IN VITRO ANTIPLASMODIAL AND ANTIOXIDANT ACTIVITIES OF

www.wjpps.com Vol 4, Issue 08, 2015.

101

Dossa et al. World Journal of Pharmacy and Pharmaceutical Sciences

IN VITRO ANTIPLASMODIAL AND ANTIOXIDANT ACTIVITIES OF

ETHANOLIC AND HYDROETHANOLIC EXTRACTS OF HYPTIS

SUAVEOLENS

S. E. Medoatinsa1,4

, C. P. Agbangnan Dossa1*

, F. Viwami2, G. S. R. Bogninou-

Agbidinoukoun1, J. P. Noudogbessi

†1, L. Lagnika

3, H. Ahissou

4, D. C. K. Sohounhloue

1

1Laboratoire D’etude Et De Recherche En Chimie Appliquee (Lerca), Ecole Polytechnique

D'abomey-Calavi (Epac), Universite D'abomey-Calavi (Uac), 01 Bp 2009 Cotonou,

Republique Du Benin. 2Centre D’etude Et De Recherche Sur Le Paludisme Associe A La Grossesse Et A l’enfance,

Institut De Recherche Pour Le Developpement (Ird), Institut Des Sciences Biomedicales

Appliquees (Isba), Cotonou, Benin. 3Laboratoire De Biochimie Et De Biologie Moleculaire, Faculte Des Sciences Et Techniques

(Lbbm/Fast/Uac). 4Laboratoire D’enzymologie Et De Biochimie Des Proteines, Faculte Des Sciences Et

Techniques (Leb/Fast/Uac).

ABSTRACT

Hyptis suaveolens (Lamiaceae) is a fragrant plant that smells

peppermint when crushed. It’s being used in the tropical countries for

its insect repellent power and scientific studies have confirmed this

property as well as its antihelminthic, hepatoprotective, antibacterial,

antifungal and antiplasmodial activities. Few studies have been

conducted on the non-volatile extract and antiplasmodial power of this

plant. The present study aims to assess "in vitro", antiplasmodial

(semi-microtest of Rieckmann) and anti-radical activities of leaves of

H. suaveolens from two localities of Benin. The phytochemical

screening carried out prior to the evaluation of the biological activities

revealed the presence of several metabolites, mainly phenolic

compounds (tannins, flavonoids) that may be responsible for

antiplasmodial and anti-radical properties observed during this study.

The quantitative analysis of the polyphenols by spectrophotometry

shows that the hydroethanolic extract (34.32 mg/g) is richer in these compounds than the

ethanolic extract (3.98 mg/g) thus confirming theyield of extraction of phenolic compounds

obtained (2.4% against 8% hydroethanolic extract). The extracts investigated contain

Article Received on

27 May 2015,

Revised on 22 June 2015,

Accepted on 15 July 2015

*Correspondence for

Author

C. P. Agbangnan Dossa

Laboratoire D’etude Et De

Recherche En Chimie

Appliquee (Lerca), Ecole

Polytechnique D'abomey-

Calavi (Epac), Universite

D'abomey-Calavi (Uac),

01 Bp 2009 Cotonou,

Republique Du Benin.

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SSJJIIFF IImmppaacctt FFaaccttoorr 55..221100

VVoolluummee 44,, IIssssuuee 0088,, 110011--111122.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357


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compounds able to trap DPPH whose color changes from purple to yellow and are endowed

with good antiplasmodial activity with an IC50 <6.25 µg/mL, the lowest concentration used

during this study.

KEYWORDS: Phenolic compounds; anti-radical; antiplasmodial, phytochemistry.

INTRODUCTION

Hyptis suaveolens (L.) Poit. (Lamiaceae) is a perennial shrubby herb fast growing that grows

along roadsides and around stockyards in tropical

regions. H. suaveolens is an aromatic annual herb

earthly, erect up to 200 cm height with cowardly

inflorescences. Its leaves are simple, entire,

opposite, stalked, ovate, and hairy on both sides. H.

suaveolens is a flowering plant, hermaphrodites,

blue corolla (6 mm long) grouped in axillary

clusters. It flowers from november to february and

fruiting from december to march. It is originates

from tropical America, although widespread in Africa and Asia tropicale.[1]

In West Africa,

the leaves of the plant are accepted as substituent infusion tea. It is renowned for its

effectiveness against benign painful attacks. In the Congo (Brazzaville), the plant is put in the

bath to wash the children against fever. In Côte d'Ivoire and Senegal, the tisane is given to

fight against coughs, bronchial disorders. In America, the poultice of the leaves is used

against cancer and tumors. In obstetrical villages in Ghana, the plant has a very important

role in the treatment of anemia during pregnancy and midwives to prescribe to induce or

provoke and facilitate the work of pregnant women during child birth. The leaves are used in

