Antimalarial and Antioxidant Potentials of Extract and Fractions of Aerial Part of Borreria ocymoides DC ( Rubiaceae ) .

Introduction: Borreria ocymoides (Burm F) DC is a weak, erect and decumbent herb that has several folkloric, ethno medicinal uses and is included in antimalarial preparations by some traditional healers. It is also used in treatment of ring worm, eczema and microbial infections. Objectives: To evaluate antimalarial activity of extract and fractions of Borreria ocymoides in Plasmodium berghei infected mice and to investigate their antioxidant activity using 1, 1-diphenyl-2-picryl-hydrazile (DPPH). Methods: The methanol extract of aerial part of B. ocymoides and the solvent fractions obtained from partition between organic solvents were assessed for antimalarial activity against chloroquine sensitive Plasmodium berghei NK65 infected mice using the suppressive and curative test procedures. Chloroquine (10 mg/ml) was used as positive control. The antioxidant activity was evaluated using DPPH radical scavenging ability and determination of total phenolic content. Results: The crude extract (250 and 500 mg kg-1) produced a dose dependent anti-plasmodial activity in the suppressive and curative tests. The chemo suppression activity was best in the ethyl acetate fraction (87.31%) and in the order ethyl acetate >dichloromethane > hexane > aqueous fraction. The DPPH radical scavenging activity of the extract increased with concentration. The antioxidant activity was less than ascorbic acid used as positive control. Oral administration up to 5 g/kg produced no noticeable deleterious effect 24 hours after dosing and up to 7 days afterwards. Conclusion: The results indicated that the extract has a potent anti-plasmodial activity against Plasmodium berghei and the activity seems to reside in the mid-polar fractions. Thus, the plant is a potential source of new antimalarial agents.

] Minimizing oxidative stress could therefore ameliorate many physical ailments and help prevent certain degenerative diseases caused by free radicals. 18Antimalarial agents with both antimalarial and antioxidant activities will be an advantage in malarial treatment or prevention. 19rreria ocymoides (Burm F) DC is a weak, erect and decumbent herb and belongs to the family Rubiaceae. 20The plant has several folkloric and ethno medicinal uses which include dermatological use in treatment of ring worm and eczema in Nigeria, antihelmintic treatment of internal worms, treatment of microbial infections (dysentery, diarrhoea, gonorrhea), aids healing of wounds, 21 gastric ulcer protective potency 22.The plant was also mentioned by some traditional healers in treatment of malaria.The aim of the present study is to determine anti-plasmodial and antioxidant activities of methanol extract of aerial part of Borreria ocymoides and its solvent fractions using mice infected with chloroquine sensitive Plasmodium berghei.

Plant material
The aerial parts of B. ocymoides was collected at Ede Oballa village, Nsukka town Anambra state, Nigeria and authenticated by a retired taxonomist of University of Nigeria, Nigeria, Mr. Ozoko and confirmed by Mr. Daramola, a curator, formerly of the University Herbarium, Department of Botany, Faculty of Science, University of Lagos Akoka.The herbarium specimen was prepared and deposited with voucher number LUH 3624.

Extract and fractions preparation
The collected plant material was cleaned, air dried under shade at room temperature and powdered by using a grinding mill (Hamburg 76 West Germany).The powdered plant material (1.3 kg) was extracted exhaustively with methanol by continuous percolation in a Soxhlet apparatus.The filtrate was concentrated using a rotary evaporator under reduced pressure (Buchi Rota vapor, Germany) at 45 rpm and 40°C to obtain dried crude extract (124 g).Extract (100 g) was suspended in 300 ml distilled water and successfully fractionated with n-hexane (3 × 300 ml), dichloromethane (3 × 300 ml) and ethyl acetate (3 × 300 ml) to yield nhexane (23.22 g), dichloromethane(16.90g), ethyl acetate (8.17 g), and aqueous (43.28 g) fractions respectively.The extract and fractions were kept in airtight sample bottles and were stored at 4°C for the assays.

