Printer Friendly

Artemisia vulgaris L. ethanolic leaf extract reverses thrombocytopenia/ thrombocytosis and averts end-stage disease of experimental severe Plasmodium berghei murine malaria.

INTRODUCTION

At present, malaria remains one of the greatest challenges of global health. Antimalarial drug resistance is thus identified as one of the major causative phenomena hindering malaria control. Artemisinin, a sesquiterpene lactone compound isolated from the plant Artemisia annua (1) containing regimens meet the urgent need of effective treatment for multidrug resistant malaria and are advocated for widespread deployment (2). Nevertheless, emergence of parasites resistant to artemisinin at the Thai-Cambodia border could seriously undermine the success of global malaria control efforts (3). At present when the control of malaria is increasingly limited by the growing resistance of the malaria parasites to available drugs, the need for much better use of existing drugs, as well as the development of new antimalarials has become an imperative need.

Anthelminthic, antiseptic, antibacterial and anti-inflammatory properties of plant species of the Genus Artemisia (family Asteraceae) is widely studied and reported (4-5). Artemisia vulgaris (English--common worm-wood or mugwort; Sinhala--Walkolondu; TamilMacipattiri), is an invasive weed, growing on nitrogenous soils, found in waste dumps and on roadsides. It is the only Artemisia species prevalent in Sri Lanka. Previous studies by our group substantiated that oral administration of organic and ethanolic extracts of A. vulgaris respectively showed moderate and high antiparasitic activity in Plasmodium yoelli (6) and in P. berghei ANKA (7) rodent malaria models.

In vivo testing of antimalarial activity of developed formulations by using P. berghei-infected mice as a suitable model for studying malaria is promising, as the infection presents structural, physiological and life cycle analo gies with the human malaria caused by P. falciparum (8).

A characteristic feature of malaria is the presence of fever episodes. Rare attempts were made to observe this phenomenon in rodent models. Mice infected with P. berghei are considered to be in the end-stage disease when their body temperature rapidly drops ([less than or equal to] 35.5[degrees]C/ 95[degrees]F), 7 or 8 days post-infection and usually succumb to the infection within 24 h, as a consequence of cerebral malaria (9). Though such observations are scarcely documented for rodent malaria, it is a well-established fact associated with other fatal diseases such as renal (10) and cardiac failure (11). End-stage disease is the manifestation of the worst condition for an organ or disease state. At this point the organ barely functions.

Several studies report that platelets may play a key role in the immune-pathophysiology of experimental, severe P. berghei malaria (12-13) as well as of falciparum malaria (14). In childhood falciparum malaria, median platelet counts were lower among severe cases than in mild cases, and in children who died than among those who recovered. Moreover, multivariate analysis identified thrombocytopenia as an independent predictor of death associated with malaria (15). Interestingly, some studies indicate a protective function for platelets in the early stages of erythrocytic infection in malaria which demonstrated that purified human platelets killed P. falciparum parasites cultured in red blood cells and increase production in early stages of the infection in in vivo models (16). It was later established that platelet factor 4 and the erythrocyte Duffy antigen receptor were required for platelet killing of P. falciparum (17).

Elevated body temperature (18), body aches and headaches (19) are classic malaria symptoms. Therefore, patient management is one of the common practices associated with malaria treatment. Thus, a plant extract manifesting a plethora of antimalarial disease activities will prove to be very useful to reduce disease complications.

Thus, we undertook a study to investigate antidisease properties of A. vulgaris ethanolic leaf extract (AVELE) in the P. berghei ANKA (lethal strain) murine malaria model that elicit pathogenesis similar to falciparum malaria. In vivo antidisease activity of AVELE was investigated using standard methodology. Especially, effects of AVELE on malaria induced critical pathologies such as thrombocytopenia/thrombocytosis and the end-stage disease were examined for the first time.

MATERIAL & METHODS

Ethical approval

Ethical clearance for this research study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo, Sri Lanka (Ref No. EC-10-132).

Collection and authentication of plant material

Leaves of A. vulgaris, were collected from the roadsides in Nuwara Eliya (altitude: 1868 m, 6.9667[degrees] N, 80.7667[degrees] E), Sri Lanka. The specimen were authenticated by the Herbal Technology Division, Industrial Technology Institute, Sri Lanka, and a voucher specimen was deposited in the museum of the Department of Zoology, Faculty of Science, University of Colombo, Sri Lanka (GY/01/2010).

