INTRODUCTION:
Thrombocytopenia and anemia is the most common clinical manifestation and plays an important role in malaria morbidity and mortality.4,5
Anemia can occur due to erythrocyte outbreaks: the degree of anemia depends on the parasitic species that causes it. Plasmodium falciparum infects young erythrocytes and mature erythrocytes. Plasmodium vivax and Plasmodium ovale only infect young erythrocytes, which account for only 2% of all erythrocytes. Anemia is evident in malaria caused by Plasmodium falciparum with rapid and severe destruction of erythrocytes, whereas anemia caused by Plasmodium vivax, Plasmodium ovale, and Plamodium malariae generally occurs in chronic conditions.6
As severe malaria is life threatening infectious disease killing more than million human beings every year, especially children, thus it becomes mandatory to have acquaintance about the disease, disease causing vectors, various categories of therapeutically active drugs, their pharmacokinetics and safety.7 In 2010, there were an estimated 216 million cases of malaria worldwide, with a mortality rate of 655,000 people. Based on data from the Indonesian Ministry of Health, until the end of 2017 there were 261,671 cases of malaria in Indonesia, 100 of which died. One of the provinces in Indonesia where there are many cases of malaria is West Nusa Tenggara Province. Based on reports from districts/cities, the number of suspected malaria in 2017 was 111,781 people and 122,326 people were tested for blood and found to be malaria positive for 1,190 cases.8 Novel therapies are needed against malaria because of emergence of multidrug resistant plasmodium falciparum to existing drugs such as Chloroquine (CQ) and Sulfadoxin-Primetamin (SP). The emergence of chloroquine-resistant P. falciparum malaria in southeast Asia in the 1960s caught the attention of the Chinese government.9 According to the molecular institute Eijkman, almost 100% of malaria parasites in Indonesia have experienced gene mutations and are immune to CQ and between 30-70% are immune to SP.10,11
The emergence of Plasmodium resistance to antimalarials led the research to find new antimalarials to replace ineffective antimalarials. One effort to find and develop new antimalarial drugs that are relatively safer and cheaper is through the exploration of active compounds from natural materials. The potential of extractive substances as a source of medicine is supported by the high biodiversity of medicinal plants in Indonesia's tropical forests which is a source of secondary metabolite compounds that can be used as traditional and modern medicines to overcome various diseases including antimalarial.12 Some medicinal plants used in the treatment of malaria include lime tree leaves, red fruit, mango parasite leaves, mangosteen rind, betel fruit, cempedak bark, mundu bark, sunflower leaves.13 Whereas some natural compounds were successfully isolated and proven antiplasmodial activity both in vitro and in vivo. These compounds are generally secondary metabolites of the alkaloids, terpenoids, cuasinoids, flavonoids, limonoids, chalcons, peptides, xantons, quinones, coumarin, and several other antimalarial drugs.14
Chalcone (C15H12O), 1,3-diphenyl-1propen-1-on or benzylideneacetophenone is an essential compound in nature. Chalcone and its derivatives have several activities such as: antibacterial, antiplatelet, antiulcerative, antimalarial, anticancer, antiviral, antileishmanial, antioxidant, antihyperglycemic, immunomodulatory, anti-inflammatory, antimicrobial agent.15,16 One of the plants that are rich in chalcone content and thrives in West Nusa Tenggara is Ashitaba. Osaka Pharmaceutical University research in 1990 stated that the amount of active ingredient in 100 g Ashitaba was Xanthoangelol 0.25%, 4-Hydroxyderricin 0.07% and total Chalcone 0.32%.17 In the last decade, several active constituents representing chalcone, flavanone and coumarin has been isolated and characterized from Ashitaba. Most of the literature on bioactive metabolites isolated from Ashitaba concerns the most abundant chalcone activity found in Ashitaba roots.18
MATERIALS AND METHODS:
Materials:
The materials in this research are micropipette, erlenmeyer, measuring cup, glass jar, analytical balance, laminar airflow (LAF), incubator, refrigerator, mix gas, pipette, petri-dish, well 96, sterile medium bottle, centrifuge, microscope, water bath, glass stir bar, oven, silica gel GF254, chamber, and microtube, ethanol, Ashitaba roots (Angelica keiskei K), parasites used are Plasmodium falciparum strain 3D7, 50% dimethyl sulfoxide (DMSO), Roswell Park Memorial Institute (RPMI), 20% Giemsa coloring and 5% sorbitol, Chloroquine.