Sierra Leone and Benin to repel insects (mosquitoes) away houses.[2]

Several scientific

studies have confirmed the traditional use made of this plant. Abagli et al, (2011)[3]

have

shown the insect repellent power of H. suaveolens while Praveen et al, (2010)[4]

; Babalola et

al, (2011)[5]

; Malele et al., (2003)[6]

and Chukujekwu et al., (2005)[7]

have shown respectively

its antihelminthic, hepatoprotective, antifungal and antiplasmodial properties.

Affecting about 216 million and killing 660.000 people in 2010, malaria remains one of the

most widespread parasitic tropical diseases.[8]

The spread of resistance of Plasmodium

falciparum to most anti-malarial drugs is an important difficulty in the treatment of this

disease. The use of Artemisinin Combination Therapy (ACT) as first-line treatment of


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uncomplicated malaria caused by P. falciparum was officially recommended by the WHO in

2006.[9]

Unfortunately, the emergence of resistance to artemisinin derivatives was recently

reported from the south east Asia.[10,11]

In this context, new anti-malarial compounds are

urgently needed to treat this major endemic disease.

The present study aims to evaluate "in vitro" antioxydant and antiplasmodial activities of

extracts from the leaves of H. suaveolens of Benin a view to validating the traditional use

made of this plant.

MATERIALS AND METHODS

Reagents

Among the reagents which have been used in this work, we can cite the positive control used

in the evaluation of the antiplasmodial activity, which is a drug owned by the range of

Artemisinin-based Combination Therapy (ACT) purchased from pharmacy

(Godomey/Benin). Gallic acid, Butyl Hydroxy Anisole (BHA), quercetin, RPMI 1640,

catechin purchased from Sigma Chemical Co. (St. Louis, MO) and 2,2-diphenyl-1-

picrylhydrazyl (DPPH) are used to test the anti-radical. The ethanol was used as solvent

during extraction.

Parasite

chloroquine-resistant Dd2 strain of Plasmodium falciparum (IC50 = 114 nM) was used as

parasite and was cultivated in human O Rh+ red blood cells.

Plant material

Leaves of wild plants of H. suaveolens were harvested from two differents locations of

Benin: Abomey-Calavi and Godomey; dried at room temperature (25°C) and pulverized. All

the collected samples were washed once with tap water and once with distilled water to

remove the adhering salts and other associated animals before drying.

Phytochemical screening

The large chemical groups contained in the powder leaves of Hyptis suaveolens have been

determined by colorimetry test tube according to the method of Houghton (1998).[12]

The

reaction with ferric chloride (FeCl3, 2%) has been used to characterize polyphenols while

flavonoids were detected by reaction with cyanidin. Catechic tannins identification was

carried out using reagent of Stiasny. For gallic tannins, we filtered the solution of catechic


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tannins identification and the filtrate was collected and saturated with sodium acetate. The

addition of 3 drops of FeCl3 (1%) allows to identify the presence of gallic tannins by the

appearance of intense blue-black coloration. Alkaloids were characterized from reagents of

Mayer (iodo-iodized reagent) and Dragendorff (iodo-reactive potassium bismuthate). Order

to identify the saponins, we based on their aphrogène power.

Extraction

20 g of air-dried leaves of Hyptis suaveolens were extracted for 24 hours by maceration with

200 mL of ethanol or ethanol-water (70:30) at room temperature under magnetic stirring. The

extracts were filtered and the filtrate was concentrated by rotary vacuum evaporation at 40 ℃

until obtaining a solid residue.

Determination of phenolic compounds

The table I present the phenolic compounds analyzed spectrophotometrically well as the

wavelength and the reagent to each specific compound.

Table I: phenolic compounds measured in a spectrophotometer.

Reagents Wavelengths References

Condensed tannins Vanillin sulfuric 500 nm Xu et Chang, (2007)[13]

Flavonoids Aluminum trichloride 510 nm Enujiugha , (2010)[14]

Anthocyanins Sodium bisulfite 520 nm Agbangnan et al., (2012)[15]

Total polyphenols Folin-Ciocalteu 765 nm Siddhuraju et al., (2002)[16]

Anti-radical activity

The antioxidant activity of a compound corresponds to its ability to resist oxidation. It was

evaluated by the method of trapping free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)[17]

,

which is a stable radical having a specific absorption at 517 nm due to its purple color but

turns yellow when it is reduced by a free radical scavenger or antioxidant. The percentage

inhibition of DPPH (I) was calculated by the following formula.