Preparation of the plant extract

The A. vulgaris ethanolic leaf extract (AVELE) was prepared essentially following the procedure described by Bamunuarachchi et al (7). Briefly, washed, air-dried leaves of A. vulgaris were powdered and soaked in an organic solvent mixture consisting of diethyl ether, methanol and petroleum ether at a 1:1:1 ratio. The resulting dark green solution was filtered and the leaves were soaked in the organic solvent mixture for a second time. The extracted solution was then evaporated at reduced pressure using a rotavapour. This crude extract was suspended in 5% ethanol. The test animals were orally treated with the AVELE at the doses of 250, 500, 750 and 1000 mg/kg of body weight that represented low, human equivalent, moderate high and high doses, respectively (7).

Experimental animals

Healthy inbred adult male ICR (Institute of Cancer Research) mice weighing 25-30 g, purchased from the Medical Research Institute, Colombo, were used in this study. All the animals were housed in plastic cages in the animal house, Department of Zoology, University of Colombo under standard conditions (temperature 28-31[degrees]C, photoperiod: approximately 12 h natural light per day, Relative humidity: 50-55%). The animals were given ad libitum access to food pellets (Master Feed Ltd., Colombo, Sri Lanka) and drinking water. Except at the time of experimental procedure, the animals were handled only during cage cleaning.

Parasite isolates

Plasmodium berghei ANKA parasites maintained through serial blood passage in mice were used to assess in vivo antimalarial activity of AVELE.

Evaluation of survival for infected mice

A 4-day suppressive assay (20) was used to evaluate the survival of infected mice (n = 6/group). AVELE (250, 500, 750, 1000 mg/kg) and control (5% ethanol) were administered to all the test animals with prior exposure to P. berghei from Day 0 through D3. Mice were observed daily for eight consecutive days and their parasitaemia was monitored (7). Mice were treated orally with Coartem [R] when parasitaemia reached 50%. The number of mice that survived in each group with levels of parasitaemia below 50% at the end of eight days was recorded.

Investigation of temperature fluctuations associated with P. berghei rodent malaria

A group (n = 8) of mice was injected intraperitoneally (IP) with 107 infected RBC on D0. The rectal temperature of mice was recorded twice a day, once in the morning (0900-1000 hrs) and repeated in the evening (1500-1600 hrs) using a digital thermometer (VT-801 series, Valeo Corporation, Taipei, Taiwan) from D0 through D8. Simultaneously, blood smears were prepared (morning and evening) from tail bleed of mice and parasitaemia levels were determined. Rectal temperature of normal, uninfected mice (n = 8) were also recorded.

Effect of AVELE on the temperature fluctuation of mice infected with P. berghei

Four groups (n = 8/group) of male ICR mice were infected with 107 P. berghei-infected RBC on D0. Daily oral administration of the AVELE (500, 750, and 1000 mg/kg) and control (5% ethanol) took place from D0 through D3. The rectal temperature of mice was determined from D0 through D8, twice daily, in the morning (0900-1000 hrs) and in the evening (1500-1600 hrs). Concurrently, blood smears were prepared from tail bleed of mice and parasitaemia levels were determined for each group.

Effect of AVELE on the thrombocyte-related pathology associated with P. berghei rodent malaria

Four groups of male ICR mice (n = 6/group) were inoculated with 10 (7) P. berghei-infected RBC on D0. AVELE (500, 750, and 1000 mg/kg) and control (5% ethanol) were administered to all from D0 through D3, where their thrombocyte counts (21) and parasitaemia were monitored from D0 through D6. Thrombocyte counts of normal, untreated mice (n = 6) were also recorded.

Antipyretic activity of AVELE investigated in the yeast-induced mice pyrexia model

Total 24 mice were randomly assigned into four equal groups (n = 6/group). The rectal temperature of mice were determined using a digital thermometer (VT-801 series, Valeo Corporation, Taipei, Taiwan). Mice were then sub cutaneously injected with 0.3 ml/kg of 15% (w/v) aqueous suspension of active dry yeast (Instant-Dry yeast, AB Mauri India Pvt. Ltd, Ratnagiri, India) to induce pyrexia. The rectal temperature of these mice was determined 19 hours later (22), and subsequently the four groups were orally treated with AVELE (500 and 1000 mg/kg body weight doses), 5% ethanol (control), and the reference drug, Paracetamol (6 mg/kg), respectively. Subsequently, the rectal temperature of test animals was determined at 1, 2, 3, 4, 5 and 6 h post-treatment.

Investigation of antinociceptive effects of AVELE

Hot plate test and tail flick methods were used, respectively, to assess the central and spinally mediated antinociceptive effects of AVELE (23). Four groups (n = 6/ group) of healthy male ICR mice were used; the first two groups were treated with 500 and 1000 mg/kg doses of AVELE, while the third group received 5% ethanol (test control group), the fourth group treated with distilled water functioned as the normal group.

