 1-5.

71. Ying C, Yonghong M, Liyong H, Juxiang L, Haiyan S, Yuanzeng Z, Ying Y, Wenke Z.

Antioxidant activies of saponins extracted from Radix trichosantyhis: an vivo and in vitro

evaluation. BMC Complementary and Alternative Medicine, 14; 2-8.

72. Zhou XI, Yang M, Xue BG, He HT, Zhang CM, Liu MM, Zhang LJ, Fei R. Anti-

inflammatory action of Ginkgo biloba leaf polysaccharide via TLR4/NF-ҡb signalling

suppression. Biomedical Research, 2014; 25(4): 449-454.

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