antimalarial effect of the root of silenemacrosolen a

Research ArticleAntimalarial Effect of the Root of Silene macrosolen A Rich(Caryophyllaceae) on Plasmodium-berghei-Infected Mice

Gebru Hagos Atsbha 12 Rajkapoor Balasubramanian 23 and Abadi Kahsu Gebre2

1Department of Pharmacy College of Health Sciences Adigrat University Adigrat Ethiopia2School of Pharmacy College of Health Sciences Mekelle University Mekelle Ethiopia3Department of Pharmacology JKK Nattraja College of Pharmacy Komarapalayam-638 183 Tamilnadu India

Correspondence should be addressed to Gebru Hagos Atsbha gebruhagos2011gmailcom

Received 16 September 2020 Revised 24 February 2021 Accepted 9 March 2021 Published 16 March 2021

Academic Editor Letizia Angiolella

Copyright copy 2021 Gebru Hagos Atsbha et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Malaria remains a major public health problem globally Poor access to antimalarial drugs compounded with rapidlyevolving drug resistance encourages researchers to continuously look for new drugs Of importance traditionally used medicinesof plant origin are the highest priority as the ethnobotanical claim can be used as an important clue for its safety and efficacyprofiles Silene macrosolenA Rich (Caryophyllaceae) has been traditionally used for malaria treatment in Ethiopiaerefore thisstudy was aimed to evaluate the in vivo antimalarial activity of the plant against Plasmodium-berghei-infected (ANKA strain) Swissalbino mice Methods e dried powdered root of Silene macrosolen was extracted using 80 methanol e crude extract wasfractionated using chloroform ethyl acetate and distilled water that have different affinities to plant phytoconstituentse in vivoantimalarial activities of the crude extract were evaluated using 4-day suppressive prophylactic and curative tests e anti-malarial activity of the solvent fractions was evaluated in a 4-day suppressive test e oral acute toxicity of the crude extract wasalso determined according to the OECD guidelines Results e percentage of parasite suppression on the crude extract was3102 3582 and 3923 in prophylactic curative and 4-day suppressive tests respectively at the tested dose level of 400mgkg e percentages of chemosuppression of the solvent fractions (400mgkg) were 4307 4261 and 3838 in aqueous ethylacetate and chloroform fractions respectively Both the crude extract and solvent fractions also significantly prolonged survivaltime except in the prophylactic test In addition prevention of weight loss and reduction in temperature and packed cell volume(PCV) were observed in crude extract as well as solvent fractions e acute toxicity test of the plant extract also exhibited no signof toxicity Conclusion e result indicated that Silene macrosolen has a significant antimalarial activity justifying the traditionaluse of the plant material for treatment of malaria

1 Introduction

Malaria is a fatal infectious disease that has a large burden ofdisease [1] Malaria infects 219 million people globally withannual death of almost half a million [2 3] Sub-SaharanAfrican countries including Ethiopia are disproportionatelyaffected by malaria morbidity andmortality with the highestburden on children under five and pregnant women [3 4]

e rapidly evolving antimalarial drug resistance alsoposes a significant challenge in reducing the burden ofmalaria Resistant parasites have been reported against allthe available antimalarial medications [5 6] is indicatesmalaria may cause unprecedented morbidity and mortality

in the future justifying the need for new antimalarial drugdevelopment [5 7]

Plants have been an important source of drugs for manycenturies [8] According to the WHO report about 80 ofAsians and Africans still rely on herbal medicine to meettheir primary health-care needs [9 10] Most of the currentlyavailable antimalarial drugs also have a plant origin [8 11]erefore plants are considered as a robust source for theevent of future effective antimalarial agents Ethiopia is richin biodiversity and the traditional medicinal plant hasplayed a significant role in malaria treatment [1 12 13]

e genus Silene is small shrubs containing about 700species that belong to the Caryophyllaceae family It has

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2021 Article ID 8833865 11 pageshttpsdoiorg10115520218833865

several reported pharmacological activities including anti-microbial antitumor phagocytic antiviral antifungal andantioxidant activities [14]

Silene macrosolen known by its vernacular name saer-osaero (Tigrigna) and wogert (Amharic) is an herbal plantbelonging to the Genus Silene that grows in different areas ofEthiopia [15] Traditionally it is used for treatment ofmalaria in Ethiopia [15 16] S macrosolen is also used as amosquito repellent and for treatment of an evil eye [17]hepatitis [18] and hemorrhoids [15] However pharma-cological activity against malaria is not scientifically sup-ported Hence the aim of this study was to evaluate in vivoantimalarial activity of the crude extract and solvent frac-tions of S macrosolen using 4-day suppressive prophylacticand curative tests in Plasmodium-berghei-infected mice

2 Materials and Methods

21 Chemicals Reagents and Drugs Absolute methanol(Loba Chemic Pvt Ltd India) chloroform (Nice ChemicalsPvt Ltd India) ethyl acetate (Research-Lab Fine ChemIndustries India) Tween 80 (Loba Chemic Pvt Ltd India)microscopic oil immersion (Laboratory reagents Ethiopia)Geimsa stain (Addis Ababa Ethiopia) trisodium citrate(Loba Chemic Pvt Ltd India) and chloroquine phosphate(APF Ethiopia) were used in this study and the chemicalsand reagents were laboratory or analytical grade

22 Plant Material Collection and Authentication Freshroots of S macrosolen were collected from Kilte AwulaeloDistrict Tigray Ethiopia located 825 km north of AddisAbaba e collected plant material was identified by abotanist (name) and the specimen was deposited at theBiology Department Gondar University with vouchernumber 001GHA2019

23 Extraction and Fractionations of Plant Material ecollected root of the plant was cleaned with tap water and airdried under shade at room temperature before it waspowdered using an electric mill Extraction of the powderedroot was carried by the maceration technique Specifically500 grams of the powdered root was mixed with 2500ml of80methanol that corresponded to one ratio five of betweenthe weight of the powder and solvent proportion emaceration continued for three days with intermediateshaking e macerate was filtered using muslin cloth andWhatman filter paper (No 1) consecutively while the marcwas remacerated twice with 80 methanol using the sameprocedure [19]e combined filtrate was dried in an oven ataround 400C

For fractionization the dried crude extract (160mg) wasdissolved in distilled water and partitioned three times withan equal volume of chloroform e chloroform layer wascollected in a beaker and dried in the oven at around 40degCafter combining together while the aqueous layer wasfurther shaken using equal volume of ethyl acetate threetimes following the same procedure as fractionation withchloroform Finally the aqueous fraction was taken and

dried in an oven at around 40degC e crude extract and thefractions were stored in the refrigerator throughout theexperiment

24 Experimental Animals and Parasite Healthy Swiss al-bino mice of both sexes aged 6 to 8 weeks obtained from theDepartment of Pharmacology and Toxicology School ofPharmacy Mekelle University were used to test the anti-malarial activity of both the crude extract and solventfractions e mice were acclimatized for seven days prior tothe experiments and housed in standard plastic cages withexposure to 12 h lightdark cycle and room temperatureemice had free access to standard pellet and water ad libitumthroughout the experiment e mice were handledaccording to the internationally accepted laboratory animaluse and care guideline [20] e experimental animal studieswere approved by the Institutional Review Committee of theCollege of Health Sciences Mekelle University (ERC15442018) before the actual commencement of the study

