MATERIALS AND METHODS

Parasite: A rodent malaria parasite, Plasmodium berghei (NK-65) was maintained in white Swiss mice, Mus musculus (BALB/c) as per guidelines of Institutional Animal Ethics Committee of Himachal Pradesh University, Shimla vide number IAEC/Bio/2-2012. The erythrocytic stages was maintained by passing the infection to normal mice intraperitioneally (i.p) with 1x10⁵ P. berghei infected erythrocytes in citrate saline (Banyal and Kumar, 1991). The course of parasitaemia was monitored by observing daily blood smears.

Preparation of extracts: The parts of the plant i.e., roots and leaves of Picrorhiza kurrooa were washed separately with distilled water, air dried and weighed. Homogenisation was done in ethanol and homogenate filtered, centrifuged (Sigma 3k-30) at 2000 rpm for 10 min. Ethanol evaporated and residual concentrated concentrated material was used as plant extract. This extract was stored at 4°C till further use. The screening of extracts of different parts of plant was carried out according to Peter’s 4 day test (Peters, 1970). Experimental as well as control groups of animals were inoculated intraperitoneally with 1x10⁵ P. berghei parasitized red blood cells suspended in 0.2 mL of 2% citrate saline, on day 0. The extract suspended in water and given in concentration of 500 mg per kg b.wt. per dose per day per mouse to the experimental animals daily from day 0 to day 3 by oral route. As standard antimalarial, chloroquine was given to another control group of mice at 4 mg kg^-1 per dose for 4 days. Untreated control receive water only. Blood smears were prepared from the tail vein of all the mice of extract treated groups as well as chloroquine control and untreated control groups, on day 4. Afterward percent infection was monitored.

RESULTS

The antimalarial effect of extracts of roots and leaves of Picrorhiza kurrooa was evaluated. These extracts showed varying degree of results. No mortality was observed, within 24 h of drug administration which indicated that the plant products were not toxic to mice. Mice of weight between 20-35 g were used in the present study. On day 4, five mice of control groups exhibited a mean parasitaemia of 23.15±1.66% and the parasitaemia ranged 21-25% (Table 1 and Fig. 1). In the chloroquine control group, no parasite was noticed. In group of mice treated with root extract of Picrorhiza kurrooa, mean parasitaemia on day 4 was 4.14±1.70% and the parasitaemia ranged between 2.50-6%. The group of mice treated with leaf extract showed mean parasitaemia of 11.4±1.66% and the percent infection ranged between 9.15-13.5% on day 4. All the treated mice with leaf or root extract finally succumbed to infection and animals died between day 10 and 15 postinoculation (Table 2). The maximum parasitaemia achieved by a mouse was observed in smears prepared on the day given in Table 2 and the animal died later on due to infection.

Table 1:	Percent infection on day 4 in mice treated with roots and leaves extracts

Fig. 1:	Parasitaemia on day 4 in control and extracts (roots and leaves) treated mice

Table 2:	Maximum parasitaemia attained by mice on postinoculation

DISCUSSION

The present study confirms the finding of Singh and Banyal (2011) that roots of Picrorhiza kurrooa inhibited P. berghei propagation in vivo, the leaves used in this study alongwith roots significantly inhibited P. berghei infection.

Picrorhiza kurrooa is widely used in traditional as well as modern system of medicine. Traditionally, the mature plants of Picrorhiza kurrooa are uprooted, rhizomes and roots are traded for preparing medicinal formulations to treat disorders of the liver and upper respiratory tract, dyspepsia, chronic diarrhoea and scorpion sting (Verma et al., 2009). The hepatoprotective action of Picrorhiza kurrooa is not fully understood but may be attributed to Picrorhiza’s ability to scavenge free radicals and to inhibit the generation of oxygen anions (Russo et al., 2001). The effect of Picrorhiza’s antioxidants has been similar to that of superoxide dismutase, xanthine oxidase inhibitors and metal-ion chelators (Chander et al., 1992). During present investigation, extracts of both the parts i.e., roots and leaves of Picrorhiza kurrooa showed antimalarial effect, however, extract of roots being more effective than that of leaves. Both the part of the Picrorhiza kurrooa have important components showing medicinal properties and antimalarial activity. However, the revelation of the chemical constituent showing the property needs to be elucidated.