Experimental animals
Swiss albino mice (20 -22 g) of both sexes were used for the study and were bred and kept in the Laboratory Animals Center, College of Medicine, University of Lagos, Nigeria.The animals were maintained on standard rodent feed and water ad libitum under 12 h light and dark cycle at room temperature.The animals were allowed to acclimatize for 14 days preceding the experiments.All experimental protocols were in compliance with internationally accepted principles for laboratory animal use and care. 25

Acute toxicity
The safety of the crude extract was evaluated by determining the oral acute toxicity using a modified Lorke s method. 26The mice were starved for 24 h prior to drug administration.They were randomly divided into six groups of 10 mice per group (one control group and five treated groups).The control group received 0.2 ml of 1% tween 80, while the treated groups were given single doses of 500, 1000, 2000, 4000 and 5000 mg/kg orally, in dose volume of 0.2 ml.The mice were observed for signs of toxicity and mortality at regular intervals for 24, 48, and 72 h then subsequently daily for 7 days. 27

Malaria parasites
The rodent parasite Plasmodium berghei berghei chloroquine sensitive strain (ANKA strain NK-65) was obtained from National Institute of Medical Research (NIMR), Yaba, Lagos and was maintained by continuous re-infestation in mice.

Inoculum preparation
Parasitized erythrocytes were obtained from a donor infected mouse by cardiac puncture.The inoculum was prepared by determining percentage parasitaemia and erythrocytes count of the donor mouse and diluting it with normal saline in proportions as indicated by the determinations. 28

Suppressive test
The Peter's 4-day suppressive test was used to determine the suppressive activity of the extract and its fractions against chloroquine sensitive Plasmodium berghei NK 65 infection in mice. 29On first day (D 0 ), 24 mice were inoculated by intraperitoneal injection (i.p.) with 0.2 ml of inoculum of Plasmodium berghei berghei containing 1 x 10 7 infected erythrocytes.The animals were randomly divided into 4 groups of six mice each and three hours later drugs were administered to the mice.Groups 1-2 received 250 and 500 mg/kg/day extract respectively, group 3 was the negative control and received 10 ml/kg/day of 1% tween 80 in distilled water while groups 4 served as positive control received 10 mg/ kg/day of chloroquine.The drug administration continued daily for 4 days (D 0 -D 3 ) and on 5 th day (D 4 ) thin blood films were prepared from tail blood obtained from each mouse then stained with Giemsa to show parasitized erythrocytes out of 500 in a random field of the microscope.The above procedure was repeated for hexane, dichloromethane, ethyl acetate and aqueous fractions respectively to evaluate their antimalarial activity (250 mg/kg).Percentage parasitaemia was calculated as % Parasitaemia = Number of parasitized RBC x 100 Total number of RBC counted % Chemosuppression = APN -APT x 100 APN Where APN is average parasitaemia in negative control and APT is average parasitaemia in test group.

Curative test
The test was carried out as described by Liu & co workers 30 with modifications.Twenty four mice were infected intrapertoneally with standard inoculum of 1x10 7 (0.2ml) Plasmodium berghei berghei NK 65 infected erythrocytes on the first day (D 0 ) and left for 72 h then mice were divided into 4 groups of six mice each.Groups 1-2 received orally 250 and 500 mg/kg/day extract respectively, group 3 was the negative control and received 10 ml/kg/day of 1% tween 80 in distilled water while group 4 served as positive control 10 mg/kg/day of chloroquine.The treatment was carried out once daily for 5 days and Giemsa's stained blood smears were prepared from the tail blood sample of each mouse collected on each day of treatment to monitor parasitaemia level.Any death that occurred during this period was noted and used to determine the mean survival time over a period of 28 days (D o -D 27 ).

DPPH radical scavenging test
The antioxidant activity of the extract and its fractions were determined by the free radical scavenging ability using DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay. 31About 1ml of sample solution of different concentrations (0.05-1.0 mg/ml) was mixed with 3 ml of methanol and 1 ml of 1 mM DPPH in methanol was added to make up to 5 ml.The mixtures were shaken, allowed to stand at room temperature in a dark chamber for 30 minutes and the absorbance was measured at 517 nm using spectrophotometer.Ascorbic acid was used as positive standard control.The percentage of inhibition DPPH (%) was calculated as follows: % inhibition = Abs of control -Abs of test sample x 100 Absorbance of control Absorbance of control The concentration of extract leading to 50% reduction of DPPH (IC50) was determined from the curve of % inhibitions plotted against the respective concentrations.

