Antiplasmodial Test of Tinospora crispa Stem Extract against Plasmodium falciparum 3D7 Strain In Vitro Uji Anti Plasmodium Ekstrak Batang Tinospora crispa terhadap Plasmodium falciparum Galur 3D7 secara In Vitro

This study aimed to prove the existence of anti-plasmodial activity from Tinospora crispa (T. crispa) stem extracts and to determine the IC values ​​as well as the best T. crispa stem extract concentration in inhibiting the growth of P. falciparum 50 3D7 strain in vitro. The degree of parasitemia was observed by counting the number of infected red blood cells in Giemsa stained blood films under a light microscope with a magnification of 1000x. The Plasmodium DNA concentration was measured using flow cytometryc with Propidium Iodide (PI) staining. Analysis of fourier transform infrared (FTIR) spectrophotometer showed that the methanol extracts of T. crispa stem contained tinocrisposide compound. From the quantitative test results of Thin Layer Chromatography (TLC), it was obtained 0,22 % alkaloids that might contain compounds of berberine and palmatine. One way ANOVA statistical analysis showed that the degree of parasitemia and the concentration parasite DNA of treatment group of dose of 2,0mg/ml was significantly lower compared to the control group after 48 hours (p=0,001) and 72 hours (p=0,001) of incubation. The T. crispastem methanol extract has antiplasmodium activity, with IC values between ​​ 0,27 mg/ml and 0,29mg/ml, and the effective dose to inhibit the growth of P. 50 falciparum 3D7 strain is 2,0 mg/ml with reducing parasitemia degree by 47,12% and 56,83% after 72 hours of incubation. From this study shown the methanol extract of T. crispastem was able to reduce the parasitemia degree of P. falciparum 3D7 strain in vitro and could to be a potential candidate for anti malarias.

test the antimalarial activity of methanol extract of T. falciparum, World Health Organization (WHO) has crispastem, we put a 200μl culture medium containing recommended the use of combination anti-malarial drug methanol extract of T. crispa stem with concentrations containing artemisinin derivatives (Artemisinin-based appropriate to treatment into sterile micro-plate (96 Combination Therapy/ACT) (5). wells), each well contained 20μl malaria culture pellet and The emergence of Plasmodium sp resistance to incubated for 48 and 72 hours. The thin blood smear was antimalaria has encouraged researchers to look for new made by giemsa staining and observed under a antimalarial drugs through research on plants that have microscope. Next, the number of erythrocytes infected by been traditionally used by people to treat malaria. We P. falciparum 3D7 strain parasite was counted in 1000 investigated the usefulness of a traditional medicine erythrocytes.Parasites Staining and Flow cytometric Tinospora crispa (T.crispa), a climberplant commonly Analysis grown in the garden, locally called Brotowali. Tinospora crispacontain; alkaloid, soft resin, starch, glycosides, bitter Pellets was suspended in 1ml PBS containing 0,25% substances, pikroretin and anti-malaria of quaternary glutaraldehyde and incubated at room temperature for 20 alkaloid derivative in the form of berberine and palmatine.
minutes and centrifuged at 450xg for 5 minutes. The pellet Tinospora crispaalso contains compounds such as was washed using cold PBS 2 times. Cells were suspended glycosides furanoditerpen in the form of tinocrisposide in 0,5ml PBS containing 0,01 % saponin and incubated at (6,7). Thus, it is supposedly able to suppress the room temperature for 5 minutes and centrifuged at 450xg development of Plasmodium. Previous study showed for 5 minutes. Then, the pellet was mixed with 40μl RNase mice that inoculated with Plasmodium yoelii then treated A and incubated in the dark at a temperature of 37 C for 40 with the crude extract of T. crispa at doses of 40 mg/kg minutes. The cells were suspended into 500ml propidium showed a surprising and interesting result, one mouse iodide and incubated in the dark at a temperature of 37 C from the group in which the dose of 80 mg/kg was for 60 minutes. Drug activity against malarial parasites was administrated was still alive and the parasite was cleared determined by measuring incorporation of PI. Flow from the blood stream (8).
cytometric data analyses were performed on a FACS caliber (Becton-Dinkinson) by using the CellQuestPro-METHODS program. Data were analyzed with normality and This study was conducted from September 2012 until July homogenity test. The statistical analysis was conducted 2013 at Central Laboratory of Biomedicine, Faculty of using SPSS software version 20 with one-way ANOVA. If Medicine, Brawijaya University. This study was an significant results were obtained, it would be conducted experimental study. The subjects used in this study were further testing using LSD. IC values analyzed using probit 50 culture of Plasmodium falciparum divided into control cuadratic analysis with SPSS software version 20. group (P. falciparum cultures without treatment), group -8 that exposed by artemisinin 10 M (an IC dose of 50 RESULTS artemisin in vitro) and groups that exposed by methanol extract of T. crispa stem with the dose of 0,5mg/ml, Analysis of Tinospora crispa Anti-malarial Compounds 1,0mg/ml, 1,5mg/ml, 2,0mg/ml, 2,5mg/ml, and Identification results of anti-malarial compound of 3,0mg/ml respectively, each of group was repeated 2 furanoditerpen glycoside in the form of tinocrisposide times (in duplicates well) (9).