The reaction times of the mice were measured one hour prior to treatment. In the hot plate test, the mouse was placed inside the hot plate analgesia meter (Model MK 35A, Muromachi Kikai Co. Ltd., Tokyo, Japan) at 52[degrees]C and the time taken for animals to lick the hind paw or to jump was recorded. A cut-off time of 20 sec was set to avoid tissue damage. In the tail flick test, 2-3 cm of the tail from the tip was immersed in a hot water bath at 54[degrees]C and time taken to flick the tail was recorded. Readings were taken one hour post treatment and then repeated at hourly intervals for 5 h.

Writhing test (24) was used to assess peripheral analgesia using four groups (n = 6/ group) of male ICR mice; two groups were treated with either 500 or 1000 mg/kg AVELE, the third group with 10 mg/kg of diclofenac sodium (reference drug) and the control group with 5% ethanol. One hour later, 0.05 ml of 10% acetic acid was injected IP to each of these mice, and the number of writhing (abdominal contractions and stretches) that occurred between 5 and 20 min were recorded. Percentage inhibition of writhing was calculated using the following formula:

% Inhibition = [[(Mean number of writhing in control group)--Mean number of writhing in test group)]/ Mean number writhing in control] x 100

Statistical analysis

Data were analyzed using the Minitab 15 statistical package for Windows. Data were expressed as mean [+ or -] SEM. Statistical analysis was performed using the Mann-Whitney U-test. Significance was set at p [less than or equal to] 0.05.

Survival of test animals

Figure 1 illustrates the effect of oral administration of AVELE on the survival of mice infected with P. berghei. The group treated with the high dose (1000 mg/ kg) of the extract and the positive control group showed maximum (100%) survival.

Effect of AVELE on rectal temperature fluctuation of P. berghei-infected mice

Body temperature associated with P. berghei infected mice showed that within 6 h (morning compared with evening), their rectal temperature fluctuated by 3[degrees]F in the control group, D1 through D6 which was a significant manifestation (p [less than or equal to] 0.01) absent in the uninfected group (Fig. 2a). Importantly, mice treated with 500, 750 and 1000 mg/kg doses of the AVELE significantly (p [less than or equal to] 0.01) deviated from this and maintained normal rectal temperatures until Days 5 and 6 post-inoculation (Fig. 2b).

[FIGURE 1 OMITTED]

[FIGURE 2a OMITTED]

[FIGURE 2b OMITTED]

Effect of AVELE on P. berghei malaria-associated thrombocytosis/thrombocytopenia in mice

On D3, at early-stage of disease establishment, profound thrombocytosis in the control was observed compared to the healthy, untreated group (p [less than or equal to] 0.01) (Fig. 3a). Importantly, 500, 750 and 1000 mg/kg doses of AVELE significantly (p [less than or equal to] 0.01) reversed this condition by 27.5, 47.3 and 41.5%, respectively, compared with the control (Table 1 and Fig. 3b).

On D6, at a much later stage of the infection, significantly (p [less than or equal to] 0.01) profound thrombocytopenia was detected in the control compared to the healthy, untreated group (Fig. 3a) which was observed to be significantly (p [less than or equal to] 0.01) reversed by the 500, 750 and 1000 mg/kg doses of AVELE, by 489.9, 922.7 and 324.6%, respectively (Table 1 and Fig. 3b).

[FIGURE 3a OMITTED]

[FIGURE 3b OMITTED]

Antipyretic activity of AVELE in the yeast-induced mice pyrexia model

AVELE at 500 mg/kg dosage significantly (p < 0.05) suppressed the yeast induced pyrexia (> 100[degrees]F) in the fourth hour which significantly lasted for only an hour. Conversely the 1000 mg/kg dose of AVELE significantly (p < 0.05) suppressed pyrexia from the second hour onwards that was sustained until fourth hour (Table 2).

Evaluation of antinociceptive activity of AVELE

When compared with the control, mice treated with the 1000 mg/kg dose of AVELE showed significantly (p [less than or equal to] 0.01) prolonged reaction times (fourth hour by 31.9%; fifth hour by 40.1%) in the hot plate test but not in the tail flick test (Table 3), indicative of the ability of AVELE to curb acute pain, central supra spinally.

The assessment of peripheral analgesic effect of both 500 and 1000 mg/kg doses of AVELE, resulted in significantly (p [less than or equal to] 0.01) reduced acetic acid induced writhing reaction in mice, the latter which in effect was comparable to that of the reference drug, Diclofenac sodium that act on sustained pain (Table 4).

DISCUSSION

Currently, the first line chemotherapeutics against malaria are fixed dose Artemisinin-based combination therapies (ACTs), which are presumed to act against the blood stages of all human malaria (25). However, emergence of parasites resistant to artemisinin at the Thai-Cambodia border (3) would seriously undermine the effective use of ACTs. Thus, the relentless pursuit of novel antimalarials is a worthy exercise.

