determination of the antioxidant and antiplasmodial

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Combassere-Cherif and al. World Journal of Pharmacy and Pharmaceutical Sciences

DETERMINATION OF THE ANTIOXIDANT AND ANTIPLASMODIAL

ACTIVITIES OF MITRAGYNA INERMIS AND SPERMACOCE

VERTICILLATA

Combassere-Cherif Kaba Mariama*1,2

, Sawadogo Assétou1, Koama K. Benjamin

1,3,

Belem Hadidiatou1, DA L.S. Nadège

1, Kagambega Wendmi

1, Drabo A. Flora

1, Meda N.

T. Roland1 and Ouedraogo G. Anicet

1

1Laboratoire De Recherche Et d’Enseignement En Santé Et Biotechnologies Animales, Unité

De Formation Et De Recherche En Sciences Et Techniques, Université Nazi Boni, Bobo-

Dioulasso, Burkina Faso.

2Groupe De Recherche Action En Santé (GRAS), Ouagadougou, Burkina Faso.

3Laboratoire De Médicine Et Pharmacopée Traditionnelle, Institut De Recherche En Sciences De

La Santé(IRSS), Direction Bobo-Dioulasso, Burkina Faso.

ABSTRACT

Mitragyna inermis and Spermacoce verticillata were two plants of the

Rubiaceae family all used by traditional medicine to treat malaria in

the western region of Burkina Faso. This study aims to evaluate the

antioxidant and antiplasmodial activities of their aqueous and ethanolic

extracts. The colorimetric methods were used to quantified total

phenolics and flavonoids. The antioxidant activity of the plant extracts

has been evaluated using DPPH, ABTS and FRAP methods. Seven

groups of six mice per each group were used to assess the

antiplasmodial activity using the suppressive test. The groups of

animals were administered 250 mg extract/kg body weight respectively

for the six extract used. While the negative control, were administered

with 200 µl of distilled water. Both, the ethanolic and aqueous extracts

of the part of plants used presented phenolic and flavonoids contents.

However, the content of flavonoids was significantly higher in

ethanolic extract compared to aqueous extract. The antioxidant activities were much better

with the aqueous extracts of the leaves of Mitragyna inermis (IC50 = 1.21 µg / ml) and

ethanolic extract of Spermacoce verticillata (IC50 = 3.04 µg / ml) in DPPH method. The

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 11, 129-143 Research Article ISSN 2278 – 4357

*Corresponding Author

Combassere /Cherif K.

Mariama

Laboratoire De Recherche Et

d’Enseignement En Santé Et

Biotechnologies Animales,

Unité De Formation Et De

Recherche En Sciences Et

Techniques, Université Nazi

Boni, Bobo-Dioulasso,

Burkina Faso.

Article Received on

10 September 2020,

Revised on 30 Sept. 2020,

Accepted on 20 Oct. 2020

DOI: 10.20959/wjpps202011-17681


130


ethanolic extracts of the leaves of Mitragyna inermis (32.9%) and Spermacoce verticillata

(27.6%) showed the best percentages reduction in parasitaemia. This study showed that the

plant parts used are assumed to have antioxidant and antiplasmodial activities that could

justify their traditional uses in the malaria treatment and that can be explored for the

management of malaria.

KEYWORDS: Mitragyna inermis, Spermacoce verticillata, antioxidant activity,

antiplasmodial activity.

INTRODUCTION

Mitragyna inermis and Spermacoce verticillata belongs to the Family Rubiaceae, are

common plants in Saharan and sub-Saharan Africa.[1,2,3]

It is a savannah plants found in the

west areas of the forest.[1]

It is often part of remedies in African human therapies particularly

it is used for treatment of malaria, bilharziose, jaundice, chilhod diarrhea.[1]

These plants

were used by traditional medicine to treat malaria in the western region of Burkina Faso.[1]