Preparation of Methanol Extract of Tinospora crispastem
can be seen in Figure 1. Brotowali (Tinospora crispa) was collected from its UPTD Materia Medica in Batu, the stems of T. crispa were minced into small pieces then dried in sunlight for a day and ground into powder with a traditional grinder. The powdered stem was macerated for 24 hours using 90 % methanol solvent. Maceration process was carried out 3 times by replacing the methanol solvent every 24 hours, then evaporated and concentrated using N2 gas. Extract was made in the organic laboratory of Faculty of Mathematics and Natural Sciences of Brawijaya University, Indonesia.

Preparation of Tinospora crispa Extract
The methanol extract of T. crispastem was made as stock

Parasitemia Degree of Parasite Culture
The activity of methanol extract of T. crispa stem antiplasmodium was obtained by calculating the degree of parasitemia examined at 0 hour, just after T. crispa extract was exposed. Next, the degree of parasitemia was examined at 48 and 72 hours after the administration of T. crispa extract. The research results show that the degree of parasitemia for all treatments were lower than the positive control. The examination results of parasitemia degree are presented in Table 1.
The data in Table 1 below shows that over the observation time accretion (48 and 72 hours), it is followed by the average reduction trend in the degree of parasitemia. The results of one way ANOVA test showed a difference in the degree of parasitemia among groups with p value of 0,000, the results of statistical analysis of LSD indicated that after incubation for 48 hours, several treatment groups were significantly different compared to the positive control group (p<0,005) and after 72 hours of incubation, all treatment groups showed significantly different compared to the positive control group (p<0,005).  The research results showed that the concentration of Plasmodium DNA for all treatments were lower than the positive control. Treatment with methanol extract of T. crispa stem administration with the dose of 2,0mg/ml provided the best result or the average DNA concentration was the lowest compared to other treatments with the percentage of DNA concentration equal to 11,38% Based on the data in Table 1 above, it can also be seen that treatment with methanol extract of T. crispa stem administration with the dose of 2,0mg/ml provides the best result or the average degree of parasitemia was the lowest compared to other treatments with the percentage of parasitaemia degree equal to 7,62% (48 hours) and 5,85% (72 hours) with reducing parasitemia degree by 56,83% after 72 hours of incubation.