Methods:
Extraction of ashitaba roots:
The making of Ashitaba root ethanol extracts used 600 gram of Ashitaba roots soaked in 1000 ml of 70% ethanol for 24 hours. The maceration process for each simplicia was remastered three times. Maserat that has been collected was thickened using a rotary evaporator at 60oC.
Phytochemical Screening:
Flavonoids: 2 ml of extract was put into a test tube, added 10 drops of 25% HCl and 0.1 mg of Magnesium powder, if there is a change in yellow to orange, indicating the presence of flavonoids (Wilstater method). Alkaloids: 2 ml of extract was put into a test tube, plus 0.5 ml of 2% HCl and the solution was divided into 2 tubes. Tube 1 was added 2-3 drops of Dragendroff reagent, and tube 2 was added 2-3 drops of Mayer reagent. The results showed positive alkaloids when brick, red, orange (with Dragendroff reagent) and white or yellowish sediments formed (with Mayer reagents). Saponin: 2 ml of the extract is put into a test tube, added with water (1: 1) and then shaken for 1 minute, if after adding HCl 1 N the foam formed can last for 10 minutes with a height of 1-3cm, then the positive extract contains saponin. Tannins: 2 ml of extract was put into a test tube, added 2-3 drops of 1% FeCl3 solution. If the solution produces a blackish or dark blue color, the extract contains catechol tannins.
Antimalarial Activity Assay:
Sample preparation: 1 mg sample was dissolved in 100 μl DMSO (stock solution, concentration of 10,000 µg/ml). The serial dilution was made from the stock solution to obtain a final concentration of 1000 μg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml. Parasite preparation: The parasites used in this test were parasites that have been synchronized (ring stage) with parasitemia ± 1%. Procedure: 2 µl of the test solution with various concentrations was taken and inserted in each microwell, then 198 μl of parasite was added to obtain the final concentration of the test sample of 100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 µg/ml, 0.01 µg/ml. The microwell then put in a chamber and given a mix gass (5% O2, 5% CO2 and 90% N2). The chamber containing was incubated 48 hours, temperature 37oC. The culture is then harvested and a thin blood smear is made with 20% Giemsa staining.
Statistical analysis:
Blood smears that have been made are calculated by counting the number of infected erythrocytes every 1000 normal erythrocytes under a microscope. The data is then used to determine percentage growth and percentage inhibition. Percent growth is obtained by the following formula:
Inhibiton percentage was calculated as follows:
Note:
D0 = Parasitemia percentage of infected red blood cell on day 0
Xu = Growth percentage of each sample
Xk = Growth percentage of negative control
Based on the percent inhibition data, an analysis was made between the test concentration of the percent inhibition with the SPSS program using probit log analysis to determine the IC50 value or the concentration of the test material that could inhibit the growth of parasites by 50%.
RESULTS AND DISCUSSION:
Extraction in this study used the maceration method because it was not heated so that it can be used to extract compounds that were thermolable. Maceration is the process of extracting simplicia using solvents with several shaking or stirring at room temperature.20 Maceration carried out in this study uses 70% ethanol solvent because according to Tiwari et al. 2011 ethanol with a concentration of 70% can attract high bioflavonoid active compounds compared to using pure ethanol. This is because 70% ethanol consists of 30% water content so that the polarity of the solvent will increase so that it will facilitate the extraction process, 70% ethanol will cause penetration into cellular membranes to extract intracellular materials.21 Data resulting from simplicia and yield from Ashitaba root extraction process is presented in table 1.
Table 1. Extract rendement of Ashitaba roots
|
Fresh roots (g) |
Simplicia (g) |
Thick extract (g) |
Rendement (%) |
|
4,100 |
1,200 |
13.23 |
1.102 |
Phytochemical Screening:
The extract was dissolved with ethanol then filtered to produce a clear yellow filtrate. Furthermore, the filtrate was divided into 6 tubes, each of 2 ml to be reacted with some reagents as a phytochemical screening test for Ashitaba roots, as shown in Figure 1.