In Burkina Faso as in most sub-Saharan countries, one of the major public health problems

and greatest health challenges faced by 40.50% of the population is the malaria disease,

which is caused by Plasmodium species, and transmitted by the bite of female Anopheles

mosquito. With 46.50% of the reasons for the consultation, 61.50% of the reasons for

hospitalization and 30.50% of deaths.[4]

Malaria caused much suffering and premature death

in the poorer region in Burkina Faso, where it is stable, seasonal transmission and

endemic.[5,6]

The current control methods of malaria are based on the reduction of human contact with

mosquitoes through the use of repellents and Long-Lasting Impregnated Mosquito Nets

(LLINs),[7]

the treatment based on the rapid and adequate diagnosis. Despite these measures

taken to prevent malaria, the drug resistance to most anti-malarial drugs, insecticide

resistance, terolism and civil disturbances, climatic changes, environmental changes,

population increase and travel constitute a major problem for the control of the disease.[8]

Therefore, the search for new anti-malarial compounds, either synthetic or natural is

important for the killing of either the vector or parasite.[5]

The use of plant-derived drugs for

the treatment of malaria has a long and successful tradition in Africa.[9]

Medicinal plants are

one of the interesting alternative avenues of investigation, such as the discovery of quinine,


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artemisinin and their derivatives derived from plants used in traditional medicine. Few cases

of resistance are observed with natural molecules, unlike synthetic antimalarial drugs.[5]

In Burkina Faso as well as in other developing countries, the poverty accentuates the

difficulties of access to primary health care reason why the first recourse as regards treatment

of malaria is the use of medicinal plants.[5,6]

Among these plants, Mitragyna inermis and

Spermacoce verticillata are two plants of the Rubiaceae family all used by traditional

medicine to treat malaria in the western region of Burkina Faso.

Kumulungui and al. (2016) showed that the tannins and alkaloids of Mitragyna inermis (M.

inermis) displayed antiplasmodial activity in vitro on Plasmodium falciparum with an IC50 of

2.36 and 2.56 µg / mL respectively.[10]

However, the antiplasmodial efficacy of this plant in

vivo has not yet been investigated. With regard to Spermacoce verticillata (S. verticillata), no

study to our knowledge has been conducted on its antiplasmodial efficacy both in vitro and in

vivo.

Considering the importance of the use of these plants in traditional medicine, it is imperative

to attach importance to their study. It is in this direction that this study was initiated in order

to assess the antioxidant and antiplasmodial activities of M. inermis and S. verticillata.

MATERIAL AND METHODS

Plants materials

The barks and the leaves of M. inermis and the whole plant of S. verticillata were collected in

Diaradougou 15 km north-west of Bobo-Dioulasso in Burkina Faso. The collection area for

these plants had the GPS coordinates N 11 ° 15 ’32.11’ ’; W 004 ° 26 ’44.7’ ’and N 11 ° 15’

50.6 ’’; W 004 ° 26 ’53.8’ ’respectively. The plants were identified by Dr Hermann Ouoba a

botanist and cytoecologist from Nazi BONI University (UNB). The plant samples were

washed and dried away from direct sunlight and humidity for two (02) weeks. The dried

samples were ground using a mixer and an aluminum mortar.

Animals and parasites

The mice used are female albino mice of NMRI (Naval Medical Research Institute) strains

with an average weight of 25 ± 3 g and 2 months old. These mice were purchased from the

animal husbandry of the International Center for Research and Development on Livestock in

Subhumid Zones (CIRDES) in Bobo-Dioulasso, Burkina Faso. They had access to a standard


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pellet diet and water ad libitum. A total of Seven groups of six mice per each group were

used to assess the antiplasmodial activity using the suppressive test.

Plasmodium berghei (strain ANKA) is continuously maintained in the entomology laboratory

of Health Sciences Research Institut at Bobo-Dioulasso in Burkina Faso by an acyclic

passage from infected mice to healthy mice through an injection of parasitized blood.

Extraction

Aqueous extraction

50 g of powder from each plant sample (leaves and stem bark of Mitragyna inermis and the

whole plant of Spermacoce verticillata) were mixed with 500 ml of distilled water. The

mixture was heated and boiled under reflux for 15 min and filtered several times. The

obtained decoctions were frozen and lyophilized.

