Anti-malarial activity of 6-(8'Z-pentadecenyl)-salicylic acid from Viola websteri in mice
© Chung et al; licensee BioMed Central Ltd. 2009
Received: 01 May 2009
Accepted: 07 July 2009
Published: 07 July 2009
Petroleum ether extracts of Viola websteri Hemsl (Violaceae) were reported to have anti-plasmodial activity against Plasmodium falciparum in vitro, with this activity being largely attributable to 6-(8'Z-pentadecenyl)-salicylic acid (6-SA).
The schizontocidal activity of 6-SA on early Plasmodium berghei infections was evaluated in a four-day test. The possible 'repository' activity of 6-SA was assessed using the method described by Peters. The median lethal dose (LD50) of 6-SA, when given intraperitoneally, was also determined using uninfected ICR mice and the method of Lorke.
In the present study, 6-SA was found to have anti-malarial activity in vivo, when tested against P. berghei in mice. 6-SA at 5, 10 and 25 mg/kg·day exhibited a significant blood schizontocidal activity in four-day early infections, repository evaluations and established infections with a significant mean survival time comparable to that of the standard drug, chloroquine (5 mg/kg·day).
6-SA possesses a moderate anti-malarial activity that could be exploited for malaria therapy.
Isolation of 6-SA from the plant material was reported previously . The test compound was prepared by suspending the 6-SA in saline containing 0.5% Tween-80.
As experimental hosts, 8-week-old outbred male ICR mice were purchased from Japan SLC (Hamamatsu, Japan). The animals were housed in standard cages, with standard feed and water given ad libitum, and they were acclimatized for 10 days prior to the experiments. All animal experiments were performed according to the guidelines for animal experimentation, Sanbon Medical Center, Wonkwang University.
The malarial parasite used was a chloroquine-sensitive strain of P. berghei (ATCC 50175; American Type Culture Collection, Manassas, VA, US), which has been maintained by serial blood passage in mice. The inoculum consisted of 5 × 107 Plasmodium berghei parasitized red blood cells per ml. This was prepared by determining both the percentage parasitaemia and the red blood cell count of the donor mouse and diluting the blood with isotonic saline in proportions indicated by both determinations. Each mouse was inoculated on day 0, intraperitoneally, with 0.2 ml of infected blood containing about 1 × 107 P. berghei parasitized red blood cell. The LD50 of the compound was determined using male ICR mice by intraperitoneal route using the method of Lorke et al .
Evaluation of schizontocidal activity on early infection (4-day test)
The schizontocidal activity of 6-SA on early P. berghei infections was evaluated in a four-day test . For this test, after determining the animal's percentage of parasitaemia and erythrocyte count, blood from an infected donor mouse was diluted with isotonic saline to obtain an inoculum containing 5 × 107 infected erythrocytes/ml. Thirty ICR mice were then (at about 07.30 hours) inoculated intraperitoneally with 0.2 ml of the inoculum before being randomly divided into six equal treatment groups of five mice each. Every morning (at 08.00–09.00 hours) from the day of infection (day 0) to 3 days later (day 3), each mouse was administered orally either 6-SA in 0.2 ml saline containing 0.5% Tween-80 (at 5, 10 and 25 mg 6-SA/kg·day), chloroquine in 0.2 ml saline containing 0.5% Tween-80 (at 5 mg chloroquine/kg·day) or 0.2 ml saline containing 0.5% Tween-80. On day 4, 24 hours after the last treatment, a thin smear was made from the tail blood of each mouse and stained with Giemsa to determine the percentage parasitaemia (by counting the number of parasitized erythrocytes per 200 erythrocytes, in random fields). For each group of mice treated with 6-SA or chloroquine, the mean percentage chemosuppression was then calculated as 100 [(A-B)/A], where A was the mean percentage parasitaemia of the mice 'treated' only with saline containing 0.5% Tween-80 (the negative controls) and B was the mean parasitaemia in the test group.
Evaluation of the repository activity
The possible 'repository' activity of 6-SA was assessed using the method described by Peters . For this, another six groups of mice (five per group) were treated pre-infection, with 0.2 ml oral doses of 6-SA in saline containing 0.5% Tween-80 (at 5, 10 or 25 mg/kg·day), pyrimethamine in saline containing 0.5% Tween-80 (at 1.2 mg/kg·day) or saline containing 0.5% Tween-80, for 4 consecutive days (days 0–3). On day 4, the mice were inoculated with P. berghei (as in the 4-day test) and on day 7 (72 hours post-infection) their parasitaemias were assessed.
Evaluation of schizontocidal activity in established infection (Rane test)
To evaluate schizontocidal activity in established infection , the 4-day test was repeated but modified so that the first treatment did not take place until 72 hours after the mice had been infected, the mice were treated daily for five (not four) days and parasitaemias were evaluated on each day of treatment. In addition, mortality and weight changes in the mice were followed-up for 30 days post-infection (day 29) and the day-29 parasitaemias of the survivors were evaluated. The median lethal dose (LD50) of 6-SA, when given intraperitoneally, was also determined using uninfected ICR mice and the method of Lorke .
Data were compared using Student's t tests, with a P value of < 0.05 being considered statistically significant.
Results and Discussion
Blood schizontocidal activity of 6-SA, as measured against Plasmodium berghei in mice
Average % parasitaemia
Average % suppression
Average % parasitaemia
Average % suppression
43.4 ± 0.25*
34.3 ± 0.37*
21.5 ± 0.34*
17.4 ± 0.28*
19.3 ± 0.09*
10.4 ± 0.09*
6.4 ± 0.13*
4.9 ± 0.32*
58.2 ± 0.23
4.9 ± 0.32*
In the toxicity tests, all the mice administered 6-SA at 5–500 mg/kg exhibited insignificant signs of toxicity, ranging from writhing and gasping (LD50 of >500 mg/kg) to decreased respiratory rate, decreased limb tone, and death. The LD50 was calculated to be >500 mg/kg. The present results indicate that 6-SA possesses useful blood schizontocidal when used at doses that cause no marked toxicity in mice. Although the mechanism of action of this compound has not been elucidated, 6-SA clearly merits further investigation.
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