%DPPH•sc = (Acont – Asamp) × 100/Acont

Where, Acont is the absorbance of the control and Asamp is the absorbance of the sample.

Antiplasmodial activity "in vitro"

Chloroquine-resistant Dd2 strain of Plasmodium falciparum was grown in 96-well plates as

described by Trager and Jensen.[18]

Blood cells were washed three times with RPMI 1640

before use in culture. Erythrocytes were then suspended in RPMI supplemented with l-


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glutamine (4.2 mM), HEPES (25 mM), bovine fœtal serum (10%: v/v), streptomycin (100

g/ml) and penicillin (100 IU/ml). Hematocrits were adjusted at 5% and parasite cultures were

used when they exhibited 2% parasitaemia.

The “in vitro” antimalarial tests were performed by light microscopy using Giemsa-stained

smears as described by Rieckmann et al.,.[19]

Plant extracts and the 'Lumate forte' were

dissolved in physiological water. The aliquots of drug solutions were added in duplicate. A

control experiment was performed separately to check the effect of solvents on parasite

maturation. Drug concentrations in the wells ranged from 6.25 µg/mL to 1 mg/mL for the

ethanolic extracts, hydroethanolic extracts and reference compound. The plates were

incubated at 37 °C in a candle jar for a total period of 96 h. Each 24 hours blood smears were

made and stained with May-Grünwald Giemsa to assess parasitemia and determine the 50%

inhibitory concentration (IC50) of the growth of P. falciparum. Finally parasitaemia was

calculated using the following formula.

RESULTS AND DISCUSSION

Phytochemical screening

The powder of Hyptis suaveolens leaves was subjected to preliminary phytochemical analysis

using standard procedures to find out the phytoconstituents present in the sample. Table II

shows the different metabolites identified in the plant material studied.

Table II: Metabolites identified in the leaves of Hyptis suaveolens

H. suaveolens (1) H. suaveolens (2)

Polyphenols + +

Tanins

Total + +

Cathechic + -

Gallic + +

Flavonoids

Total + +

Anthocyanin + +

Leucoanthocyanin - -

Saponosids - +/-

Mucilages - -

Alkaloids - -

Coumarins - +

Anthraquinones

Total - +

Free - +/-

Reducing sugar - +

+: Presence; +/-: Doubt; - : Absence


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The phytochemical screening test revealed the presence of phenolic compounds (gallic

tannin, anthocyanin) within two samples of Hyptis suaveolens studied. They do not contain

leucoanthocyanins, mucilages and alkaloids. In contrast to the sample 1, sample 2 contains

coumarins, anthraquinones, reducing sugars, cathechic tannins and saponins. This sample was

therefore selected for further analysis in the present work.

According to Praveen et al.,[4]

the phytochemical analysis of Hyptis suaveolens showed the

presence of phenolic compounds is well noted. Gavani and Paarakh[20]

were noticed that

tannins, flavonoids and carbohydrats were found in the extracts of Hyptis suaveolens leaves

harvested at Bangalore, Karmataka (India).[20]

Extraction yield of phenolic compounds

The percentage of extraction was calculated by using the following formula:

Weight of the extract (g)100

Weight of the plant material (g)Y X

Y: Yield (%).

The mixture ethanol-water (30/70) showed a yield of extraction three times higher than that

of ethanol. The values obtained are respectively (2.4%) for ethanol and (8%) for the mixture

ethanol-water. Thus we note that ethanol has a low extraction yield of phenolic compounds

unlike the extract made with the hydroethanolic solvent (30/70). This observation was made

by Agbangnan et al.[21]

and Naczk et al.[22]

who showed that the mixture water/alcohol extract

phenolics better than each solvent considered separately. They attributed this affinity of

phenolic compounds to the binary ethanol-water to the fact that it has a higher polarity than

ethanol or water taken alone.

Phenolic content

Figure 1 shows the levels of phenolic compounds in ethanolic and hydroethanolic extracts of

leaves of H. suaveolens.


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PT: Total Polyphenols; Fla: Flavonoids; Ant: Anthocyanins; TaC: Condensed Tannins

Figure 1: Content of phenolic compounds of ethanolic and hydroethanolic extracts.

The hydroethanolic solvent was extracted respectively over eight times and over three times

the total polyphenols and flavonoids as ethanol. However, the condensed tannin content of

the ethanolic extract is three times greater than that of the hydroethanolic extract. Both

solvents extract anthocyanins in the same proportions.

