malaria is one of the world 's leading killers , having a greater morbidity and mortality than any other infectious diseases , with greater mortality in children and pregnant women .
malaria is caused by a parasite of the genus plasmodium and transmitted by female anopheles mosquito .
renal damage and hemolysis induced by malaria , especially p. falciparum and p. vivax , occur between 2 and 6% of hospitalized patients with a mortality that can reach up to 50% .
the pathogenesis of renal damage and hemolysis during malaria infection is multifactorial and not well characterized , but several studies suggest involvement of cytoadherence of parasitized erythrocytes and proinflammatory response as well as oxidative stress [ 35 ] .
this has prompted research towards the discovery and development of new , safe , and affordable antihemolysis drugs during malaria infection . in this
plant extracts are commonly investigated in several parts of the world for their possible activities for using in malaria [ 710 ] . moreover , many studies recently endorsed the use of traditional medicinal plants to treat diseases [ 1116 ] .
this plant is widely found in asia and southeast asia including thailand and used for general weakness , malaria fevers , and chronic rheumatism .
this result is confirmed by the existence of phenolic and flavonoids such as catechin , luteolin , and borapetoside .
these are responsible for high antioxidant activity and other activities including anti - inflammation , antimicrobials , and anticancer [ 1921 ] .
the antioxidant properties of t. crispa stem extract make it useful for the treatment of diseases resulting from oxidative stress such as cardiovascular disease , cancer , liver damage , and renal disease .
it also has been used as an antimalarial agent in vivo [ 22 , 23 ] .
however , it has not been reported yet to use t. crispa stem extract to treat renal damage and hemolysis induced malaria infection .
hence , the present study was aimed to investigate the protective effects of t. crispa stem extract on renal damage and hemolysis induced by p. berghei infection in mice .
fresh stems of tinospora crispa were collected from natural environment in suphanburi province , thailand , from march to april 2015 .
stems were minced into small pieces and dried in hot - air oven at 60c for 72  h , after which the dried plants were ground and then extracted in distilled water at the proportion of 1  :  10  ( w / v ) using microwave at 360  w for 5  min .
the extract was filtered through whatman number 1 filter paper , and evaporation was then performed to remove solvent .
powder extract was dissolved in distilled water to obtain appropriate doses for using in mice and volume should not be higher than 1  ml .
drug doses , expressed in mg / kg of body weight , were adjusted at the time of administration according to the weight of the mice .
pathogen - free , female icr mice , aged 46 weeks and weighting 2530  g , were purchased from the national laboratory animal center , mahidol university , bangkok , thailand .
they were kept at 2225c with 12  h light / dark cycle and given standard mouse pellet and water ad libitum .
all procedures of the animal experiments were approved by the ethical committee on animal experimentation , faculty of medical technology , western university . 

pbanka was kept alive by intraperitoneal ( ip ) passage of 1  10 parasitized erythrocytes in nave mice .
in addition , hematocrit ( % hct ) was also monitored by collecting tail vein into heparinized hematocrit tubes and centrifuged at 10,000  g for 10  min .
blood urea nitrogen ( bun ) and creatinine were used as indicators of renal function .
mouse blood was collected from tail vein into heparinized hematocrit tubes and centrifuged at 10,000  g for 10  min .
plasma was subsequently collected and used as subjects for measurement of bun and creatinine using an available commercial kit ( biosystems ) according to manufacturer 's instruction .
protective effect of aqueous crude extract of t. crispa on renal damage and hemolysis during malaria infection was done based on standard 4-day suppressive test .
groups of icr mice ( 5 mice of each ) were randomly divided , and infection was then performed with 1  10 parasitized erythrocytes of pbanka by ip injection .
two hours after infection , they were given orally by gavage with 500 , 1000 , and 2000  mg / kg of the extracts and every 24  h twice a day for 4 consecutive days .
three control groups were used ; normal controls were given either distilled water or the extract while untreated control was given only distilled water . on day 4 of experiment , tail blood was collected to assess hemolysis by measurement of % hct .
parasitemia was first detectable on day 1 after infection with a parasitemia of 0.5% and reached 65% on day 10 ( figure 1(a ) ) .
next , we observed that bun and creatinine levels were markedly increased in infected mice , and first significant ( p < 0.05 and p < 0.001 ) increases were found on day 4 after infection ( figures 1(b ) and 1(c ) ) .
additionally , we also observed progressive decreases of % hct in infected mice in response to the presence of parasitemia ( figure 1(d ) ) .