Artemisia vulgaris is not commonly used to treat malaria but is reported to possess many other ethnopharmaceutical properties (4-5). Our group previously substantiated safe and highly potent antiparasitic activity of A. vulgaris ethanolic leaf extract when administered orally, against P. berghei ANKA rodent malaria parasites (7).

The rodent malaria model used in this study, the lethal strain of P. berghei ANKA, as mentioned before, causes severe malaria in mice and the infection elicits structural, physiological and life-cycle analogies with the human disease (8). These parasites elicit experimental severe malaria (ESM), and comparatively similar pathogenesis characters with P. falciparum human malaria, where infected mice exhibit a marked systemic inflammatory response, profound thrombocytopenia, and cerebral malaria (12). Plasmodium berghei ANKA infection in inbred ICR mice as a model of cerebral malaria (CM) can reproduce many of the important features of CM and, therefore, can be used as a tool to advance our understanding of the disease pathogenesis (26-27). Cerebral malaria, the major severe form of the infection, is lethal and in humans results from P. falciparum infections. Therefore, any potential antidisease activity of the AVELE manifested in the P. berghei in vivo model may be presumed as potential activity against pathological complications of P. falciparum malaria, though such interpretation should be made with caution.

Patient management is one of the common practices associated with malaria treatment for the reason that malaria depicts several disease complications such as cerebral malaria, profound thrombocytosis and thrombocytopenia, fever episodes, elevated body temperature and body aches and headaches (12, 14-16, 19)

In human malaria pathology, a characteristic feature is the presence of fever episodes. Rare attempts have been made to observe this phenomenon in murine models. Mice infected with P. berghei parasites exhibit a rapidly decreasing body temperature and these succumb to the disease within 24 h, 8 or 9 days into the infection. Mice are considered to be in end-stage disease as a consequence of CM (9). We too observed this phenomenon of end-stage disease in mice infected with P. berghei but importantly, those test animals treated with 500, 750 and 1000 mg/kg doses of AVELE significantly (p [less than or equal to] 0.01) deviated from this debacle and clearly maintained normal rectal temperature until 5 and 6 days post-inoculation. Thus, it may be a clear indication that AVELE alleviate the cerebral complications associated with rodent malaria.

One of the hallmarks of CM, caused by infection with P. falciparum, is petechial haemorrhaging in the brain, indicating that platelets may play a crucial role in malarial pathogenesis (28). Both thrombocytopenia and platelet-induced clumping of P. falciparum-parasitized RBCs is a common feature of acute malaria and occurs in both P. falciparum and P. vivax infections regardless of the severity of infection (14). The current study for the first time demonstrated that the oral administration of crude AVELE significantly reversed both thrombocytosis and thrombocytopenia that occurred in P. berghei murine malaria, which reiterated that AVELE may have the potential to lessen cerebral complications and haemorrhages associated with CM.

At least three different types of antimalarial activity can be evaluated. First, activity is considered to be prophylactic if the action is exerted against the sporozoites or the parasites of the initial tissue phase of the disease; suppressive, if against the parasites of the asexual erythrocytic phase of the disease; and curative, if against the parasites of the persisting tissue phase (2). The most widely used initial test for in vivo screening of antimalarial com pounds, which uses P. berghei or less frequently P. chabaudi, is a 4-day suppressive assay (29). We previously demonstrated that the 1000 mg/kg dose of the AVELE possess antiparasitic activity in the 4-day suppressive assay but not in the curative assay, which suggests that this crude plant extract in this study manifested suppressive but not curative activity (7). Thus, it may be presumed that the concentration of the active compound in the crude extract may not have sufficed to eliminate an established infection. This highlights the need for future in vitro and in vivo investigations using the isolated active component(s) of A. vulgaris to substantiate its potential as a therapeutic agent against malaria and also to elucidate its mode of action.

Furthermore, the 1000 mg/kg dose of A. vulgaris leaf extract exhibited significant antipyretic activity in the yeast-induced mice pyrexia model. Elevated body temperature is a classic malaria symptom. As the malaria parasites enter the blood stream they infect and destroy red blood cells. Destruction of these essential cells lead to fever (12). Therefore, fever management is one of the common practices associated with malaria treatment. Despite widespread use of Paracetamol in fever management, a study on humans infected with malaria concluded that Paracetamol can prolong malaria parasitaemia (30). Thus, the anti pyretic property of A. vulgaris crude plant extract in concert with established antimalarial properties, further corroborates the potential of this plant to be developed in to an effective antimalarial.