Ethanolic extraction

The plant samples (20 g) were sequentially extracted with 200 ml of petroleum ether and

ethanol using a soxhlet apparatus. The extracts were first concentrated to maximum using

soxhlet dispositive and then to dryness at room temperature in the Petri dishes.

Determination of Total Phenolics and Total Flavonoids

Total phenolics

Total phenolics were evaluated according to the colometric method described by Meda and

al. (2010).[11]

The absorbances were read at 760 nm against a standard curve of gallic acid

( . The results are expressed in mg of gallic acid

equivalent per 100 milligrams of the extract (mg EAG / 100 mg).

Total flavonoids

The method described by Meda and al. (2010) was used to measure total flavonoids

extracts.[11]

To 0.625 ml of aluminum chloride (2%) added 0.625 ml of the sample solution of

extracts and the whole incubated for 10 minutes in the dark. The absorbance read against a

standard curve of quercetin ( ) at 415 nm. The results expressed

in mg of quercetin equivalent per 100 mg of the extract (mg EQ / 100 mg).


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Antioxydant Activities

The evaluation of the antioxidant activity was carried out according to the method described

by Meda and al. (2010) and the results expressed in μmol equivalent ascorbic acid per 1 gram

of extract (μmol EAA / g of extract).[11]

Iron (III) to iron (II) activity (FRAP)

The sample solution of the extracts (0.250 ml) was mixed with 0.625ml of the phosphate

buffer solution (0.2M; pH 6.6) and 0.625ml of potassium hexacyanoferrate (1%). The whole

was incubated at 50°C for 30 min in a sonicator. After incubation, 0.625 ml of trichloroacetic

acid (10%) is added and the mixture is centrifuged at 3000 rpm for 10 min. Once the

centrifugation is complete, 0.3125 ml of the supernatant is added to 0.3125 ml of distilled

water and 0.0625 ml of freshly prepared iron chloride (0.1%). The absorbances of the

reducing power by the FRAP read at 700 nm against a standard curve of ascorbic acid

( ). A series of 03 readings were carried out.

ABTS radical cation decolorization assay

The cation radical ABTS ● +

was regenerated by mixing an aqueous solution of ABTS (7

mM) with 2.5 mM potassium persulfate (final concentration) and the mixture is kept in the

dark at room temperature for 12 hours before use. The mixture was then diluted with ethanol

to give an absorbance of 0.70 ± 0.02 at 734 nm using the spectrophotometer. 10 µl of the

sample solution of extracts added to 990 µl of the ABTS reagent. The whole incubated for 15

min, protected from light. The absorbances read three times at 734 nm against a standard

curve of ascorbic acid ( ).

DPPH radical scavenging activity

To a volume of 0.75 ml of the sample solution of the extracts dilued to 1/2 was mixed with

1.5 ml of the DPPH solution (20 mg / l) and the whole is incubated for 15 min in the dark.

The absorbances were read at 517 nm with a spectrophotometer. Three absorbance readings

of the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method at 517 nm are required with a

spectrophotometer. The capacity of the extracts to trap free radicals determined using the

Methanol like white sample. The inhibition percentage (PI) of each extract according to the

following formula: PI = (Absorbance of white-Absorbance of the sample) / (Absorbance of

white) * 100. The IC50 were determined graphically by doing 03 readings.


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Antiplasmodial Activities

The method of Peter Robinson, 1975 used to assess the antiplasmodiale activity of our

extracts.[12]

Infestation

Parasitized blood from donor mice constantly maintained in the laboratory is used to infest 06

test mice for each batch due 07 batches including one control and 06 for extracts. To do this,

the parasitaemia of each donor mouse read in order to obtain 107 parasitized globules to be

injected into the test mice by the intraperitoneal route.