Chloroquine-sensitive P berghei ANKA strain obtainedfrom Ethiopian Public Health Institute (EPHI) Addis AbabaUniversity was used to infect the mice e parasites weremaintained by passage of blood from infected to normalmice on a weekly basis

25 Acute Oral Toxicity Test An acute oral toxicity test wasconducted in accordance to the Organization for EconomicCo-operation and Development (OECD) 425 guidelines[21] Accordingly healthy and nonpregnant 5 female micewere used and the first mouse was given a limited dose of2000mgkg orally with an oral gavage after it was restrictedfrom food access for 4 hoursemouse was observed for 24hours for any signs of toxicity and mortality Afterward theremaining 4 mice were treated with the same dose andobserved strictly for the first one hour and intermittently forthe subsequent three hours for any signs of toxicity likediarrhea loss of appetite hair erection lacrimation con-vulsions salivation lethargy and paralysis Follow-up wascontinued once daily for 14 days

26 Grouping and Dosing In each of the test models (4-daysuppressive test prophylactic test and curative test) thirtymice were divided randomly into five groups consisting ofsix mice

e groups were categorized as groups I (treated withvehicle) and II (treated with chloroquine phosphate) ethird fourth and fifth groups (group IIIndashV) were treatedwith three different oral doses of the crude extract (Table 1)or solvent fractions

27 Inoculation of Parasite Mice with a parasitemia level ofapproximately 30 were used as a donor of chloroquine-sensitive strain of P berghei ANKA throughout the exper-iment [22] e donor mice were sacrificed by decapitationand blood was taken by severing the jugular vein [23] Bloodwas collected from all the donormice and it was diluted with09 normal saline to obtain uniform 5times107 P berghei-

2 Evidence-Based Complementary and Alternative Medicine

infected red blood cells (IRBC) in 1ml of blood [24] In eachof the antimalarial model used each mouse was inoculatedwith 02ml of aliquot that contains 1times 107 P berghei-in-fected erythrocyte intraperitoneally [25]

28 In Vivo Antimalarial Activity Screening

281 Four-day Suppressive Test e 4-day suppressive testwas used to measure the schizonticidal activity of the crudeextract and fractions against P berghei-infected mice [26]Accordingly thirty mice were inoculated with P berghei onday 0 and randomly divided into five groups e micereceived different doses of the crude extract or solventaccording to the grouping and dosing protocol described inTable 1 Treatment was continued for 4 days from day 0 today 3 and on the 5th day a thin blood film was preparedfrom each mouse by taking blood through tail snip to de-termine the parasitemia level and percentage of inhibitionRectal temperature body weight and packed cell volume(PCV) were recorded at day 0 (before parasite inoculation)and day 4

282 Prophylactic Activity (Peterrsquos Repository Test)Evaluation of the prophylactic potential of the crude extractwas carried out according to the method described by Petersin 1975 [26] Accordingly 30 mice were randomly dis-tributed into 5 groups (Table 1) e mice were treated dailyfor four consecutive days On the 5th day the mice in allgroups were infected with inoculums of 1times 107 P berghei-infected erythrocyte On the 8th day blood smears wereprepared from each mouse and the parasite level was de-termined Body weight temperature and PCV wererecorded at day 5 before infection and at the end oftreatment

283 Test on Established Malaria Infection (Ranersquos Test)e curative potential of the crude extract was also evaluatedusing the method described by Ryley and Peters [27] In thisexperiment 30 mice were inoculated with an inoculum of1times 107 P berghei-infected erythrocyte intraperitoneallySeventy-two hours after infection the mice were randomlydivided into 5 groups (Table 1) and treated daily for 4 days AGiemsa-stained thin blood film was prepared from the tail ofeach mouse at the 4th day (before the first dose) and on the8th day (24 hours after the last dose) to determine theparasitemia level e body weight packed cellular volume

(PCV) and temperature of the mice were also recorded onthe 4th (before the first dose) and 7th days (24 hours after thelast dose)

29MeanSurvivalTime Mortality of each mouse in three ofthe models was monitored every day for a month and thenumber of days from inoculation of the parasite up to deathwas recorded to evaluate the effect of the crude extracts andsolvent fractions on survival time e Mean Survival Time(MST) for each mouse was calculated using the followingformula [8]

MST Sumof the survival time of mice in a groupdays

Total number of mice in that group

(1)

210 Parasitemia Level In all models thin smears of bloodfilm were made on coded microscope slides by taking bloodfrom the tail of each mouse e smears were fixed usingabsolute methanol and stained with 10 of Giemsa stain for15 minutes [24]e slides were then washed using tap waterand dried at room temperature e numbers of parasitizedred blood cells were examined under a light microscopeusing an oil immersion objective of times100 magnificationAfter microscopic evaluation of the parasitized red bloodcells the percentages of parasitemia and parasitemia sup-pression were calculated [8]

Parasitemia Number of parasitized red bloodcells

Total number of red bloodcells

times 100

Suppression A minus B

A1113890 1113891 times 100

(2)

where A is the mean percentage of the parasitemia level ofthe mice in negative controls while B is the mean parasitemialevel of mice in the treatment groups

211 Packed Cell Volume Blood samples were collectedfrom the tail of each mouse using heparinized capillary tubesbefore and after treatment of the crude extract and fractionsand PCV was measured e capillary tubes were filled withblood and sealed and centrifuged for 5minutes at 10000gPCV was then calculated using a microhematocrit readerthat takes the following formula into consideration [28]

PCV Volume of erythrocytes in a given volume of blood

Total blood volume

times 100

(3)

2111 Determination of Body Weight and TemperatureChange Mean percentage changes in body weight and rectal

Table 1 Animal grouping and dosing

GroupsDose (mgkg)

Crude extract FractionsI 10mlkglowast 10mlkglowastlowastII 25lowastlowastlowast 25lowastlowastlowastII 100 100IV 200 200V 400 400lowastDistilled water lowastlowast3 tween 80 in the fractions lowastlowastlowastChloroquine phosphate

Evidence-Based Complementary and Alternative Medicine 3

temperature before and after treatment were measured andrecorded [12 22]

212 Phytochemical Screening e presence of differentsecondary metabolites such as flavonoids alkaloids terpe-noids tannins saponins and steroids in the crude extractand solvent fractions of S macrosolen root was testedaccording to previously established protocols [29]

213 DataManagement andAnalysis e data were enteredinto Statistical Package for Social Sciences (SPSS) version 23e results were summarized as meanplusmn standard error ofmean (SEM) One-way analysis of variance (ANOVA) fol-lowed by Tukeyrsquos post hoc test was used to compare dif-ferences in mean among the groups A P value of less than005 was considered statistically significant

3 Results

31 Percentage Yield of the Plant Material e percentageyield obtained from the S macrosolen crude extract was 28ww with an actual yield of 420mg e highest percentageyield of the solvent fraction was obtained from the aqueousfraction (9370) while the lowest yield was from thechloroform fraction (125) (Table 2)