Determination of Total Phenolic Content
The total phenolic content of the extract was measured according to the Folin-Ciocalteu method. 32The extract solution (0.25% w/v; 1 ml) was mixed with 5 ml of 1 in 10 aqueous solution of Folin-Ciocalteu reagent and 5 min later 4 ml of 7.5% w/v sodium carbonate was added then the mixture was incubated at room temperature for 1 h.The absorbance was measured at 760 nm with a spectrophotometer.Gallic acid solutions with concentration ranging from 5-200 µg/ml were used for calibration.A dose response linear regression was generated by using the gallic acid standard absorbance and the level in the extract was expressed as gallic acid equivalent (mg of GAE/g of extract).The procedure was done in triplicate and results were expressed as mean ± SEM.

Statistical analysis:
Results were expressed as mean ± S.E.M.The significance of difference was determined using one-way analysis of variance (ANOVA).P < 0.05 was considered to be statistically significant.

Preliminary phytochemical analysis
The phytochemical screening of the crude extract of B. ocymoides indicated the presence of phenolic compounds like flavonoidsand tannins, steroidal compounds and saponins while alkaloids, cyanogenetic glycosides and anthraquinones werenot detected.

Acute toxicity
There was no mortality recorded in the mice upon oral administration even at doses as high as 5,000 mg/kg.This indicates that the experimental doses used are relatively safe.

Suppressive activity
The results of the study showed that the extract displayed schizonticidal activity at the studied doses.Percentage inhibition analysis indicated that the extract produced a significant (p<0.05)dose-dependent decrease in parasitaemia at 250 and 500 mg kg -1 compared to the negative control group (Table 1).The hexane, dichloromethane and ethyl acetate fractions Parasitaemia expressed as mean ± SEM, n = 5, results are expressed as the percent suppression of parasitaemia with reference to non-treated mice, *significant difference from control at p < 0.05 also reduced parasitaemia level significantly (p < 0.05) compared to the negative control.The order of chemo suppression of the fractions was ethyl acetate > dichloromethane > hexane > aqueous (Table 2).

Curative activity:
The result of the curative test showed extract of B. ocymoides demonstrated a dose-dependent reduction in mean parasitaemia (p < 0.05) in mice by 76.2 and 84.6% at 250 and 500 mg/kg respectively like chloroquine treated groups while the negative control group showed a daily increase in parasitaemia (Table 3).Parasitaemia reduction was also observed for chloroquine, the standard drug, where reduction started from D 1 , (3.11%) to D 5 , where total clearance was recorded (100%).Death did not occur in the chloroquine treated groups but occurred at 250 and 500 mg/kg of extract at 40 and 20% respectively.The survival was 0, 60, 80 and 100% for negative control, extract 250, 500 mg/kg and chloroquine group respectively.

Antioxidant activity
The extract of B. ocymoides exhibited a concentration dependent DPPH radical scavenging activity which was significantly less (P > 0.05) than ascorbic acid used as positive control with IC 50 value 1.85 and 0.05 re-spectively (Figure 1).The total phenolic content of methanol extract of B. ocymoides was calculated as 48.0 ± 2.49 mg GAE/g of extract.