Treatment

After two hours of waiting, the groups of animals were administered 250 ug extract/kg body

weight respectively for the six extract used. While the negative control, were administered

with 200 ul of distilled water. The treatment was done once a day for 04 days. On the 5th day,

the dried thin smears fixed in methanol to be stained with 10% Giemsa for 10 min.

Parasite diagnostic

After staining, the rinsed slides dried in the open air before the reading. The parasitaemia

reading did on 03 fields under the optical microscope at the 100X objective by depositing

immersion oil on the slide. The reduction percentages (PR) calculated using the formula

below:

We used the rating scale of in vivo antiplasmodial activity from Rasoanaivo et al. (2004) to

assess the parasitological efficacy of our extracts.[13]

The extract was considered at a dose of

250mg / Kg depending on the percentage reduction in parasitaemia:

Very active, if the percentage reduction of the parasitaemia is between 100 and 90;

Active to moderate, if the percentage reduction of the parasitaemia is between 90 and 50;

Moderate to low, if the percentage reduction in parasitaemia is between 50 and 10;

Inactive if the percentage reduction in parasitaemia is 0.

Statistical Analysis

The statistical analysis (calculation of means, standard deviations and P-values) was done

with the R software.


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RESULTS AND DISCUSSION

Results

Total phenolics

The total phenolics contents of different plant extracts were presented in the Table I. For the

leaves of M. inermis, the total phenolics content of the aqueous extract (39.84 ± 13.84 mg

EAG / 100 mg) was highest than that of the ethanolic extract (18.13 ± 7.2 mg EAG / 100

mg). As regards the barks of M. inermis and of the whole plant of S. verticillata, the ethanolic

extracts have shown the best contents with the values of 20.56 ± 6.68 mg EAG / 100 mg and

28.92 ± 1.90 mg EAG / 100 mg, respectively. This obtained results showed that there is no

significant difference between the total phenolics contents of the ethanolic extracts and those

of the aqueous leaves and barks of M. inermis as well as those of the whole plant of S.

verticillata.

Table I: Total phenolics of S. verticillata, leaves and barks of M. inermis extracts.

Plants Extracts Total phenolics (mg EAG/100

mg of extract) P-value

Leaves of M. inermis Ethanolic 18.13± 7.20

0.07 Aqueous 39.84± 13.84

Barks of M. inermis Ethanolic 20.56 ± 6.68

0.73 Aqueous 10.35 ± 1.09

Whole plant of S. verticillata Ethanolic 28.92 ± 1.90

0.85 Aqueous 20.35 ± 0.42

M. inermis: Mitragyna inermis, S. verticillata: Spermacoce verticillata, values are Mean ±

standard deviation (n=3), comparison of ethnaolic and aqueous extracts from each part of

plants.

Total flavonoids

The table II indicated that the flavonoids were in the all extracts of the plants. The flavonoids

contents of the aqueous extracts were varied from 0.40 to 5.10 mg EQ/100 mg of extract and

those of the ethanolic extracts from 2.14 to 6.80 mg EQ / 100mg of extract. However, a

significant difference was observed in the ethanolic and aqueous extracts of the leaves of M.

inermis (P-value = 0.0154). The same applies to the extracts of the barks of M. inermis and

the whole plant of S. verticillata. The highest content of M. inermis total flavonoids was

observed with the ethanolic extract of the leaves of 5.53 ± 0.04 mg QE / 100mg. For S.

verticillata, it was always the ethanolic extract with 6.80 ± 0.27 mg QE / 100mg which had

the highest content of flavonoids. Of all the extracts, aqueous extract to the barks of M.

inermis were contained the lowest content of total flavonoids (0.40 ± 0.01mg QE / 100mg).


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Table II: Total flavonoids of S. verticillata, leaves and barks of M. inermis extracts.