Concentration of parasite DNA
Flow cytometry analyzes using propidium iodide (PI) was conducted to determine the concentration of parasite DNA. Flow cytometry examination was conducted at 48  Quantitatively conducted Thin Layer Chromatography test results indicate that the methanol extract of T. crispa stem contains alkaloid compounds equal to 0,22%, in line with several studies concluding that T. crispa contains several alkaloid derivatives compounds such as aporfin, pikoretin, berberine and palmatine, pikroretosid tinocrisposide and triterpenoids (11,12).
Reduction in the percentage of parasitemia degree of P. falciparum 3D7 equal to 47,12% and parasite DNA concentration to 56,83% after 72 hours of incubation parasite culture in the treatment of administration methanol extract of T. crispa stem is suspected to be caused by the active ingredient of T. crispa as an antimalarial compound form; berberine, palmatine and tinocrisposide. Similar research data were revealed by previous study using Plasmodium falciparum treated with the crude extract of Tinospora crispa at doses of 2,5 mg/ml showed reducing parasitemia degree equal to 100% after 72 hours treatment with methanol extract of T. crispa stem in vitro (9).
Berberine constitutes quaternary alkaloid compound, which is compound structure containing quaternary nitrogen which is known to be able to inhibit the growth of Plasmodium by blocking/inhibiting intracellular transport of choline (13). Choline compounds are required for phospholipid biosynthesis in the parasite membrane formation to close the parasitophorous vacuole, the cytosol and various subcellular compartement. Thus, inhibition of choline causes the failure of the formation of a new parasite. The choline transport blocking has been Figure 3. Concentration of parasite DNA as the results used as one of malaria treatment strategies (14). Berberine of analysis of flow cytometry 72 hours after treatment.
is an inhibitor of nucleic acids and proteins biosynthesis of Note: ET= Extract of Tinospora crispa. (Continued) P. falciparum, which is in vitro showed a strong interaction with DNA (15).Similar with berberine, palmatine also constitutes quaternary alkaloid compound and quinoline The research results showed that the concentration of whose chemical structure only differs in their methoxyl Plasmodium DNA for all treatments were lower than the and hydroxyl groups (16). Therefore, the two active positive control. Treatment with methanol extract of T.
ingredients are thought to have similar activities, crispa stem administration with the dose of 2,0 mg/ml mechanism, and inhibition target as antimalaria. provided the best result or the average DNA concentration According to tinocrisposide, it is a compound that has was the lowest compared to other treatments with the structure of glycosides furanoditerpen (7). This structure is percentage of DNA concentration equal to 8,73% similar to the structure of nimbolid compound that has the effect of antimalaria. Although up to recently, the Similar to the microscopic analysis, flow cytometric inhibition mechanism of the two compounds on the analysis results showed significant differences in mean growth of Plasmodium is still unknown, but due to the DNA concentration among groups with the value of similarity of molecular structure between the two p=0,002 (48 hours) and the value of p=0,000 (72 hours). Of compounds, it is assumed that these two compounds have probit test results based on the observation of the same mechanism in inhibiting the growth of P. parasitemia degree using light microscope, it is obtained falciparum. IC value equals to 0,29mg/ml, and of probit test results of 50 Neem seed extract contains antimalarial compounds of concentration of DNA parasites based on flow cytometryc azadirachtin and nimbolid that are able to reduce the analysis, it is obtained IC value equals to 0,27mg/ml. 50 degree of parasitemia, and the number of P. falciparum hemozoin is better than chloroquine (17). It is also DISCUSSION explained that although the mechanism of inhibiting the The identification of T. crispa above indicate the same formation of hemozoin is unknown, it is suspected that pattern of absorption similar with the results of research nimbolid compound interact with heme such as conducted which is suspected as the absorption of OH, CH artemisinin, due to the similarity between the molecular and ester C=O (7). It is supported by the statements that structure of artemisinin active substance (sesquiterpene lactone) and molecular structure of nimbolid compound OH group absorption is in the range of wave numbers of -1 (triterpenoid) (17). 2700-3800cm , CH group absorption is in the range of -1 wave numbers of 2850-3000cm and C=O group is in the Based on the average degree of parasitemia (Table 1) and -1 range of wave numbers of 1600-1850cm (10). Thus, it the concentration of parasite DNA ( figure 2 & 3), it can be might be that the methanol extract of T. crispa stem seen that the inhibitory effect of T. crispa extract on the contains the antimalarial compounds of glycoside growth of P. falciparum tends to follow the magnitude of furanoditerpen in form of tinocrisposide.
the dose, at doses more than 2.0 mg/ml, an increase in the inhibitory effect is no longer visible, even tends to rise modifying a wide range of molecules in the parasite cause although still lower than positive control. Moreover, the the parasite dies (21). In addition, the mechanism of correlation test results between the magnitude of T. crispa parasite growth inhibition by artemisin is due to the methanol extract dose and inhibition percentage and covalent bond between drugs and the parasite protein will concentration of parasite DNA have no significant damage the parasite membrane (22). relationship. This phenomenon is expected because of the The use of single drug, including artemisinin, can lead to role of antioxidants contained in T. crispa. Brotowali Plasmodium resistance on anti-malarial drugs. The use of (Tinospora crispa (L.) Miers), besides containing artemisinin as a single drug is only weakening but not antimalarial compound, also contains antioxidant killing the parasites. To prevent this, WHO (World Health compound. A previous research showed that the Organization) recommends the use of the antimalaria drug ethanolic extract of brotowali stem has an effect as an of artemisinin in combination with other antimalaria drugs antioxidant (18). Other research showed that brotowali through Artemisinin Combination Therapies (ACTs) plant contains flavonoid compound, epigenin, which has method which has been shown effective to achievecure up an activity as antioxidant (19). Antioxidant has an to 95% (23). important role for parasite survival in particularly at high The curve of parasitemia degree and the concentration of concentrations or under certain conditions. parasite DNA (the data are not shown) show that they are Another possibility is the influence of metabolic enzymes.
in line with the increase of observation time (48 and 72 Tinocrisposide in small dose has not been able to induce hours), it is followed by the downward trend in the average metabolic enzymes so that it remains active against degree of parasitemia and parasite DNA concentration Plasmodium growth, whereas larger dose of although the correlation test results provide insignificant tinocrisposide will stimulate metabolic enzymes, resulting correlation. in tinocrisposide compound that will be decomposed, Based on the results above, it can be concluded that the oxidized or conjugated, thus, the anti malaria activity will methanol extract of T. crispa stem has anti-plasmodium be reduced or become inactive (7). It has been explained activity, with IC value between 0,27mg/ml and 50 that it is predicted due to biphasic nature of tinocrisposide 0,29mg/ml, and the dose effective to inhibit the growth of compound, of which the interaction with the receptor is P. falciparum 3D7 strain is 2.0 mg/ml by decreasing not directly proportional to the concentration (20). It also parasitemia degree equal to 47,12% and DNA may be due to the physical nature of tinocrisposide itself concentration to 56,83% after 72 hours of incubation. In constituting a compound group of diterpene glycosides. this research, it can be concluded that the methanol The artemisinin treatment group can reduce the extract of T. crispa stem is able to reduce the parasitemia parasitemia degree of parasite culture, it is presumably degree of P. falciparum 3D7 Strain in vitro and could to be a because the artemisinin is known to have the bridge potential candidate for anti malarias. structure of peroxide (C-O-O-C), which plays an important role in the mechanism of drug action. This peroxide bridge