32 Acute Toxicity Study e acute toxicity study revealedthe safety of the crude extract and solvent fractions at a doseof 2000mgkg No gross behavioral and physical change andany sign of toxicity such as appetite loss lacrimationtremors salivation hair erection and diarrhea wereobserved

33 Antimalarial Studies

331 Effect of the Crude Extract and Solvent Fractions ofS macrosolen on the 4-Day Suppressive Test Mice treatedwith 200mgkg and 400mgkg doses of the crude extractshowed a significant (Plt 001 at 200mgkg and Plt 0001 at400mgkg) level of parasite suppression compared to thenegative control (Table 3) But none of the doses of the extractcompletely cleared the parasite Mice treated with 400mgkgalso exhibited a significant parasite suppression compared tothe mice which received the lowest two doses (Plt 001)

In the solvent fractions all tested doses of the aqueousfraction (Plt 005 at 100 and Plt 001 at 200 and 400mgkg)400mgkg dose of the chloroform fraction and 200 and400mgkg doses of the ethyl acetate fraction showed sig-nificant (Plt 005) parasitemia suppression compared to thenegative control e highest percentage of chemo-suppression of the solvent fractions was observed in theaqueous fraction (4307) followed by the ethyl acetatefraction (426) and chloroform fraction (3838)

With regard to MST the crude extract at 200mgkg(Plt 005) and 400mgkg (Plt 001) significantly increasedthe survival time of mice In the solvent fractions micetreated with 200 and 400mgkg doses (Plt 001) of aqueous

chloroform and ethyl acetate fractions and standard drug(Plt 0001) significantly increased mean survival time whencompared to the negative control (Table 3)

e middle and highest doses of the crude extractresulted in lower body weight loss associated with infectioncompared to the negative control (Plt 0001) In the case ofthe fractions 200 and 400mgkg (Plt 005) doses of theaqueous fraction 200 (Plt 005) and 400 (Plt 0001) mgkgdoses of the chloroform fraction and 200 (plt 001) and 400(Plt 005) mgkg doses of the ethyl acetate fraction signif-icantly prevented infection-induced body weight losscompared to the negative control A significant weight in-crement was observed among chloroquine-treated groups(Plt 0001) compared to both the fraction- and vehicle-treated groups (Table 4)

As indicated in Table 4 analysis of percentage change inrectal temperature also revealed that mice treated with 200(Plt 005) and 400 (Plt 0001) mgkg doses of the crudeextract significantly prevented temperature reductioncompared to the untreated group In the solvent fractions200 (Plt 005) mgkg doses of the aqueous fraction as well asthe intermediate and highest doses (Plt 005) of the ethylacetate fraction attenuated infection-induced reduction inrectal temperature compared to their respective negativecontrol e effects are comparable with the standard drug

In case of PCV the 200 and 400mgkg doses of the crudeextract showed comparable activity with the standard drugin preventing reduction in PCV (Plt 0001) associated withmalarial infection (Figure 1) All groups treated with thesolvent fractions have also progressively improved PCVehighest prevention in PCV was observed in the highest doseof the ethyl acetate fraction (Figure 2)e effect produced bythe fractions is comparable with the standard drug

332 Effect of the Crude Extract of S macrosolen on theProphylactic Test e percentages of inhibitions of thecrude extract were 1248 2123 and 3102 in micetreated with 100 200 and 400mgkg doses respectively andonly the 400mgkg dose showed a significant (Plt 005) levelof parasite suppression compared to the negative controlHowever there was no significant difference in mean sur-vival time among the groups (Table 5)

e mean percentage change in body weight and rectaltemperature between D4 and D7 is summarized in Table 6Only the highest dose (400mgkg) significantly prevented theloss of body weight compared to the negative control(Plt 0001) e groups that received the different doses of thecrude extract and the standard drug had also significantly lowerreductions in temperature compared to the negative control

As indicated in Figure 3 all groups showed reduction inPCV But the middle and the highest doses of the crudeextract (Plt 005) and standard drug (Plt 001) significantlyprevented a reduction in PCV compared to the negativecontrol

333 Effect of the Crude Extract of S macrosolen on RanersquosTest In the curative test the crude extract of S Macrosolenhad a considerable parasite suppressive activity against


Table 4 Effect of the crude extract and solvent fractions of S macrosolen on body weight and rectal temperature of P berghei-infected micein the 4-day suppressive test

GroupsWeight (g) Temperature

D0 D4 change D0 D4 changeDW10mlkg 2917plusmn 060 2473plusmn 074 minus152 3635plusmn 018 3367plusmn 037 minus738CQ 25 2785plusmn 085 2825plusmn 079 150a3 3615plusmn 008 3607plusmn 015 minus023a3

SM100 2867plusmn 085 2548plusmn 072 minus1105b3 3623plusmn 022 3373plusmn 037 minus690b3

SM200 2945plusmn 079 2742plusmn 068 minus686a3b3 3635plusmn 009 3487plusmn 015 minus407a1b2

SM400 2933plusmn 061 2708plusmn 072 minus772a3b3 3575plusmn 031 3488plusmn 031 minus242a3

AF100 2813plusmn 079 2482plusmn 072 minus1179b2d1 3670plusmn 035 3498plusmn 039 minus467b1

AF200 2700plusmn 057 2493plusmn 065 minus768a2b2c1 3682plusmn 046 3568plusmn 047 minus308a1

AF400 2768plusmn 052 2510plusmn 054 minus934a2b2 3667plusmn 020 3512plusmn 051 minus421b1

TE10mlkg 2760plusmn 068 2490plusmn 068 minus978 3655plusmn 013 3445plusmn 040 minus575CQ25 2658plusmn 045 2833plusmn 043 655a3 3625plusmn 021 3640plusmn 017 041a3

CF100 2740plusmn 077 2527plusmn 093 minus779b3d1 3647plusmn 027 3502plusmn 015 minus398b1

CF200 2740plusmn 077 2620plusmn 083 minus438a1b3 3663plusmn 025 3512plusmn 029 minus414b2

CF400 2690plusmn 055 2618plusmn 056 minus265a3b3c1 3683plusmn 021 3575plusmn 040 minus294EF 100 2712plusmn 075 2565plusmn 094 minus550b3 3670plusmn 031 3515plusmn 033 minus422b2

EF200 2720plusmn 068 2652plusmn 094 minus259a2b3 3653plusmn 033 3597plusmn 033 minus155a2


Data are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water(negative control for the crude and AF fraction) SM Silene macrosolenD0 day 0 D4 day 4 a compared to the negative control b compared to the positive control c compared to 100mgkg of CF d compared to 400mgkgof CF AF aqueous fraction TE 2 Tween 80 (negative control for CF and EF fractions) CF chloroform fraction EF ethyl acetate fraction 1 Plt 0052 Plt 001 3 Plt 0001

Table 2 Percentage yield and physical properties of the crude extract and solvent fractions of S macrosolen

Extractfractions Nature of extractfractions Color of extractfractions Actual yield (g) yield (ww)Chloroform Viscous Brown 2 125Ethyl acetate Viscous Brown 4 25Aqueous Solid powder Whitish yellow 150 937Crude extract Solid powder Whitish yellow 420 28

Table 3 Effect of the crude extract and solvent fractions of S macrosolen on the parasite level parasite suppression and MSTof P berghei-infected mice in the 4-day suppressive test