Supra spinally mediated acute pain killing effect of an organic extract of A. vulgaris leaves was previously reported by us in a P. yoelli mouse model (6). More importantly, when we evaluated the antinociceptive potential of AVELE, acute (supra spinally mediated) as well as peripheral analgesic effects were manifested where the effect of the high dose of AVELE was comparable to that of the standard peripheral analgesic drug, Diclofenac sodium. The peripheral analgesic effects were monitored using acetic acid induced writhing of mice (24). The quantification of prostaglandins by radio immuno assay in the peritoneal exudates of rats, obtained after intraperitoneal injection of acetic acid, found high levels of prostaglandins, PGE2 and PGF2 alpha during the first 30 min after acetic acid injection (31). Nevertheless, it was also recorded that the intraperitoneal administration of acetic acid induces the liberation of not only of prostaglandins but also of the sympathetic nervous system mediators (32). Thus, it may be assumed that A. vulgaris ethanolic leaf extract may significantly act either by inhibiting the action of prostaglandin enzyme, prostaglandin E synthases, or by blocking the prostaglandin receptor. It may also act on the sympathetic nervous system mediators. As malaria elicits body aches and pain, this effective systemic antinociceptive (peripheral pain killing) property of the AVELE, that reiterates the findings of a study by Pires et al (23), will be exceptionally potent in the treatment of malaria. It may also be presumed that the responsible active ingredients of the crude plant extract may form the basis for an excellent novel analgesic.

Whether for chemotherapy or chemoprophylaxis, toxicity of drugs prescribed for malaria is a closely monitored factor. Toxicity has been singled out as the main drawback of traditional herbal antimalarial preparations (6). Thus, we previously evaluated 30 days chronic oral administration of the 1000 mg/kg dose of AVELE, which showed no overt signs of toxicity or stress. Hepatotoxicity (evaluated in terms of serum GOT and GPT levels), renotoxicity (in terms of serum urea and creatinine) and haematotoxicity (in terms of RBC, WBC and DC counts) were also ruled out. Weight and gross morphology of vital organs (liver, spleen, kidneys, heart, and lungs) were also not affected. Also continuous weight gain of animals was observed. Thus, the A. vulgaris extract was well-tolerated by mice (7).

Our previous study demonstrated that the oral administration of a crude leaf extract of A. vulgaris, possess safe and potent (87.3% parasitaemia inhibition) antimalarial effects, in terms of antiparasitic activity in a P. berghei ANKA lethal murine malaria model (7). In conclusion, this study for the first time demonstrated that the oral administration of a crude leaf extract of A. vulgaris, possesses potent antidisease action (antipyretic activity, peripheral and central antinociception, and reversal of thrombocytopenia/thrombocytosis and of end-stage disease), in a P. berghei murine malaria model. Thus, the antidisease properties of A. vulgaris crude plant extract collectively with established antiparasitic properties, further corroborate the potential of this plant to be developed into an effective antimalarial.

Activity directed fractionation and further investigations on antiparasitic and antidisease activity of the purified components may hopefully lead to significant scientific drug discovery. More importantly, A. vulgaris is a weed distributed in the hilly parts of Sri Lanka in high abundance, and thus has the potential to be developed into a cheap source of plant-based antimalarial in future.

ACKNOWLEDGEMENT

University of Colombo is acknowledged for financial assistance.

REFERENCES

(1.) World Malaria Report 2001. Geneva: WHO Press 2001. Available from: http://www.who.int/malaria/world_malaria_report_2001/en/index.html.

(2.) Woodrow CJ, Haynes RK, Krishna S. Artemisinins. Postgrad Med J 2005; 51(952): 71-8.

(3.) World Malaria Report 2009. Geneva: WHO Press 2009. Available from: http://www.who.int/malaria/world_malaria_report_2009/en/index.html

(4.) Tigno XT, de Guzman F, Anna MA, Flora TV. Phytochemical analysis and hemodynamic actions of Artemisia vulgaris L. Clin Hemorheol Microcirc 2000; 23: 167-75.

(5.) Govindaraj S, Kumari BDR, Cioni PL, Flamini G. Mass propagation and essential oil analysis of Artemisia vulgaris. J Biosci Bioeng 2008; 105(3): 176-83.

(6.) Kodippili K, Ratnasooriya WD, Premakumara S, Udagama PV. An investigation of the antimalarial activity of Artemisia vulgaris leaf extract in a rodent malaria model. Int J Green Pharm 2011; 5: 276-81.

(7.) Bamunuarachchi GS, Ratnasooriya WD, Premakumara S, Udagama PV. Antimalarial properties of Artemisia vulgaris L ethanolic leaf extract in a Plasmodium berghei murine malaria model. J Vector Borne Dis 2013; 50: 278-84.

(8.) Carter R, Diggs CL. Plasmodium of rodents. In: Kreier JP, editor. Parasitic Protozoa, v 3. New York: Academic Press 1977; p. 359-465.

