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Antiplasmodial activity of Ajuga bracteosa against Plasmodium berghei infected BALB/c mice.

About 300-500 million clinical cases and 1.2-2.8 million deaths due to malaria occur each year (1,2). The rising problem of resistance to the classical drugs (chloroquine and sulphadoxine pyrimethamine) (3,4) and the problem of recrudescence of artemisinin stress the need to look for new antimalarial agents (5). Plants and plant products have always been used for the treatment of various ailments (6). Quinine and artemisinin both are plant derivatives and have been obtained from Cinchona species (7,8) and Artemisia annua (9,11) respectively.

Ajuga bracteosa Wall ex Benth. Lamiaceae (Syn. A. remota) is a perennial herb growing wild from Kashmir to Nepal in western Himalaya at an altitude of 1300 m. Leaves of this plant are used as stimulant, diuretic and in the treatment of various diseases like rheumatism, gout, palsy and amenorrhoea (12). Its clinical efficacy as an antimalarial has not been established yet. The present study was undertaken to evaluate antiplasmodial activity of ethanolic leaves extract of Ajuga bracteosa against Plasmodium berghei infected BALB/c mice during early and established infection. Repository activity has also been checked to support its traditional use as a malaria remedy.

Material & Methods

Plant materials: Present study was undertaken in Parasitology Laboratory, Department of Zoology, Panjab University, Chandigarh. The leaves of A. bracteosa were collected in the month of September 2007 from the Mandi district of Himachal Pradesh, India. Voucher specimen (Voucher No. 8895) was deposited in the herbarium of Horticulture University, Solan, where identification of plant was confirmed by Dr N.S. Chauhan (a Taxonomist in the Department of Horticulture and Forestry, Nauni University, Solan).

The dried and powdered leaves of A. bracteosa (350 g) were subjected to Soxhlet extraction to prepare ethanolic extract. The solvent extract was evaporated to dryness in Rota evaporator. The dried residue (42 g) was stored in screw capped vials at -4[degrees]C. Phytochemical screening of the extract was carried out employing standard procedures (13,14).

Animals: Swiss albino mice (25-30 g) of either sex were obtained from the Central Animal House, Panjab University, Chandigarh, India. The animals were housed in standard plastic cages and acclimatized for a period of 30 days. The mice were maintained on standard feed and water ad libitum. Approval for the study was obtained from the Animal Ethics Committee (CPCSEA/45/1999), Panjab University, Chandigarh.

Parasite inoculation: The chloroquine sensitive Plasmodium berghei NK-65 strain was maintained in vivo in BALB/c mice in our laboratory by weekly inoculation of 1x[10.sup.7] infected red blood cells in naive mice. Experimental mice were inoculated on day 0, intraperitoneally, with 0.2 ml of infected blood in citrate saline having 1x[10.sup.7] P. berghei parasitized red blood cells.

Acute toxicity: The [LD.sub.50] of the extract was determined by administration of different concentration of extract by oral route in BALB/c mice using the limit test of Lorke (15). Extract, (5g/kg body weight/mouse) was administered orally to 5 female mice after a 4 h fast. This concentration is considered as the highest dose to be administered in rodents for the evaluation of acute toxicity of any drug (15). Test animals were observed for 14 days for mortality including various sign of toxicity. Histopathological examination of organs (liver, spleen and kidney) was done at the end of study.

Evaluation of schizontocidal activity on early infection (4-day test): Knight & Peters 4-day test was employed to evaluate schizontocidal activity in early infection (16). Four groups of mice (6 mice in each group) were orally administered 250, 500, 750 and 1000 mg/kg/day of ethanolic leave extract of A. bracteosa (ELEAB) in each group respectively and two groups of control mice, were administered chloroquine (5 mg/kg/day) as positive control in one group and equivalent volume of distilled water (0.2ml/mouse/day) as negative control in another group for 4 consecutive days; 24 h after the administration of the last dose, thin blood films were made from the tail of each mouse on fifth day. Smears were fixed in methanol and stained with Giemsa stain. The percentage of parasitaemia suppression was determined by counting the number of parasitized erythrocytes out of 500 red blood cells in random fields under light microscope. Average percentage of parasitaemia suppression was calculated as 100[(A-B)/ A], where A is the average percentage parasitaemia in the negative control group and B is the average parasitaemia in the test group (17).