Group Doses (mgkg) parasitemia (Mplusmn SEM) suppression Mean survival time (days)DW 10mlkg 5482plusmn 297 0 717plusmn 031CQ 25 000plusmn 000a3 100a3 3000plusmn 000a3SM 100 4697plusmn 097b3e2 1433b3e2 767plusmn 021SM 200 4278plusmn 244a2b3e1 2197a2b3e1 833plusmn 033a1SM 400 3332plusmn 208a3b3c2d1 3923a3b3c2d1 867plusmn 033a2AF 100 3848plusmn 566a1b3 2981a1b3 833plusmn 033b3AF 200 3263plusmn 323a2b3 4048a2b3 900plusmn 037a2b3AF 400 3121plusmn 402a2b3 4307a2b3 933plusmn 042a2b3

TE 10mlkg 4885plusmn 734 0 667plusmn 033CQ 25 029plusmn 012a3 9941a3 3000plusmn 000a3CF 100 4179plusmn 391b3 1446b3 800plusmn 052b3CF 200 3971plusmn 410b3 1872b3 817plusmn 040b3CF 400 3010plusmn 390a1b2 3838a1b2 850plusmn 034a1b3EF 100 3520plusmn 319b3 2795b3 850plusmn 034a1b3EF 200 2946plusmn 372a1b2 3970a1b2 867plusmn 042a2b3EF 400 2803plusmn 461a1b2 4261a1b2 900plusmn 052a2b3

Data are expressed as meanplusmn SEM (n 6) AF aqueous fraction CQ chloroquine DW distilled water (negative control for the crude extract and AFfraction) TE 2 Tween 80 (negative control for CF and EF fractions) CF chloroform fraction EF ethyl acetate fraction a compared to the negativecontrol b compared to the positive control c compared to 100mgkg crude extract d compared to 200mgkg crude extract e compared to 400mgkgcrude extract SM Silene macrosolen 1 Plt 005 2 Plt 001 3 Plt 0001


P berghei-infected mice (Table 7) Although it is lower thanthe positive control (Plt 0001) all the evaluated dose levelsshowed a significant activity in parasite suppression

compared to the negative control group ere was a sig-nificant difference in parasitemia suppression among400mgkg- (Plt 001) and 100mgkg-treated groups

D0D4

GroupsEF 400EF 200EF 100CF 400CF 200CF 100CQ 25TE 10

10

0

70

60

50

40

30

20

PCV

()

a2a2 a1 a1a2

Figure 2 Effect of chloroform and ethyl acetate fractions of the S macrosolen extract on PCV of P berghei-infected mice in the 4-daysuppressive test Data were expressed as meanplusmn SEM (n 6) CQ chloroquine TE 2 Tween 80 (negative control for CF and EFfractions) CF chloroform fraction EF ethyl acetate fraction D0 day 0 D4 day 4 a compared to the negative control 1 Plt 0052 Plt 001

Table 5 Effect of the crude extract of S macrosolen on the parasite level parasite suppression and mean survival time of P berghei-infectedmice in the prophylactic test

Group Doses (mgkg) parasitemia (Mplusmn SEM) suppression Mean survival time (days)DW 10mlkg 5571plusmn 114 0 667plusmn 033CQ 25 038plusmn 014a2 9932a2 2917plusmn 083a2SM 100 4875plusmn 489b2 1248b2 733plusmn 021b2SM 200 4388plusmn 559b2 2123b2 750plusmn 022b2SM 400 3843plusmn 393a1b2 3102a1b2 833plusmn 021b2

Data are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water SM Silene macrosolen a compared to the negative controlb compared to the positive control 1 Plt 005 2 Plt 0001

70

60

50

40

30

D0D4

20

10

0DW 10 CQ 25 SM 100 SM 200 SM 400 AF 100 AF 200 AF 400

a2a1a1 a1

a1a2 a2

Figure 1 Effect of the crude extract and aqueous fraction of S macrosolen on PCV of P berghei-infected mice in the 4-day suppressive testData are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water AF aqueous fraction SM Silene macrosolenD0 day 0 D4 day a compared to the negative control 1 Plt 005 2 Plt 0001


As indicated in Table 7 the two higher doses of thecrude extract exhibited a significant increment in survivaltime as compared to the negative control (Plt 001) e200 and 400mgkg (Plt 005) doses of the crude extractalso significantly prevented body weight loss and tem-perature reduction as compared to the negative control(Table 8)

As indicated in Figure 4 PCV determination in thecurative test revealed that 100 (Plt 001) 200 and 400mgkg(Plt 0001) doses of the crude extract and chloroquine(Plt 0001) significantly prevented the reduction in PCVcompared to the negative control

34 Phytochemical Screening Result of phytochemicalscreening revealed that the crude extract and solvent frac-tions of S macrosolen contain secondary metabolites such asalkaloids saponins phenols terpenoids and flavonoidsHowever tannins and anthraquinones were absent from thecrude extract and solvent fractions of the plant

4 Discussion

Developing new drugs is absolutely critical to fight againstthe challenge in malaria treatment Herbal products have anundeniable situation in the development of antimalarial

a1 a170605040302010

0

D4D7

DW10 CQ25 SM100 SM200 SM400Group

PCV

()

a2

Figure 3 Effect of the crude extract of S macrosolen on PCV of P berghei-infected mice in the prophylactic test Data are expressed asmeanplusmn SEM (n 6) CQ chloroquine DW distilled water SM Silene macrosolen D4 day 4 D7 day 7 a compared to the negativecontrol 1 Plt 005 2 Plt 001

Table 7 Effect of the crude extract of S macrosolen on the parasite level parasite suppression and mean survival time of P berghei-infectedmice in Ranersquos test

Group Dose (mgkg) parasitemia (meanplusmn SEM)

suppression Mean survival time (days)D3 D7

DW 10mlkg 4989plusmn 184 5665plusmn 319 0 617plusmn 031CQ 25 4859plusmn 225 003plusmn 001a3 9995a3 3000plusmn 000a3SM 100 5043plusmn 329 4520plusmn 275a2b3d1 2023a2b3 683plusmn 031b3SM 200 4707plusmn 149 4341plusmn 117a2b3 2338a2b3 750plusmn 022a2b3SM 400 4783plusmn 099 3636plusmn 17a3b3c1 3582a3b3c1 767plusmn 021a2b3

Data are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water D3 day 3 D7 day7 SM Silene macrosolen a compared to thenegative control b compared to the positive control c compared to 100mgkg crude extract d compared to 400mgkg crude extract 1 Plt 0052 Plt 001 3 Plt 0001

Table 6 Effect of the crude extract of S macrosolen on body weight and rectal temperature of P berghei-infected mice in the prophylactictest


D4 D7 changes D4 D7 changesDW10mlkg 2827plusmn 049 2433plusmn 030 minus1389 3597plusmn 016 3307plusmn 023 minus806CQ25 2928plusmn 072 2852plusmn 072 minus252a2 3623plusmn 008 3570plusmn 018 minus147a2



SM400 2785plusmn 071 2578plusmn 057 minus735a2b1 3648plusmn 014 3437plusmn 028 minus580b2