(9.) Hermsen CC, Mommers E, van de Wiel T, Sauerwein RW, Eling WMC. Convulsions due to increased permeability of the blood-brain barrier in experimental cerebral malaria can be prevented by splenectomy or anti T-cell treatment. J Infect Dis 1998; 178 (4): 1225-7.

(10.) Kamath P, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, et al. A model to predict survival in patients with end-stage liver disease. Hepatology 2001; 33 (2): 464-70.

(11.) Friedrich EB, Bo"hm M. Management of end stage heart failure. Heart 2007; 93: 626-31.

(12.) Van der Heyde HC, Gramaglia I, Sun G, Woods C. Platelet depletion by anti-CD41 (aIIb) mAb injection early but not late in the course of disease protects against Plasmodium berghei pathogenesis by altering the levels of pathogenic cytokines. Blood 2004; 105(5): 1956-63.

(13.) Gramaglia I, Sahlin H, Nolan JP, Frangos JA, Intaglietta M, van der Heyde HC.). Cell- rather than antibody-mediated immunity leads to the development of profound thrombocytopenia during experimental Plasmodium berghei malaria. J Immunol 2005; 175 (11): 7699-770.

(14.) Climent CP, Oscar K, Charles RJCN, Norbert P, David JR. Short report: Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10. Am J Trop Med Hyg 2006; 75(3): 434-6.

(15.) Gerardin P, Rogier C, Amadou S, Jouvencel P, Brousse V, Imbert P. Prognostic value of thrombocytopenia in African children with falciparum malaria. Am J Trop Med Hyg 2002; 66(6): 686-91.

(16.) McMorran BJ, Marshall VM, de Graaf C, Drysdale KE, Shabbar M, Smyth GK, et al. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science 2009; 323 (5915): 797-800.

(17.) McMorran BJ, Wieczorski L, Drysdale KE, Chan JA, Huang HM, Smith C, et al. Platelet factor 4 and duffy antigen required for platelet killing of Plasmodium falciparum. Science 2012; 338 (6112): 1348-51.

(18.) Kwiatkowski D, Perlmann P. Inflammatory processes in the pathogenesis of malaria. In: Wahlgren M, Perlmann P, editors. Malaria: Molecular and Clinical Aspects. Harwood Academic Publishers 1999; p. 329-62.

(19.) Gurpreet K. Prevalence of clinical malaria among an orang asli community in Malaysia. Southeast Asian J Trop Med Public Health 2009; 40(4): 665-73.

(20.) Peters W, Protus JH, Robinson BL. The chemotherapy of rodent malaria, XXII, The value of drug resistant strains of P. berghei in screening for blood schizonticidal activity. Annals Trop Med Parasitol 1975; 69(2): 155-71.

(21.) Brecher G, Cronkite EP. Morphology and enumeration of human blood platelets. J Appl Physiol 1950; 3: 365-71.

(22.) Hullatti KK, Sharada MS. Comparative antipyretic activity of patha: An ayurvedic drug. Pharmacogn Mag 2007; 11: 173-6.

(23.) Pires JM, Mendes FR, Negri G, Duarte-Almeida JM, Carlini EA. Antinociceptive peripheral effect of Achillea millefolium L. and Artemisia vulgaris L.: Both plants known popularly by brand names of analgesic drugs. Phytother Res 2009; 23(2): 212-9.

(24.) Ojewole JAO. Antinociceptive, antiinflammatory and antidiabetic effect of Bryphyllem pinnatum leaf aqueous extract. J Ethnopharmocol 2005; 99: 13-9.

(25.) Jansen O, Tits M, Angenot L, Nicolas JP, De Mol P, Nikiema JB, et al. Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15- O-acetate as the main active compound. Malar J 2012; 11: 289.

(26.) Basir R, Rahiman SSF, Hasballah K, Chong WC, Talib H, Yam MF, et al. Plasmodium berghei ANKA infection in ICR mice as a model of cerebral malaria. Iran J Parasitol 2012; 7(4): 62-74.

(27.) Wang HZ, Xin He Y, Yang C, Zhou W, Zou CG. Hepcidin is regulated during blood-stage malaria and plays a protective role in malaria infection. J Immunol 2011; doi:10.4049/jimmunol. 1101436.

(28.) Fidock DA, Rosenthal PJ, Croft SL, Brun R, Nwaka S. Antimalarial drug discovery: Efficacy models for compound screening. Nature Rev 2004; 3: 509-20.

(29.) Brandts CH, Ndjave M, Graninger W, Kremsner PG. Effects of paracetamol on parasite clearance time in Plasmodium falciparum malaria. Lancet 1997; 350: 704-9.

(30.) Derardt R, Jougney S, Delevaliee F, Flahaut M. Release of prostaglandins E and F in an algogenic reaction and its inhibition. Eur J Pharmacol 1980; 61: 17-24.