The hydroethanolic extract of the leaves of H. suaveolens is richer in flavonoids (12.12 mg/g)

than condensed tannins (0.99 mg/g); which is in consistent with the results of Edeoga et

al.,[23]

(flavonoids: 12.54%, condensed tannin: 0.52%). This is not the case at the level of

ethanolic extract that we note that the content in flavonoids (3.76 mg/g) is slightly higher than

that of the condensed tannins (3.58 mg/g).

It emerges from results that the hydroethanolic extract is rich in phenolic compounds that

ethanolic extract, confirming the extraction of phenolic compounds obtained yields. The

differences observed between our findings and those of the literature may be related to the

extraction and quantification methods, two factors that can influence the estimate of the

content of a plant species phenolic compounds.[24]

Anti-radical activity

Figure 2 shows the 50% Inhibitory Concentrations of DPPH ethanolic and hydroethanolic

extracts studied compared with those of standards.


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AG: Gallic acid; BHA: Butylated Hydroxy Anisole; Q: Quercetin; HsET: Ethanolic

extract; HsHET: Aqueous ethanolic extract.

Figure 2: Inhibitory Concentrations 50% of DPPH ethanolic and hydroethanolic

extracts of leaves of Hyptis suaveolens.

Compared to standards used in the evaluation of the anti-radical property of our extracts, we

note that our extracts do not possess promising activity especially as the antioxidant capacity

of an extract is considerable when its IC50 is low. Nevertheless, they contain compounds

capable of trapping DPPH and this is explained by the fact that the purple color of DPPH

turned yellow. In view of the results obtained, the antioxidant activity is linked to the levels

of phenolic compounds in the extracts tested meaning that the IC50 of the hydroethanolic

extract which has high content of phenolic compounds is lower than that of the ethanolic

extract.

These observations corroborate those already made earlier by Wojdylo et al., 2007[25]

and

Djeridane et al., 2006.[26]

Antiplasmodial activity

The test focused both on the extracts of leaves of Hyptis suaveolens and a positive control

which is an Artemisinin-based Combination Therapy (Lumate forte), in vogue medicine for

the treatment of uncomplicated malaria in malaria endemic areas with presence of strains

chloroquine-resistant.

Table IV shows the concentrations (µg/mL) have reduced by at least 50% parasite density

Dd2 strain of P. falciparum in culture.


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Table III: Inhibitory Concentrations 50% of parasite density Dd2 strain of P.

falciparum in culture.

H. suaveolens

Positive control Ethanolic Hydroethanolic

Day 1 54 280 110

Day 2 < 6,25

Day 3 240 14 11

Day 4 < 6,25

After 48 hours of incubation, the extracts (ethanolic, hydroethanolic) of Hyptis suaveolens as

the positive control showed good antiplasmodial activity, according to the classification of

the antiplasmodial activity made based on the IC50 Mbatchi et al.,[27]

which states that when:

IC50 <10μg/mL good antiplasmodial activity; IC50 <50 µg/mL moderate antiplasmodial

activity; IC50 ≥ 50 µg/mL low antiplasmodial activity and IC50 ≥ 100 µg/mL no

antiplasmodial activity. The antiplasmodial activity observed is due to flavonoids and/or

coumarins identified during the screening phytochemical.

Chukwujekwu et al.,[7]

showed in 2005 that the ether extract of H. suaveolens of Nigeria has

good antiplasmodial activity with an IC50 = 2.µg /mL. Several factors could be the basis of

the differences observed between the IC50 extracts studied and observed values in the

literature. According to some authors, the polarity of the solvent used in conjunction with its

solubilizing capacity of some plant substances, the period of collection of plant material, the

vegetative stage of the plant and soil[28]

are all factors capable of influencing the power

antiplasmodial extracts.

CONCLUSION

In view to enhance the medicinal plants of beninese flora, our choice fell on the leaves of

Hyptis suaveolens of two localities of Benin. The phytochemical screening showed that the

powder of the two samples investigated contain phenolic compounds (gallic tannin,

anthocyanins), and that in contrast to the sample 1, sample 2 contains coumarins,

anthraquinones, reducing sugars and saponins. The pharmacological study on ethanolic and

hydroethanolic extracts of sample 2 showed that contains molecules capable of trapping

DPPH and endowed with antiplasmodial properties at low doses. The results obtained at the

end of this study justify the use made of this plant in traditional african medicine.


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IN VITRO ANTIPLASMODIAL AND ANTIOXIDANT ACTIVITIES OF