Data are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water SM Silene macrosolen D4 day 4 D7 day7 a compared to thenegative control b compared to the positive control 1 Plt 005 2 Plt 0001


chemotherapeutic drugs Along these lines plants areconsidered as a solid hotspot for the advancement of futurecompelling antimalarial operators

In the acute oral toxicity test in this study any sign oftoxicity and mortality was not observed is indicates thatthe lethal dose (LD50) of the plant extract could be higherthan 2000mgkg body weight as per the OECD guidelineNo 425 [21]

Even though P berghei is not exactly similar to that of thehuman Plasmodium parasites it is the first step to screen invivo antimalarial activities of test substances Moreoverseveral conventional antimalarial drugs have been identifiedusing this rodentmalaria parasite Chloroquine was used as areference drug in the present study because the parasite usedin this study is chloroquine sensitive [24 28]

Models such as the 4-day suppressive test prophylactictest and Ranersquos test were used to investigate the antimalarialeffect of S macrosolen In all these three models the de-termination of percentage inhibition of the parasite is themost reliable parameter [13 24]e efficacy of a compoundis assessed by comparison of the parasitemia level and MSTbetween treated and untreated mice [28] PCV body weightchange and change in body temperature are the otherparameters used An ideal antimalarial agent is expected toprevent the reduction in PCV bodyweight loss and

reduction in rectal temperature in infected mice due to therise in parasitemia [3]

As it can be seen from the results the two higher doses ofS macrosolen in the crude extract significantly (Plt 001 at200mgkg and Plt 0001 at 400mgkg) suppressed theparasitemia level compared to the negative control in the 4-day suppressive test e 3923 parasite suppressionexerted by 400mgkg in this test is comparable with anotherstudy [30] which has shown 4073 parasitemia suppres-sion is shows the potential of the plant extract to preventthe increment in the parasite load in the blood e anti-plasmodial activity seen might be also due to the immu-nomodulatory effect of the plant extract [28 31]

In the prophylactic test only the 400mgkg dose showeda significant parasite suppressive activity (Plt 005) againstP berghei infection is might be due to the accumulationof the active constituent in the highest dose Similarly thecrude extract also exerted a significant parasite suppressionin Ranersquos test Ranersquos test is a standard test commonly usedfor antimalarial screening It relies on the ability of thestandard inoculum of P berghei to kill the recipient mousethereafter

In this model the highest percentage of suppression was3582 at the 400mgkg (Plt 0001) dose level is iscomparable with another study [23] which has shown

a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

Figure 4 e effect of the crude extract of S macrosolen on PCV of P berghei-infected mice in Ranersquos test Data are expressed asmeanplusmn SEM (n 6) CQ chloroquine DW distilled water SM Silene macrosolen D3 day 3 D7 day 7 a compared to the negativecontrol 1 Plt 001 2 Plt 0001

Table 8 Effect of the crude extract of S macrosolen on body weight and rectal temperature of P berghei-infected mice in Ranersquos test



SM100 2783plusmn 054 2528plusmn 048 minus915b1 3502plusmn 026 3352plusmn 033 minus426SM200 2712plusmn 059 2515plusmn 071 minus727a1 3525plusmn 018 3410plusmn 016 minus326a1

SM400 2677plusmn 082 2473plusmn 091 minus766a1 3525plusmn 019 3428plusmn 029 minus273a1

Data are expressed as meanplusmn SEM (n 6) CQ chloroquine DW distilled water SM Silene macrosolen D3 day 3 D7 day 7 a compared to thenegative control b compared to the positive control 1 Plt 005 2 Plt 001 3 Plt 0001


3579 parasite suppression e parasitemia suppressiveeffect of the crude extract in all the three models increasedwith doses is might be due to the higher concentration ofthe active secondary metabolites in higher doses [13]

As discussed above the crude extract exhibited thehighest chemosuppressive effect in the 4-day suppressive testwith the lowest effect in the prophylactic test e lowerchemosuppressive effect in the prophylactic test might bedue to the rapid clearance of the active component since itwas given before infection at which the metabolic rate of themice is active [32] is finding is similar to other studies inwhich 4-day suppressive tests have a higher chemo-suppressive effect than curative tests and prophylactic tests[22 25]

e antimalarial effect of S macrosolen seen may be dueto the presence of alkaloids saponins phenols terpenoidsand flavonoids is is in agreement with other works inwhich the antiplasmodial activity of alkaloids [17] saponins[13] phenols [27] terpenoids [31] and flavonoids [33] hasbeen reported Phenolic compounds flavonoids alkaloidsand terpenoids have an antioxidant or free-radical scav-enging effect so these phytochemicals can prevent oxidativestress induced by the parasite [34] Antioxidants can alsoinhibit heme polymerization and unpolymerised heme istoxic for the parasite [35] e proved immunomodulatoryeffect of terpenoids and flavonoids also might have animpact on the host-parasite interrelationship [34]

e three fractions of S macrosolen also showed aparasite-suppressive effect with varying degree of suppres-sion e aqueous fraction (4307) and ethyl acetatefractions (4261) were found to possess higher bloodschizontocidal activity than the chloroform fraction(3838) is could be attributed to the high concentrationof phytochemicals in the aqueous fraction and the leastnumber of phytochemicals in the chloroform fraction eresults are similar to former studies in which the aqueousfraction had higher activity than chloroform and ethyl ac-etate fractions [2 31]

In vivo antiplasmodial activity can be classified asmoderate good and very good if an extract displayed apercent parasite suppression equal to or greater than 50 ata dose of 400 200 and 100mgkg respectively [13 34] edetermination of percentage inhibition of parasitemia is themost reliable parameter in antimalarial screeningereforeit is clear from the result (Table 3) that the P berghei-infectedmice treated with the crude extract of S macrosolen reducedin percentage parasitemia compared to those of the un-treated control animals

Based on this classification the crude extract and solventfraction showed moderate antimalarial activity Further-more a test substance is considered as active when it showspercentage suppression of parasitemia greater than 30 andprolongs survival time of infected mice [17 22] is resulthas supports of our study

Regarding MST the crude extract in the 4-day sup-pressive and curative tests increased survival time of micesignificantly (Plt 001) as compared to the negative controlOn the other hand the survival time did not increase sig-nificantly in the prophylactic test e difference in survival

time in the three models could be due to the differences inparasitemia inhibition [13 36] Similarly the three fractionsof S macrosolen also increased the survival time of micesignificantly compared to the negative control Mice treatedwith the aqueous fraction lived a longer time followed byethyl acetate and chloroform fractions is might be due tothe localization of active metabolites in the aqueous fraction[31] Prolongation of the MST of mice indicates that thecrude extract and solvent fractions suppressed P berghei andreduced the pathologic effect of the parasite [37]

However the crude extract in the three models and thesolvent fractions in a 4-day suppressive test significantlyattenuated body weight loss compared to their respectivenegative control e improvement in body weight might bedue to the parasite suppressive effect of the extract andsolvent fractions It might be also due to the effect of thecrude extract and solvent fractions on the other aspects ofmalaria illness such as PCV and rectal temperature [23 38]