Plants Extracts Total flavonoids (mg EQ/100

mg of extract) P-value

Leaves of M. inermis Ethanolic 5.53± 0.04

0.0154 * Aqueous 4.31 ± 0.04

Barks of M. inermis Ethanolic 2.14± 0.07

<0.001 *** Aqueous 0.40± 0.01

Whole plant of S. verticillata Ethanolic 6.80± 0.27

<0.001 *** Aqueous 5.10± 0.12

M. inermis: Mitragyna inermis, S. verticillata: Spermacoce verticillata, values are Mean ±

standard deviation (n=3), comparison of ethanolic and aqueous extracts from each part of

plants. P-value *: difference is significant, P-value **: difference is very significant, P-value

**: difference is very very significant.

Antioxidant Investigations

DPPH (2, 2-diphenyl-1-picrylhydrazyl)

The figure 1 presents antioxidant activity results of plant extracts through DPPH method. The

aqueous extracts from the leaves of M. inermis and ethanolic extracts of S. verticillata

showed the best capacities to scavenge free radical’s DPPH with IC50 values of 1.21 µg / ml

and 3.04 µg / ml respectively. These extracts exhibited an excellent ability to scavenge DPPH

free radicals.

Figure 1: Capacity antioxidant values with DPPH method of aqueous and ethanolic

extracts of Whole plant of S. verticillata, leaves and barks of M. inermis.


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ABTS (2, 2’-azinobis- [3-ethylenzothiazoline-6-sulfonic acid])

The ability of the various extracts to reduce ABTS ● +

radical cations was indicated in the

Figure 2. The values were varied from 41.06 to 67.73 µmol EAA / g for M. inermis. Those of

S. verticillata were varied from 47.84 to 98.21 µmol EAA / g. Among all the extracts tested,

the best activities were obtained with the aqueous extract of the leaves of M. inermis (67.73

µmol EAA / g) and the ethanolic extract of S. verticillata (98.21 µmol EAA / g).

Figure 2: Capacity antioxidant of the different extracts with ABTS method from M.

inermis (Mitragyna inermis), S. verticillata (Spermacoce verticillata).

FRAP (Ferric Reducing Antioxidant Power)

The Figure 3 illustrated the results of the ferric reducing power of the different extracts by the

FRAP method. The values varied between 41.06 µmol EAA / g and 67.73 µmol EAA / g for

extracts of M. inermis and from 47.84 µmol EAA / g to 98.21 µmol EAA / g for extracts of S.

verticillata. The lowest activity of 41.06 µmol EAA / g was recorded with the aqueous

extract of the bark of M. inermis. All extracts had the ability to reduce ferric iron to ferrous

iron.


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Figure 3: capacity antioxidant of the different extracts with FRAP method from M.

inermis, S. verticillata.

Antiplasmodial Activities and the Impact of Extracts on Number of White Blood Cells

The results obtained showed significant decrease in parasitaemia of P. berghei infected mice

treated with the ethanolic and aqueous extract used (Table III). The ethanolic extracts of M.

inermis and S. verticillata were presented the highest parasitaemia reduction percentages 32.9

and 27.6%, respectively. These results indicated that all the extracts reduce the parasitaemia.

All the aqueous extracts had a lower percentage of parasitaemia reduction than the ethanolic

extracts. However, there was no significant difference regarding the impact of the ethanolic

and aqueous extracts on the number of white blood cells counted compared to the control

group (Table IV).

Table III: Antiplasmodial activity of the different extracts from M. inermis and S.

verticillata.

Plants Extracts

Extract

concentration

(mg/kg)

Meam

parasitaemia

counts

%

Inhibition

Control Water - 29.33±4.80 0

Leaves of M.

inermis

Aqueous 250 27.67±3.56 11.8

Ethanolic 250 21.50±3.99 32.9

Barks of M.

inermis

Aqueous 250 29.00±5.22 9.9

Ethanolic 250 24.50±4.09 22.4

Whole plant of

S. verticillata

Aqueous 250 23.33±5.05 27.6

Ethanolic 250 23.50±4.85 27.6

M. inermis: Mitragyna inermis, S. verticillata: Spermacoce verticillata.


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Table IV: Impact of aqueous and ethanolic extracts of Whole plant of S. verticillata,

leaves and bark of M. inermis on number of white blood cells.