Evaluation of the repository activity: Peters (18) method was used to assess the repository activity. Five groups of mice (6 mice in each group), were administered orally with 250, 500 and 750 mg/kg/day doses of the extract and 1.2 mg/kg/day pyrimethamine (positive control) and 0.2ml/mouse/day distilled water (negative control) for 4 consecutive days (D0-D3) respectively. On day 5 (D4), the mice were inoculated with P. berghei infected red blood cells. Seventy two hours later, the parasitaemia level was assessed by studying Giemsa stained blood smears.

Evaluation of schizontocidal activity in established infection (Rane test): Rane test was employed to evaluate schizontocidal activity of extract in established infection (19). On the first day (D0), standard inoculum of 1x[10.sup.7] P. berghei infected erythrocytes were injected intraperitoneally into mice. Seventy two hours later, the mice were divided into five groups of six mice each. Different doses of ELEAB (250, 500 and 750 mg/kg/day) were administered orally to these groups. Chloroquine (5 mg/kg/day) was given to the positive control group and an equal volume of distilled water to the negative control group. The drug/extract was given once daily for 5 days. Thin blood smears were prepared from tail of each mouse for 5 days, to monitor the parasitaemia level. The mean survival time for each group was determined arithmetically by finding the average survival time (days) of the mice (post-inoculation) in each group over a period of 30 days (D0-D29).

Statistical analysis: Data were statistically analyzed using Student's-test and P<0.05 was considered significant.

Results & Discussion

Plants have remained the ultimate source for the treatment of various ailments since ever (20). ELEAB was found to exert significant in vitro antiplasmodial activity with an [IC.sub.50] value of 10[micro]g/ml against chloroquine sensitive P. berghei isolates, during schizont maturation inhibition assay as reported in our previous study. Therefore, in vivo antiplasmodial study along with phytochemical screening was undertaken to show its antimalarial efficacy in present study.

Phytochemical screening of ELEAB has shown the presence of alkaloids, flavonoids, steroids, triterpenoids, saponins and tannins like phenolic compounds. Although mechanism of action of these secondary metabolites has not been evaluated in the present study, some of these metabolites have been found to exert their antiplasmodial effect either by elevating red blood cell oxidation or by inhibiting protein synthesis (21). The phytochemical investigation and isolation of various compounds from A. remota has led to the identification of various compounds like ajugarin I, II an III along with ajugasterone C, ajugalactone, cyasterone, [beta] ecdysone and ergosterol 5, 8-peroxide. Among these compounds ergosterol 5, 8-peroxide has been found to exert significant [IC.sub.50] (8.2 [+ or -] 1.1[micro]M) value against chloroquine sensitive (FCA20/GHA) strain of P. falciparum (22). Further, triterpenoid ergosterol 5, 8-peroxide acts as an antimicrobacterial compound (23). This compound has been found to inhibit the growth of protozoan parasite of Trypanosomatidae family such as typanosome cruzi and various Leshmania species by interfering with the integrity of the cell membrane (24). The active involvement of this compound in the plant extract to inhibit plasmodial parasite growth cannot be denied.

The [LD.sub.50] of ELEAB was found to be more than that 5g/kg body weight of naive mice, which proves the clinical safety of extract. Histopathological examination of liver, spleen and kidney did not reveal significant pathological changes. No hepatomegaly/ splenomegaly was observed. Size and colour of organs was also comparable to normal tissues. The concentration of 5g/kg in mouse is prescribed upper limit for administration of test material in one dose in rodents according to limit test of Lorke (15).

During early infection, oral administration of 250, 500 and 750mg/kg/day concentration of extract caused chemosuppression of 42.4, 63.2 and 77.7 per cent respectively, which was statistically significant (P<0.001) as compared to negative control. The standard drug chloroquine (5mg/kg/day) caused 86.2 per cent chemosuppression which was more significant as compared to extract treated groups (Table). Chloroquine has been used as a standard schizontocidal drug in early and established infection, as it interrupts with the heme polymerization by forming FP-chloroquine complex. This complex is highly toxic to the cell, thus disrupts membrane function which ultimately leads to parasite cell autodigestion (25). The highest concentration of extract used (1000 mg/kg/day) showed 86.6 per cent chemosuppression which was similar to that of standard drug chloroquine (5 mg/kg/day), however, 60 per cent mortality was observed with this concentration on day 7. As all natural plant product work in synergistic manner with immune system of host, the higher extract concentration might be downmodulating the natural immune resistance of host to malaria (26). This concentration was not tested further for repository and curative activity of ELEAB.