Hypothermia is one of the manifestations of P berghei-infected mice e mechanism for fall in temperature mayinclude the general debilitating effect of the infection such asanemia on heat production andor heat conservation insmall animals Hypothermia in Plasmodium berghei-infectedmice is also associated with increased turnover in brainserotonin the neurotransmitter which reduces both foodintake and body temperature in rodents [39]

Analysis of percentage change in rectal temperature inour study revealed that the mice treated with the crudeextract and solvent fractions except in the prophylactic testand the chloroform fraction showed a significant preventionin the reduction of rectal temperature compared to theirrespective negative controls e improvement in rectaltemperature is probably due to a significant parasite-sup-pressive effect of an improvement in pathological conditionsassociated with parasite infection that causes a reduction inbody temperature [13] e observed antimalarial activitywas dose dependent but at a dose of 400mgkg body weighta reduction in parasitemia was recorded with 3582 percentchemosuppression on day 8 (Table 7) in Ranersquos test

is may be due to the immunosuppressive activity ofthe extract It had been reported that oral administration ofphytochemicals such as saponin tannins and phenolspossessed the ability to suppress cellular immunity [40]

Malaria-induced reduction in PCV is another mani-festation of P berghei-infected mice It is caused by RBCdestruction as a result of parasite multiplication reducederythropoiesis and dyserythropoiesis [23] PCV was mea-sured to estimate the efficacy of the crude extract and solventfractions in averting hemolysis due to rising parasitemialevels [34] Analysis of the mean percentage change in PCVin this study revealed that the crude extract and solventfractions significantly prevented a reduction in PCV ascompared to their respective negative control e relativereversal of the reduction in PCV might be due to the sig-nificant reduction in the parasite level of infected mice andor prevention in hemolysis [13 41] In both the crude extractand solvent fractions the highest improvement in PCV wasobserved in the higher doses From the three models thelowest reduction in PCV was seen in the 4-day suppressive


test followed by curative test and prophylactic test iseffect in PCV agreed with the results of parasite suppression[12]

e potential of plant-derived natural products forantimalarial drug discovery has been examined in severalreview papers [42ndash44] Previous studies have shown thatplant-derived alkaloids have a great potential for antima-larial drug development [42ndash44] Triterpenoid and steroidsaponins have been found to be detrimental to several in-fectious protozoans such as Plasmodium falciparum [45]eantimalaria activity of S macrosolen might be due to thepresence of one of these constituents based on phyto-chemical analysis of the root of S macrosolen e resultsfrom our studies support the scientific credential to thefolkloric use of the root extract of S macrosolen for thetreatment of malaria in Ethiopia

5 Conclusions

e results of the present study confirmed that S macrosolenis safe and possesses moderate antimalarial activity esolvent fractions also showed an antimalarial activity withvarious degrees of chemosuppression with an aqueousfraction being relatively effective Our results support thetraditional use of plants in the treatment of malaria bytraditional users in Ethiopia In the future subacute andchronic toxicity tests should be conducted to check its long-term safety profile It is also important to identify and isolateactive constituents that explain the observed antimalarialactivity on the root of S macrosolen

Data Availability

All the data used to support findings of this study areavailable with the corresponding author when requests comefrom concerned bodies

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this research paper

Authorsrsquo Contributions

GH and AKG designed the study analyzed the data pre-pared the first draft and acquired the fund GH conductedthe study and AKG and BR supervised the study All authorsread and approved the final manuscript

Acknowledgments

is study was supported with the Mekelle University small-scale grant with registration number CRPOCHSsmallyoungrec0122011 e authors gratefully acknowledgeAdigrat University and Mekelle University for allowing touse their laboratory facilities Addis Pharmaceutical Factory(APF) for providing the standard drug and the EthiopianPublic Health Institute (EPHI) for giving the parasite-in-fected mice

References

[1] WHO World Malaria Report World Health OrganizationGeneva Switzerland 2013

[2] S Asnake T Teklehaymanot A Hymete B Erko andM Giday ldquoEvaluation of the antiplasmodial properties ofselected plants in southern Ethiopiardquo BMC Complementaryand Alternative Medicine vol 15 pp 1ndash12 2015


[4] S Borgella N Fievet B T Huynh et al ldquoImpact of preg-nancy- associated malaria on infant malaria infection insouthern Beninrdquo PLoS One vol 8 no 11 Article ID e806242013

[5] Z Petros ldquoe need of standardized herbal remedies as al-ternate sources of antimalarial products in Ethiopiamdashupdatedreviewrdquo Pharmacology Online vol 3 pp 1440ndash1447 2011

[6] A P Phyo and L V Seidlein ldquoChallenges to replace ACT asfirst-line drugrdquo BMC Malaria Journal vol 16 p 291 2017

[7] E M Bobasa B G Alemu S T Berkessa et al ldquoAntimalarialactivity of selected Ethiopian medicinal plants in micerdquoJournal of Pharmacy amp Pharmacognosy Research vol 6pp 57ndash64 2018

[8] M Rudrapal and D Chetia ldquoPlant flavonoids as potentialsource of future antimalarial leadsrdquo Systematic Reviews inPharmacy vol 8 no 1 pp 13ndash18 2016

[9] Z Getnet S Chandrodyam and G Masresha ldquoStudies ontraditional medicinal plants in ambagiorgis area of Wogeradistrict Amhara regional state Ethiopiardquo InternationalJournal of Pure amp Applied Bioscience vol 4 pp 38ndash45 2016

[10] J W Gathirwa G M Rukunga P G Mwitari et al ldquoTra-ditional herbal antimalarial therapy in Kilifi district KenyardquoJournal of Ethnopharmacology vol 134 no 2 pp 434ndash4422011

[11] L Cui and X-Z Su ldquoDiscovery mechanisms of action andcombination therapy of artemisininrdquo Expert Review of Anti-infective Cerapy vol 7 no 8 pp 999ndash1013 2009

[12] W Y Belay A Endale Gurmu and Z B Wubneh ldquoAnti-malarial activity of stem bark of periploca linearifolia duringearly and established plasmodium infection in micerdquo Evi-dence-Based Complementary and Alternative Medicinevol 2018 Article ID 4169397 7 pages 2018

[13] L Bantie S Assefa T Teklehaimanot and E Engidawork ldquoInvivo antimalarial activity of the crude leaf extract and solventfractions of Croton macrostachyus Hocsht (Euphorbiaceae)against Plasmodium berghei in micerdquo BMC Complementaryand Alternative Medicine vol 14 pp 1ndash10 2014

[14] N Kucukboyaci B Ozcelik N Adiguzel and A C GorenldquoFatty-acid compositions of Silene vulgaris and S csereisubsp aeoniopsis seeds and their antimicrobial activitiesrdquoChemistry of Natural Compounds vol 46 no 1 pp 88ndash912010

[15] Y S Birhan S L Kitaw Y A Alemayehu andM N Mengesha ldquoEthnobotanical study of medicinal plantsused to treat human diseases in enarjenawga district eastGojjam zone Amhara region Ethiopiardquo Journal of MedicinalPlants Studies vol 1 pp 1ndash20 2017

[16] A Teklay B Abera and M Giday ldquoAn ethnobotanical studyof medicinal plants used in Kilte Awulaelo district Tigrayregion of Ethiopiardquo Journal of Ethnobiology and Ethno-medicine vol 9 no 1 p 65 2013