 |
Figure 1. Phytochemical Screening Results
Note: (a) Ashitaba Root Extract that has been dissolved with ethanol (b) Flavonoids (c) Saponins (d) Tanin (e) Alkaloids (dragendroff) (f) Alkaloids (Mayer)
Table 2. Phytochemical Screening Results Data
|
S. No |
Secondary metabolites |
Result |
||
|
Positive |
Negative |
Note |
||
|
1 |
Flavonoid |
ü |
|
Orange-red |
|
2 |
Saponin |
|
ü |
No foam |
|
3 |
Tanin |
ü |
|
Blackish green |
|
4 |
Alkaloid (Dragendroff) |
|
ü |
No deposits |
|
|
Alkaloid (Mayer) |
|
ü |
No deposits |
In vitro Antimalarial Activity Test:
Research on the antimalarial activity of Ashitaba root ethanol extracts (Angelica keiskei K.) was carried out on Plasmodium falciparum strain 3D7 in vitro. The in vitro activity test with P. falcifarum was carried out as a preliminary test to evaluate the biological material which was suspected of having antimalarial activity. Screening extracts from plants used for traditional medicine, in vitro testing offers the advantage of being able to use P. falciparum which causes malaria in humans who have several strains that are resistant to many antimalarial drugs as a parasitic test.24 In vitro antimalarial testing was done at various concentrations through several stages which are sample preparation, parasite check and parasitic inhibition assay.
Figure 2. The first ten visual fields (a) observation of 1000 erythrocytes (b) parasitic syzon stage (c) ring parasites stage
The parasites that were counted were parasites with ring-stage indicated by black dots or small circles inside the erythrocytes, the parasitemia as calculated per 1000 erythrocytes to find the percent of inhibition that occurred. The culture check was done by making a thin blood smear colored with 10% giemsa. Negative control in this study using DMSO aim to ensure that antimalarial activity at the time of the study came from test samples. The choice of DMSO as a negative control is because DMSO was a solvent that can dissolve polar and nonpolar compounds. In addition, DMSO did not provide parasitic growth inhibition.25 While positive control used chloroquine. Based on its chemical structure, chloroquine phosphate is a 4-aminoquinoline derivative. The 4-aminoquinoline derivative had relatively high antimalarial activity compared to quinine. The toxicity is relatively low, long-term use with large doses can affect hearing and vision. The way the chloroquine drug works is by destroying the form of sexual erythrocytes (gametocytes) from the malaria parasite thereby preventing the spread of plasmodium to the anopheles mosquitos.26
According to research conducted by Antonius et al. (2001) stated that "The prevalence of chloroquine resistant cases was found to be 40.8%”. The area declared chloroquine-resistant and the treatment replaced with alternative antimalarial treatment, the results of the WHO meeting in Kuala Lumpur, Malaysia in 1981. The meeting determined the number of subjects that needed to be tested for at least 30 patients, endogenous falciparum and in vivo test results were found to be 5% degrees RI (resistant I) and or R II or R III, or 1% R II and or R III.
In vitro antimalarial activities are experimental models for detecting antiplasmodial activity of plant extracts in the erythrocytic stage of malaria parasites.27 In vitro antimalarial activity test results of Ashitaba root extracts with 5 concentrations obtained growth rate, inhibition and IC50 values (Table 3). Percentage of growth is obtained from the percentage of infected erythrocytes per 1000 erythrocytes. Ethanol extract of Ashitaba root showed inhibition on the growth of P. falciparum strain 3D7 in concentrations of 100 µg/ml (66.36%), 10 µg/ml (42.90%), 1 µg/ml (29.32%), 0.1 µg/ml (12.81%) and 0.01 µg/ml (2.62%). According to Pouplin et al. 2007, and extract was said to be active in reducing parasitemia if the extract can reduce parasitemia >30%.28 Based on this, in conjunction with 100 μg/ml and 10 μg/ml, Ashitaba root extracts could be said to be active in reducing parasitemia.
Table 3 Data on Antimalarial Activity Test Results in vitro
|
Sample |
Concentration (µg/ml) |
Growth (%) |
Inhibition (%) |
IC50 (µg/ml) |
|
Extract |
100 |
1.09±0.03 |
66.36±0.72 |
16.09 |
|
10 |
1.85±0.01 |
42.90±0.18 |
||
|
1 |
2.29±0.01 |
29.32±0.13 |
||
|
0.1 |
2.82±0.02 |
12.81±1.04 |
||
|
0.01 |
3.15±0.01 |
2.62±0.21 |
||
|
Positive control (Chloroquin) |
100 |
0 |
100 |
0.007 |
|
10 |
0.28±0.28 |
91.35±0.91 |
||
|
1 |
0.55±0.49 |
82.86±1.60 |
||
|
0.1 |
0.97±0.21 |
70.22±0.53 |
||
|
0.01 |
1.42±0.01 |
56.17±0.63 |
||
|
Negative control (DMSO) |
|
3.24±0.01 |
|
|