it was found that t. crispa stem extract showed protective effect on renal damage induced by pbanka infection in mice , and the highest activity was found at dose of 2000  mg / kg of extract treated group ( figure 2 ) .
the bun and creatinine levels were increased significantly ( p < 0.01 and p < 0.001 , resp . ) by malaria infection ( untreated group ) and infected mice treated with 500  mg / kg of the extract , compared to normal control .
interestingly , after oral administration of t. crispa stem extract , bun and creatinine levels declined significantly ( p < 0.01 and p < 0.001 , resp . ) , especially at doses of 1000 and 2000  mg / kg , compared to untreated control .
however , t. crispa stem extract could not be able to decrease parasitemia levels . 

t. crispa stem extract exerted antihemolytic effect against pbanka infected mice . as shown in figure 3 ,
hemolysis was observed in untreated control and infected mice treated with 500  mg / kg of the extract as indicated by decreasing of % hct with significance ( p < 0.01 ) , compared to normal control .
interestingly , % hct was significantly ( p < 0.01 ) increased in infected mice treated with 1000 and 2000  mg / kg of the extract , compared to untreated control .
the highest effect was found at dose of 2000  mg / kg of the extract .
moreover , prolonged survival time of infected mice treated with this extract was also observed .
in the current study , we provide evidence that describes the protective effect of t. crispa stem extract on renal damage and hemolysis induced by p. berghei infection in mice .
the onset of renal damage and hemolysis in icr mice infected with pbanka come out from day 4 after infection and the incidence of renal damage and hemolysis were confirmed through manifestations such as increasing of bun and creatinine as well as decreasing of % hct in infected mice .
renal damage and hemolysis are proposed to be a consequence of parasite adhesion and exacerbated immune response against products of oxidative stress released during infection .
in addition , parasitized erythrocyte sequestration at the microvascular site of renal has also been discussed to induce renal damage .
the destruction of erythrocytes during blood stage infection accumulates high levels of toxic - free heme that has ability to induce oxidative stress from production of hydroxyl radicals via fenton / haber - weiss reaction .
moreover , heme - derived oxidative stress is considered to be a main factor in the iron - induced lipid peroxidation resulting in the formation of oxidized ldl .
therefore , these conditions derived by blood stage of infection result in renal damage and hemolysis .
however , protective effects of renal damage and hemolysis induced by malaria infection were found in infected mice treated with t. crispa stem extract .
polyphenols and flavonoids show high antioxidant and anti - inflammation properties and then protect oxidative stress development .
t. crispa stem extract has been shown to contain high amounts of polyphenols and flavonoids .
thus , the potent antioxidant activity of this extract might play a central role in protecting renal damage and hemolysis from oxidative stress induced by malaria infection .
moreover , t. crispa stem extract has been reported to have antimalarial activity against p. yoelii infection in mice .
so , antimalarial activity to inhibit p. berghei growth and subsequently inhibit renal damage and hemolysis might also be mentioned .
taken together , our results suggest that t. crispa stem extract shows protective effects against p. berghei - induced renal damage and hemolysis .
appropriate pharmaceutical strategies might now be devised to increase the low bioavailability of this extract and to protect against rapid in vivo metabolic transformation , in such a way to make them more amenable as alternative antimalarial drug in combination therapies .
 renal damage and hemolysis induced by malaria are associated with mortality in adult patients . 
 it has been speculated that oxidative stress condition induced by malaria infection is involved in its pathology . 
 thus , we aimed to investigate the protective effects of tinospora crispa stem extract on renal damage and hemolysis during plasmodium berghei infection 
 . t. crispa stem extract was prepared using hot water method and used for oral treatment in mice . 
 groups of icr mice were infected with 1  107 parasitized erythrocytes of p. berghei anka by intraperitoneal injection and given the extracts ( 500 , 1000 , and 2000  mg / kg ) twice a day for 4 consecutive days . to assess renal damage and hemolysis , blood urea nitrogen ( bun ) , creatinine , and hematocrit ( % hct ) levels 
 were then evaluated , respectively . 
 malaria infection resulted in renal damage and hemolysis as indicated by increasing of bun and creatinine and decreasing of % hct , respectively . 
 however , protective effects on renal damage and hemolysis were observed in infected mice treated with these extracts at doses of 1000 and 2000  mg / kg . in conclusion , 
 t. crispa stem extract exerted protective effects on renal damage and hemolysis induced by malaria infection . 
 this plant may work as potential source in the development of variety of herbal formulations for malarial treatment . 