DISCUSSION
In many parts of the world where malaria is endemic, herbal preparations are used to treat the disease.Plants produce phytochemicals which have protective or preventive properties against human diseases.Antimalarial activities of many medicinal plants have been reported [33][34][35] .In this study anti-malarial activity of the extract and fractions of B. ocymoides were tested using in vivo anti-plasmodial effect against chloroquinesensitive Plasmodium berghei NK 65-infected mice.The results obtained showed that the extract elicited significant (P < 0.05) dose dependent suppressive effect against early Plasmodium infection (suppressive effect) and established infection (curative effect) (Table 1 & 3).The extract fractions also exhibited different degrees of parasitaemia clearance in the 4-day suppressive schizontocidal activity test.The ethyl acetate and the dichloromethane fractions were found to possess higher blood schizontocidal activity (87.31 and 42.19% respectively) than the hexane and the aqueous fractions (27.8 and 14.22% respectively) (Table 2).This suggests the possible localization of the active ingredients in these two fractions, particularly the ethyl acetate fraction.The anti-plasmodial effect may be due to one of the active compounds of the extract or a combined effect Parasitaemia expressed as mean ± SEM, n = 5, results are expressed as the percent suppression of parasitaemia with reference to non-treated mice. of many.Previous studies on individual effects of Cinchona alkaloids and synergistic effect between the various alkaloids has shown improved activity over quinine against resistant P. falciparum in vitro, 36 particularly when quinine is combined with cinchonine. 37The synergistic effects of the active compounds has numerous benefit since it could lead to enhanced efficacy, decreased dosage or increased level of target inhibition, reduced or delayed development of drug resistance and reduction of toxic effects. 38he Rane's test evaluates the curative ability of compounds on established infection and is commonly used for antimalarial drug screening.In this test, extract of B. ocymoides has better parasitaemia clearance than in the suppressive test.In the 4-day suppressive study, the percentage parasitaemia was lower (69.33%) at the dose of 500 mg/kg than the reduction recorded in the curative test (84.61%) at the same dose.This lower activity may probably be due to specificity of the crude extract on established infection.The mean survival time of the infected mice was prolonged as 20% death was observed at higher dose of the extract increased while 100% mice-mortality occurred in the untreated control.
The study on acute toxicity revealed absence of mortality even up to the dose of 5000 mg/kg body weight of extract administered orally, which indicates the safety of the extract at the various test doses.
The preliminary phytochemical analysis of the crude extract of B. ocymoides showed the presence of phenolics, steroidal compounds and saponins.Phenolic compounds like flavonoïds and tannins are good antioxidant substances which have been reported to have biological activities and prevent or control oxidative stress related disorders. 39The crude extract tested negative to alkaloids.This finding is contrary to report of Conserva and Ferreira et al. in 2012 that alkaloids were present in species of Borreria. 40] This suggests that an increase in the body antioxidant can initiate the decrease in parasite number and severe infection at the long run. 17,46 xidative stress has been implicated in the pathophysiology of malaria. 47The efficacy of antioxidants is usually associated with their ability to inhibit oxidative damage by scavenging free radicals. 48DPPH is a very useful reagent for investigating free radical scavenging ability of compounds.B. ocymoides extract demonstrated good antioxidant activity by reducing the purple colored DPPH radical solution to yellow colored diphenylhydrazine due to the presence of hydrogen-donating antioxidants. 49The extract also has phenolic compounds that are antioxidant agents which also act as free radical terminators. 50The Folin-Ciocalteau reagent method used measures the level of oxidizable or phenolic compounds in the extract. 513] The phenolic compounds in the extract may be involved in the antimalarial activity exhibited by the B. ocymoides crude extract.The antimalarial activity of phenolic compounds was reported to be due to elevation of red blood cell oxidation, inhibition of parasite's protein synthesis and also by counteracting the oxidative damage induced by the malaria parasite. 47Plants and compounds with antioxidant activity may ameliorate the progress of malarial infection and possibly prevent the development of cerebral complications and other malaria related complications. 54However, the challenge here is that some antimalarial drugs generate the free radicals to produce their effect suggesting that their activity may be reduced in the presence of antioxidants.The anti-plasmodial activity observed in this study is consistent with the traditional use of the B. ocymoides as herbal medications against malaria in Nigeria.

CONCLUSION
This study indicates that methanol extract and solvent fractions of B. ocymoides have good anti-plasmodial and antioxidant activities.The extract appeared to be superior in curative parasitaemia reduction than suppressive activity.The findings suggested that the phytochemicals responsible for antimalarial activity of the plant are polar in nature with antioxidant properties.Future studies on the plant regarding antimalarial activity should be conducted to isolate compounds responsible.

Figure 1 :
Figure 1: Radical Scavenging Effects of B. ocymoides and Ascorbic acid on DPPH Radical