Plants Extracts

Extract

concentration

(mg/kg)

Mean white

blood cells

counts

P-value

Control Water - 24.33±13.25 -

Leaves of M.

inermis

Aqueous 250 33.33±12.31 0.99

Ethanolic 250 27.33±16.03

Barks of M.

inermis

Aqueous 250 45.17±34.78 0.207

Ethanolic 250 18.33±6.68

Whole plant of

S. verticillata

Aqueous 250 32.50±24.02 1

Ethanolic 250 29.17±7.78

M. inermis: Mitragyna inermis, S. verticillata: Spermacoce verticillata

DISCUSSION

Phenolics and flavonoids were the compounds with biological activity.[14,15]

The results

obtained in this study demonstrated that all extracts from M. inermis and S. verticillata

contained both the phenolics and flavonoids in variable contents. This variability could be

due to biotic (species, organ and physiological stage) and abiotic (edaphic factors) conditions,

the nature of the soil and the type of microclimate and also the bioclimatic stages where these

plants growed.[16]

Antioxidants were the substances capable of neutralizing or reducing damage caused by free

radicals.[17,28]

All extracts of two plants had an antioxidant activity. This activity could be due

to the phenolics and flavonoids.[18,19,20,21,22]

All extracts were contained these compounds.

The phenolic compounds especially flavonoids were endowed of the antioxidant

activity.[23,24]

All the extracts could reduce the parasitaemia. This reduction of parasitaemia could be due to

their composition. The phenolic compounds and flavonoids had antiplasmodial

properties.[26,27]

Malaria induced the production of hydroxyl radicals in the liver, which were

probably the main reason for the induction of oxidative stress and apoptosis.[25]

Antioxidants

were a source of defense against radicals.[17,28]

However, there was no significant difference regarding the impact of the ethanolic and

aqueous extracts on the number of white blood cells counted compared to the control group.


140


The immune system protects the body from possibly harmful substances by recognizing and

responding to antigens. Antigens are substances (usually proteins) on the surface of parasites,

viruses, fungi, or bacteria. Nonliving substances such as toxins, chemicals, drugs, and foreign

particles (such as a splinter) can also be antigens. The immune system recognizes and

destroys, or tries to destroy, substances that contain antigens. The aqueous and ethanolic

plants extracts did not reduce the number of white blood cells after administration. Due to

their composition and their antioxidant activity.

CONCLUSION

It is evident based our findings that M. inermis and S. verticillata possess potent antioxidant

and antiplasmodial effect justifying their usage in the traditional medicine as anti-malarial.

However, the active principle(s) are not yet identified, and there is a need for their

identification. In view of this fact, attempts are being made to carry out antiplasmodial

curative test, prophylactic test as well as guided fractionation of the extract to isolate the

active compounds and also to test for the cytotoxicity of the extract.

ACKNOWLEDGMENTS

We thank the traditional healers, field workers and the laboratory team of Laboratoire de

médicine et pharmacopée traditionnelle of Institut de Recherche en Sciences de la Santé,

Direction Bobo-Dioulasso, Burkina Faso. Laboratory work leading to the results presented in

this article was made possible by the BKF5021 project, supported by the International

Atomic Energy Agency (IAEA) for providing the facilities and the Islamic Educational,

Scientific and Cultural Organization (ISESCO) for its support to help for research in health

biotechnology.

AUTHORS’ CONTRIBUTIONS

C.K.M. was involved in the conception of the study, participated in study design, laboratory

analysis and manuscript preparation, editing and review. S.A. participated to samples

collection and analyzing, data analyzing and wrote the initial drafts of the manuscript. K.K.B.

were involved in study design and critical review of manuscript. D.S.N., K.W. and D.A.F.

contributed to the samples analyzing and to the manuscript writing. Both M.N.R. and O.G.A

supervised the project and ensured the quality of developed study concept and design and

results. All authors participated in the writing and review of the manuscript.


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CONFLITS OF INTEREST/COMPETING INTERESTS

All authors read and approved the final manuscript. They declared that they have no

competing interests.

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