[17] L B Animut ldquoUse of epidemiological and entomologicaltools in the control and elimination of malaria in EthiopiardquoMalaria Journal vol 17 pp 1ndash8 2018


[18] H Yineger E Kelbessa T Bekele and E Lulekal ldquoEthno-veterinary medicinal plants at bale mountains national parkEthiopiardquo Journal of Ethnopharmacology vol 112 no 1pp 55ndash70 2007

[19] S S Lulekal S P S Khanuja G Longo and D D RakeshExtraction Technologies for Medicinal and Aromatic Plants 1stedition Italy United Nations Industrial Development Or-ganization and the International Centre for Science and HighTechnology Vienna Austria 2008

[20] J Abdela E Engidawork and W Shibeshi ldquoIn vivo anti-malarial activity of solvent fractions of the leaves of Justiciaschimperiana hochst ex nees against Plasmodium bergheiinmicerdquo Ethiopian Pharmaceutical Journal vol 30 no 2pp 95ndash108 2014

[21] OECD Test No 425 Acute Oral Toxicity Up-and-DownProcedure OECD Guidelines for the Testing of ChemicalsSection 4 OECD Publishing Paris France 2008

[22] A Krettli J Adebayo and L Krettli ldquoTesting of naturalproducts and synthetic molecules aiming at new antimalar-ialsrdquo Current Drug Targets vol 10 no 3 pp 261ndash270 2009

[23] B Mengiste E Makonnen and K Urga ldquoInvivo antimalarialactivity of dodonaea angustifolia seed extracts against plas-modium berghei in mice modelrdquo Momona Ethiopian Journalof Science vol 4 no 1 pp 47ndash63 2012

[24] D A Fidock P J Rosenthal S L Croft R Brun andS Nwaka ldquoAntimalarial drug discovery efficacy models forcompound screeningrdquo Nature Reviews Drug Discovery vol 3no 6 pp 509ndash520 2004

[25] D Nureye S Assefa T Nedi and E Engidawork ldquoIn vivoantimalarial activity of the 80 methanolic root bark extractand solvent fractions of gardenia ternifolia Schumach amponn (Rubiaceae) against plasmodium bergheirdquo Evidence-Based Complementary and Alternative Medicine vol 2018Article ID 9217835 11 pages 2018

[26] W Peters J H Portus and B L Robinson ldquoe chemo-therapy of rodent malaria XXIIrdquo Annals of Tropical Medicineamp Parasitology vol 69 no 2 pp 155ndash171 1975

[27] J F Ryley and W Peters ldquoe antimalarial activity of somequinolone estersrdquo Annals of Tropical Medicine amp Parasitologyvol 64 no 2 pp 209ndash222 1970

[28] A Toma EA DeynoS and A F Mechesso ldquoIn vivo anti-malarial activity of solvent fractions of Echinopskeberichoroots against Plasmodium berghei infected micerdquo EC Mi-crobiology vol 12 pp 204ndash212 2017

[29] G E Trease and W C Evans Pharmacognosy BailliereTindall London UK 13th edition 1989

[30] D Dikasso E Makonnen A Debella et al ldquoIn vivo anti-malarial activity of hydroalcoholic extracts from Asparagusafricanus Lam in mice infected with Plasmodium bergheirdquoEthiopian Journal of Health Development vol 20 pp 112ndash1182006

[31] A Kefe M Giday and H Mamo ldquoAntimalarial properties ofcrude extracts of seeds of Bruceaantidysenterica and leaves ofOcimumlamiifoliumrdquo BMC Complementary and AlternativeMedicine vol 16 pp 1ndash8 2016

[32] A B Muluye E Melese and G M Adinew ldquoAntimalarialactivity of 80 methanolic extract of Brassica nigra (L) Koch(Brassicaceae) seeds against Plasmodium berghei infection inmicerdquo BMC Complementary and Alternative Medicinevol 15 pp 1ndash8 2015

[33] B Regasa ldquoMagnitude of malaria infection in EthiopiardquoGlobal Journal of Medical Research vol 14 pp 17ndash21 2014

[34] M Adugna T Feyera and A Taddese ldquoIn vivo antimalarialactivity of crude extract of aerial part of artemisia abyssinica

against plasmodium berghei in micerdquo Global Journal ofPharmacology vol 8 pp 460ndash468 2014

[35] S E Lindner J L Miller and S H I Kappe ldquoMalaria parasitepre-erythrocytic infection preparation meets opportunityrdquoCellular Microbiology vol 14 no 3 pp 316ndash324 2012

[36] N Mohammed M Abdulwuhab and F Mohammed ldquoAn-timalarial activity of crude extract of buddleja polystachyafresen (buddlejacea) against plasmodium berghei in micerdquoIOSR Journal of Pharmacy and Biological Sciences vol 11no 5 pp 27ndash35 2016

[37] R Basir S F Rahiman K Hasballah et al ldquoPlasmodiumberghei ANKA infection in ICR mice as a model of cerebralmalariardquo Iranian Journal of Parasitology vol 7 no 4pp 62ndash74 2012

[38] M Eyasu and W Shibeshi ldquoIn vivo antimalarial activity ofhydromethanolic leaf extract of Calpurnia aurea (Fabaceae) inmice infected with chloroquine sensitive Plasmodium ber-gheirdquo International Journal of Pharmacology vol 2 pp 131ndash142 2013

[39] M J Dascombe and J Y Sidara ldquoe absence of fever in ratmalaria is associated with increased turnover of 5-hydroxy-tryptamine in the brainrdquo in Temperature RegulationA S Milton Ed pp 47ndash52 Advances in PharmacologicalSciences Birkhauser Basel Switzerland 1994

[40] M Sankari V Chitra R Jubilee P Silambu Janaki andD Raju ldquoImmunosuppressive activity of aqueous extract ofLagenaria sicerarian (standley) in micerdquo Scholars ResearchLibrary vol 2 pp 291ndash296 2010

[41] W H Tesfaye ldquoIn vivo antimalarial activity of the crudeextract and solvent fractions of the leaves of Zehenriascabra(Cucurbitaceae) against Plasmodium bergheiin micerdquo Journalof Medicinal Plant Research vol 8 pp 1230ndash1236 2014

[42] S Bhatnagar and P Das ldquoAntimalarial activity in tropicalplants a reviewrdquo Journal of Herbs Spices amp Medicinal Plantsvol 13 no 1 pp 103ndash132 2007

[43] S Schwikkard and F R Van Heerden ldquoAntimalarial activityof plant metabolites electronic supplementary information(ESI) available IC50 values of plant metabolitesrdquo NaturalProduct Reports vol 19 no 6 pp 675ndash692 2002

[44] A B Oliviera M F Dolabela F C Braga R L R P JacomeF P Varotti and M M Povoa ldquoPlant derived antimalarialagents new leads and efficient phytomedicines PartI Alkaloidsrdquo Anais da Academia Brasileira de Cienciasvol 81 pp 715ndash740 2009