ELEAB produced a dose dependent repository activity as 250, 500 and 750 mg/kg/day concentrations and exhibited 32.7, 60.5 and 68.8 per cent chemosuppression respectively, significantly (P<0.05) compared to negative control group. However, standard drug pyrimethamine (1.2 mg/kg/day) was found to exert a considerable higher (76.2%) chemosuppression (P<0.001) as compared to extract treated groups (Table). For the evaluation of repository activity, the standard drug pyrimethamine was used as reference drug as it prevents DNA replication of parasite by binding to the dihydrofolate reductase (DHFR), which interferes with folic acid mechanism necessary for DNA and RNA synthesis leading to parasite death (27).

[FIGURE OMITTED]

In established infection, the percentage of parasitaemia on day 7 was 15.0, 11.0 and 9.1 per cent for 250, 500 and 750 mg/kg/day concentrations of the ELEAB treated groups respectively, while 55.1 per cent in control and 6.8 per cent in chloroquine treated groups (Fig.). The mean survival time (MST) of the mice in various groups was 15.2 [+ or -] 0.19, 21.6 [+ or -] 0.48, 27.4 [+ or -] 0.46, 29.6 [+ or -] 0.14 and 7.2 [+ or -] 0.19 days for 250, 500 and 750 mg/kg/day of extract, chloroquine and control groups respectively (Table).

In conclusion, our results showed that ELEAB was not only found to inhibit parasitaemia in dose dependent manner but also enhanced the mean survival time period of treated mice. These findings support the traditional use of plant for the treatment of malaria. It would be worthwhile to isolate its active constituents and characterize their exact mode of action which can be exploited for the treatment of malaria.

Acknowledgment

The first author (SC) is thankful to University Grants Commission (CAS programme) for providing fellowship. Authors thank Prof. Karan Vashist and Dr. Maninder Vashist of U.I.P.S, Panjab University, Chandigarh for preparing extract of plant and phytochemical analysis.

Received August 14, 2008

References

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S. Chandel & U. Bagai

Department of Zoology, Panjab University, Chandigarh, India

Reprint requests: Dr Upma Bagai, Department of Zoology, Panjab University, Chandigarh 160 014, India

e-mail: upmabagai@yahoo.co.in
Table. Evaluation of schizontocidal, repository activity and mean
survival time (MST) of ELEAB treated BALB/c mice infected
with P. berghei

Drug/extract Dose Schizontocidal activity
 (mg/kg/day)
 Average % Average %
 parasitaemia suppression

ELEAB 250 31.3 [+ or --] 0.44 ** 42.4
 500 20.0 [+ or --] 0.76 ** 63.2
 750 12.1 [+ or --] 2.80 ** 77.7
 1000 7.2 [+ or --] 0.36 ** 86.6
Chloroquine 5 7.5 [+ or --] 0.44 ** 86.2
Pyrimethamine 1.2 -- --
Control 0.2ml 54.4 [+ or --] 0.73 --
 (distilled
 water)

Drug/extract Dose Repository test
 (mg/kg/day)
 Average % Average %
 parasitaemia suppression

ELEAB 250 12.1 [+ or --] 2.19 32.7
 500 7.1 [+ or --] 2.05 * 60.5
 750 5.6 [+ or --] 1.17 * 68.8
 1000 -- --
Chloroquine 5 -- --
Pyrimethamine 1.2 4.3 [+ or --] 1.55 * 76.2
Control 0.2ml 18 [+ or --] 3.35 --
 (distilled
 water)

Drug/extract Dose Mean Survival
 (mg/kg/day) Time
 (in days)

ELEAB 250 15.2 [+ or --] 0.19 **
 500 21.6 [+ or --] 0.48 **
 750 27.4 [+ or --] 0.46 **
 1000 --
Chloroquine 5 29.8 [+ or --] 0.14 **
Pyrimethamine 1.2 --
Control 0.2ml 7.2 [+ or --] 0.19
 (distilled
 water)

Data are expressed as standard mean error (SEM) [+ or -] for six mice
per group when compared with control.

P * <0.05, ** <0.001 compared to control; ELEAB-etanolic leaves
extract of Ajuga bracteosa
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Author:Chandel, S.; Bagai, U.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Mar 1, 2010
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