(31.) Hokansan GC. Acetic acid for analgesic screening. J Nat Prod 1978; 41: 497-8.

(32.) Duartei G, Nakamura M, Ferreira SH. Participation of the sympathetic system in acetic acid-induced writhing in mice. Brazilian J Med Biol Res 1998; 21: 341-3.

Gayan S. Bamunuarachchi [1], Wanigasekara D. Ratnasooriya [1], Sirimal Premakumara [2] & Preethi V. Udagama [1]

[1] Department of Zoology, Faculty of Science, University of Colombo, Colombo; [2] Herbal Technology Division, Industrial Technology Institute, Colombo, Sri Lanka

Correspondence to: Prof. Preethi V. Udagama, Department of Zoology, Faculty of Science, University of Colombo, Colombo 03, Sri Lanka. E-mail: dappvr@yahoo.com; preethi@zoology.cmb.ac.lk

Received: 29 November 2013

Accepted in revised form: 6 August 2014
Table 1. Effect of AVELE on thrombocyte-related pathology associated
with P. berghei rodent malaria

                       Treatment Platelet count x
                          [10.sup.3]/[micro]l

                     Day 0                  Day 1

Healthy       484.2 [+ or -] 22.1    458.5 [+ or -] 34.9
Control       564.8 [+ or -] 48.3    578.3 [+ or -] 52.5
  (5% EtoH)
500 mg/kg      531 [+ or -] 27.7     518.3 [+ or -] 12.8
750 mg/kg     464.8 [+ or -] 38.8     437 [+ or -] 35.2
1000 mg/kg    320.7 [+ or -] 58.6   333.5 [+ or -] 55.6 *

                  Treatment Platelet count x [10.sup.3]/[micro]l

                     Day 2                      Day 3

Healthy        817 [+ or -] 45.2          556 [+ or -] 46.8
Control       957.7 [+ or -] 86.9       1724 [+ or -] 128 (#)
  (5% EtoH)
500 mg/kg     946.8 [+ or -] 68.6   1249.3 [+ or -] 18 ([dagger])
750 mg/kg      479.2 [+ or -] 35     908.3 [+ or -] 74 ([dagger])
1000 mg/kg    355.8 [+ or -] 29.9   1007 [+ or -] 42.8 ([dagger])

                   Treatment Platelet count x [10.sup.3]/[micro]l

                     Day 4                        Day 5

Healthy        712 [+ or -] 55.1           583.8 [+ or -] 22.3
Control       1041.2 [+ or -] 78.6         517.2 [+ or -] 44.4
  (5% EtoH)
500 mg/kg      1223 [+ or -] 54.9    1193.2 [+ or -] 89.8 ([dagger])
750 mg/kg      1135 [+ or -] 63.7      1773 [+ or -] 113 ([dagger])
1000 mg/kg     1040 [+ or -] 102            904 [+ or -] 146 *

                   Treatment Platelet
               count x [10.sup.3]/[micro]l

                          Day 6

Healthy             655 [+ or -] 11.9
Control          197.8 [+ or -] 26.7 (#)
  (5% EtoH)
500 mg/kg     1167 [+ or -] 175 ([dagger])
750 mg/kg     2023 [+ or -] 119 ([dagger])
1000 mg/kg     840 [+ or -] 107 ([dagger])

Values are expressed as means [+ or -] SEM (n = 8); (#) p < 0.01 as
compared with the healthy, untreated group; * p <0.05 and <0.01,
as compared with the control (Mann-Whitney U-test).

Table 2. Effect of oral administration of AVELE (500, 1000 mg/kg),
and paracetamol on yeast-induced pyrexia in mice

Treatment                 Rectal temperature ([degrees]F)

                          Before                 After
                       yeast-pyrexia            induced

Control (5% EtoH)   98.16 [+ or -] 0.56   100.13 [+ or -] 0.54
AVELE 500 mg/kg     98.08 [+ or -] 0.34   99.63 [+ or -] 0.37
  1000 mg/kg        98.35 [+ or -] 0.40   100.15 [+ or -] 0.37

Paracetamol         97.9 [+ or -] 0.52    99.65 [+ or -] 0.54
  (6 mg/kg)

Treatment                  Rectal temperature ([degrees]F)

                                     Post-treatment

                         1st hour               2nd hour

Control (5% EtoH)   99.05 [+ or -] 0.34    99.58 [+ or -] 0.45
AVELE 500 mg/kg     98.16 [+ or -] 0.52    98.33 [+ or -] 0.42
  1000 mg/kg        97.61 [+ or -] 0.60   97.36 [+ or -] 0.59 *

Paracetamol         95.70 [+ or -] 0.16    96.2 [+ or -] 0.24
  (6 mg/kg)              (dagger])              (dagger])