[45] F Delmas C Di Giorgio R Elias et al ldquoAntileishmanialactivity of three saponins isolated from ivy α-hederinβ-hederin and hederacolchiside A1 as compared to theiraction on mammalian cells cultured in vitrordquo Planta Medicavol 66 no 4 pp 343ndash347 2000


several reported pharmacological activities including anti-microbial antitumor phagocytic antiviral antifungal andantioxidant activities [14]

Silene macrosolen known by its vernacular name saer-osaero (Tigrigna) and wogert (Amharic) is an herbal plantbelonging to the Genus Silene that grows in different areas ofEthiopia [15] Traditionally it is used for treatment ofmalaria in Ethiopia [15 16] S macrosolen is also used as amosquito repellent and for treatment of an evil eye [17]hepatitis [18] and hemorrhoids [15] However pharma-cological activity against malaria is not scientifically sup-ported Hence the aim of this study was to evaluate in vivoantimalarial activity of the crude extract and solvent frac-tions of S macrosolen using 4-day suppressive prophylacticand curative tests in Plasmodium-berghei-infected mice

2 Materials and Methods

21 Chemicals Reagents and Drugs Absolute methanol(Loba Chemic Pvt Ltd India) chloroform (Nice ChemicalsPvt Ltd India) ethyl acetate (Research-Lab Fine ChemIndustries India) Tween 80 (Loba Chemic Pvt Ltd India)microscopic oil immersion (Laboratory reagents Ethiopia)Geimsa stain (Addis Ababa Ethiopia) trisodium citrate(Loba Chemic Pvt Ltd India) and chloroquine phosphate(APF Ethiopia) were used in this study and the chemicalsand reagents were laboratory or analytical grade

22 Plant Material Collection and Authentication Freshroots of S macrosolen were collected from Kilte AwulaeloDistrict Tigray Ethiopia located 825 km north of AddisAbaba e collected plant material was identified by abotanist (name) and the specimen was deposited at theBiology Department Gondar University with vouchernumber 001GHA2019

23 Extraction and Fractionations of Plant Material ecollected root of the plant was cleaned with tap water and airdried under shade at room temperature before it waspowdered using an electric mill Extraction of the powderedroot was carried by the maceration technique Specifically500 grams of the powdered root was mixed with 2500ml of80methanol that corresponded to one ratio five of betweenthe weight of the powder and solvent proportion emaceration continued for three days with intermediateshaking e macerate was filtered using muslin cloth andWhatman filter paper (No 1) consecutively while the marcwas remacerated twice with 80 methanol using the sameprocedure [19]e combined filtrate was dried in an oven ataround 400C

For fractionization the dried crude extract (160mg) wasdissolved in distilled water and partitioned three times withan equal volume of chloroform e chloroform layer wascollected in a beaker and dried in the oven at around 40degCafter combining together while the aqueous layer wasfurther shaken using equal volume of ethyl acetate threetimes following the same procedure as fractionation withchloroform Finally the aqueous fraction was taken and

dried in an oven at around 40degC e crude extract and thefractions were stored in the refrigerator throughout theexperiment

24 Experimental Animals and Parasite Healthy Swiss al-bino mice of both sexes aged 6 to 8 weeks obtained from theDepartment of Pharmacology and Toxicology School ofPharmacy Mekelle University were used to test the anti-malarial activity of both the crude extract and solventfractions e mice were acclimatized for seven days prior tothe experiments and housed in standard plastic cages withexposure to 12 h lightdark cycle and room temperatureemice had free access to standard pellet and water ad libitumthroughout the experiment e mice were handledaccording to the internationally accepted laboratory animaluse and care guideline [20] e experimental animal studieswere approved by the Institutional Review Committee of theCollege of Health Sciences Mekelle University (ERC15442018) before the actual commencement of the study

Chloroquine-sensitive P berghei ANKA strain obtainedfrom Ethiopian Public Health Institute (EPHI) Addis AbabaUniversity was used to infect the mice e parasites weremaintained by passage of blood from infected to normalmice on a weekly basis

25 Acute Oral Toxicity Test An acute oral toxicity test wasconducted in accordance to the Organization for EconomicCo-operation and Development (OECD) 425 guidelines[21] Accordingly healthy and nonpregnant 5 female micewere used and the first mouse was given a limited dose of2000mgkg orally with an oral gavage after it was restrictedfrom food access for 4 hoursemouse was observed for 24hours for any signs of toxicity and mortality Afterward theremaining 4 mice were treated with the same dose andobserved strictly for the first one hour and intermittently forthe subsequent three hours for any signs of toxicity likediarrhea loss of appetite hair erection lacrimation con-vulsions salivation lethargy and paralysis Follow-up wascontinued once daily for 14 days

26 Grouping and Dosing In each of the test models (4-daysuppressive test prophylactic test and curative test) thirtymice were divided randomly into five groups consisting ofsix mice

e groups were categorized as groups I (treated withvehicle) and II (treated with chloroquine phosphate) ethird fourth and fifth groups (group IIIndashV) were treatedwith three different oral doses of the crude extract (Table 1)or solvent fractions

27 Inoculation of Parasite Mice with a parasitemia level ofapproximately 30 were used as a donor of chloroquine-sensitive strain of P berghei ANKA throughout the exper-iment [22] e donor mice were sacrificed by decapitationand blood was taken by severing the jugular vein [23] Bloodwas collected from all the donormice and it was diluted with09 normal saline to obtain uniform 5times107 P berghei-











(1)




times 100


A1113890 1113891 times 100

(2)




Total blood volume

times 100

(3)



GroupsDose (mgkg)






3 Results









































D0D4


10

0

70

60

50

40

30

20

PCV

()

a2a2 a1 a1a2





70

60

50

40

30

D0D4

20

10


a2a1a1 a1

a1a2 a2






4 Discussion


a1 a170605040302010

0

D4D7


PCV

()

a2























a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References


























































(1)




times 100


A1113890 1113891 times 100

(2)




Total blood volume

times 100

(3)



GroupsDose (mgkg)






3 Results









































D0D4


10

0

70

60

50

40

30

20

PCV

()

a2a2 a1 a1a2





70

60

50

40

30

D0D4

20

10


a2a1a1 a1

a1a2 a2






4 Discussion


a1 a170605040302010

0

D4D7


PCV

()

a2























a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References




















































3 Results









































D0D4


10

0

70

60

50

40

30

20

PCV

()

a2a2 a1 a1a2





70

60

50

40

30

D0D4

20

10


a2a1a1 a1

a1a2 a2






4 Discussion


a1 a170605040302010

0

D4D7


PCV

()

a2























a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References










































































D0D4


10

0

70

60

50

40

30

20

PCV

()

a2a2 a1 a1a2





70

60

50

40

30

D0D4

20

10


a2a1a1 a1

a1a2 a2






4 Discussion


a1 a170605040302010

0

D4D7


PCV

()

a2























a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References




















































4 Discussion


a1 a170605040302010

0

D4D7


PCV

()

a2























a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References

























































a1 a2 a2

70

60

50

40

30

20

D3D7

10

0DW 10 CQ 25 SM 100 SM 200 SM 400

Groups

PCV

()

























5 Conclusions


Data Availability






Acknowledgments


References

































































5 Conclusions


Data Availability






Acknowledgments


References

















































antimalarial effect of the root of silenemacrosolen a

Documents