Treatment                   Rectal temperature ([degrees]F)

                                     Post-treatment

                          3rd hour               4th hour

Control (5% EtoH)   100.00 [+ or -] 0.45   100.18 [+ or -] 0.35
AVELE 500 mg/kg     98.96 [+ or -] 0.33    98.46 [+ or -] 0.34 *
  1000 mg/kg        97.92 [+ or -] 0.59    98.27 [+ or -] 0.34 *
                         (dagger])

Paracetamol         96.61 [+ or -] 0.41     96.96 [+ or -] 0.38
  (6 mg/kg)              (dagger])               (dagger])

Treatment                  Rectal temperature ([degrees]F)

                                     Post-treatment

                          5th hour              6th hour

Control (5% EtoH)   100.22 [+ or -] 0.31   99.96 [+ or -] 0.22
AVELE 500 mg/kg     99.25 [+ or -] 0.36    99.71 [+ or -] 0.25
  1000 mg/kg        99.25 [+ or -] 0.63    99.41 [+ or -] 0.63

Paracetamol         97.35 [+ or -] 0.43    97.85 [+ or -] 0.44
  (6 mg/kg)              (dagger])              (dagger])

Values are expressed as means [+ or -] SEM (n = 8); * p < 0.05
and ([dagger]) p < 0.01, as compared with the control
(Mann-Whitney U-test); Ambient temperature = 84[degrees]F.

Table 3. Reaction times of mice in the hot plate test following
oral administration of AVELE (500, 1000 mg/kg doses), distilled
water and control (5% ethanol)

Experimental       Reaction times for hot plate test (sec)
group

                        Initial              1st hour

Normal group       6.37 [+ or -] 0.48   5.98 [+ or -] 0.56
  (DW)
Control group      7.16 [+ or -] 0.52   6.50 [+ or -] 0.66
  (5% EtoH)
Test group (500    6.36 [+ or -] 0.55   7.72 [+ or -] 0.54
  mg/kg AVELE)
Test group (1000   6.88 [+ or -] 0.30   6.36 [+ or -] 0.81
  mg/kg AVELE)

Experimental       Reaction times for hot plate test (sec)
group

                        2nd hour             3rd hour

Normal group       6.13 [+ or -] 0.75   6.77 [+ or -] 0.79
  (DW)
Control group      6.02 [+ or -] 0.69   6.03 [+ or -] 0.78
  (5% EtoH)
Test group (500    6.23 [+ or -] 0.53   4.83 [+ or -] 0.45
  mg/kg AVELE)
Test group (1000   6.08 [+ or -] 0.48   6.35 [+ or -] 0.75
  mg/kg AVELE)

Experimental       Reaction times for hot plate test (sec)
group

                        4th hour             5th hour

Normal group       6.02 [+ or -] 0.65   5.91 [+ or -] 0.59
  (DW)
Control group      6.71 [+ or -] 0.35   6.32 [+ or -] 0.35
  (5% EtoH)
Test group (500    4.38 [+ or -] 0.47   5.41 [+ or -] 0.41
  mg/kg AVELE)
Test group (1000   8.85 [+ or -] 0.57   8.86 [+ or -] 0.58
  mg/kg AVELE)         ([dagger])           ([dagger])

Values are expressed as means [+ or -] SEM (n = 6; ([dagger]) p
[less than or equal to] 0.01 as compared with the control (Mann-
Whitney U-test).

Table 4. Analgesic activity of AVELE in acetic acid induced
writhing response in mice

Treatment             No. of writhing/      Percentage
                          unit time         inhibition

Control (5% EtoH)    25.57 [+ or -] 1.29        --
AVELE
  500 mg/kg         16.86 [+ or -] 1.34 *      34.1
  1000 mg/kg        13.57 [+ or -] 0.86 *      46.9
Diclofenac sodium   13.28 [+ or -] 0.68 *      48.1
  (10 mg/kg)

Values are expressed as means  [+ or -]  SEM (n = 6/group);
* p < 0.01 as compared with the control (Mann-Whitney U-test).
COPYRIGHT 2014 Indian Council of Medical Research
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Bamunuarachchi, Gayan S.; Ratnasooriya, Wanigasekara D.; Premakumara, Sirimal; Udagama, Preethi V.
Publication:Journal of Vector Borne Diseases
Article Type:Report
Geographic Code:9SRIL
Date:Dec 1, 2014
Words:5739
Previous Article:Detection of point mutation in Plasmodium falciparum ATPase6 gene associated with artemisinin resistance from Assam and Arunachal Pradesh.
Next Article:Evaluation of antileishmanial activity and cytotoxicity of the extracts of Berberis vulgaris and Nigella sativa against Leishmania tropica.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters