In vitro antimalarial activity of Calophyllum bicolor and hemozoin crystals observed by Transmission Electron Microscope (TEM)

Objective : In continuation of our antimalarial candidate drug discovery program on Indonesia medicinal plants especially from stem bark of Calophyllum bicolor. Metode : We extracted of bioactive crude extract with hexane, acetone and methanol from stem bark of Calophyllum bicolor and evaluated their antimalarial activity by using parasite Plasmodium falciparum in vitro. Results: Methanol fraction showed most active and potent antimalarial activity dose dependent in in vitro experiments with IC50 = 0.52 ppb, and hexane fraction showed moderate antimalarial activity, but dose not dependent, while acetone fraction have not antimalarial activity. TEM (Trasmission Electron Microscopy) analysis confirmed a remarkable reduction of hemozoin in the presence of the bioactive fraction. Conclusion: The results suggest that the antimalarial activity of Calophyllum bicolor is due, methanol fraction showed the best in vitro antimalarial activity then another fraction.


Introduction
Malaria is a serious parasitic disease transmitted by the bites of the female Anopheles mosquites , there are 4 species of plasmodium, e.i. Plasmodium falciparum, P vivax, P malaria, and P ovale, which P falciparum the most lethal [1,2,3]. Parasites belonging to Phyllum protozoa and genus Plasmodium are the most important causal pathogens and cause several human infection with globally massive impact [4]. Malaria remain one of the most significant world-wide public health problem, especially in tropical and subtropical regions [5]. Malaria is endemic to 90 counties world health and effects nearly 40% of the the world's population. Malaria is responsible for the death of 1-2 million each year with more than 90% of cases found in sub Saharan Africa and ≥1 case per 1000 population, and 80% of such cases are concentrated in 13 cauntires, over half in Negeria, Congo, Ethiopia, Tanzania and Kenya. [6,7,1]. Nigeria accounts for a quarter of all malaria cases in Africa. In the southern part of the country, transmission occurs all year round while in the north it is more seasonal [1].
One major virulence factor of this parasite is the highly variant P. falciparum erythrocyte membrane protein-1 (PfEMP) family. The malaria parasite in its blood stage is a voracious consumer of the globin component of hemoglobin, which causes the release of copious amounts (> 4000 mM) of toxic heme. Proteolysis of hemoglobin yields amino acid for protein synthesis as well as toxic heme. However, the parasite withstands heme toxicity by employing efficeinets mechanisms of heme detoxification. The conversion of heme to the relative benign heme polymer, hemozoin, is achieved by a process akin to template-mediated biomineralization. Templates for heme polymerization include performance hemozoin, histidin rich protein, and lipid or parasite cannot enzymatically cleave the porphyrin ring, heme is'detoxified' by conversion to an insolubly polymer, hemozoin [8 ,9].
The emergence and rapid spread of multidrugresistant strains of Plasmodium, particularly Plasmodium falciparum has led to serious problem for prophylaxis and treatment malaria, which becomes more difficult and limits the ability to control this disease and also limits the choice of drugs used. This has been identified as the current primary cause of control failure, this highlights the need to develop quickly more effective and less toxic new antimalarial drugs with different mechanism of action [9. 10]. It has been suggested that hemozoin formation is inhibited by the extract Calophyllum. Transmission electron microscope (TEM). TEM is one of type of electron microscope that has three essential systems: (1) an electron gun, generating the electron beam, and the condenser system, which focusing the beam onto the object, (2) the image-producing system, consisting of the objective lens, movable specimen stage, and intermediate and projector lenses, focusing the electrons passing through the specimen to form a real, highly magnified image, and (3) the image-recording system, converting the electron image toward some form perceptible to the human eye. The image-recording system usually covers of a fluorescent screen for showing and focusing the image and a digital camera for permanent records. Bisides,on, a vacuum system, consisting of pumps and their associated gauges and valves, and power supplies are needed.see Figure 4

Sample
C. bicolor P.F Stevent (2.858 kg) and C. europhyllum (1.555 kg) were collected in Bulungan, Kalimantan Island, in Indonesia. The plant were determined by staff from Herbarium Research Centre for Biology -LIPI and voucher specimen was deposited at the Herbarium Research Centre for Biology-LIPI. Indonesia.

Extraction
The air-dired C. bicolor P.F Stevent (2.858 kg) and C. europhyllum Lauter (2,555 kg) were successively extracted with n-hexane, acetone and methanol at room temperature for 4 day and such was repeated three times, the solvent were evaporated under reduced pressure to give hexane extract. The residue was extract successively with acetone (10 lt x 3) and methanol (10 lt x 3), then acetone and methanol were evaporated under reduced pressure to give a acetone and methanol extracts.

Antimalarial test
Erythrocytes infected with Plasmodium falciparum strain 3D7, from cultures obtained using the method Trager and Janson up to density of parasites 2%, were suspended in complete culture medium at a hematocrit of 5%. The suspension parasite was distributed in 96-well micro titer plates (200 µl per well). To determine the antimalarial activity against plasmodium falciparum, each extract (5 mg) was first dissolved in dimethyl sulfoxide (100 µl DMSO mix with vortek until all samples dilution completely, and then diluted with completed medium (RPMI) to obtain the desired concentration (5 mg x 10 -2 µl until x 5 mg x10 -9 µl), total volume in each well plate are 1 ml) and cloroquin was used as a positive control.
Compounds were tested in duplicate in 2% parasitemia cultures mostly at ring stage. For each assay, a parasite culture was incubated with the compound at 37 o C for 48 h in 5% CO2 at 95% relative humidity and frozen until the biochemical assay could be run. After 48 hours of incubation ach well was harvested to a glass were then fixation by methanol, also added Geimsa reagent.
Concentration of extract required to inhibit 50% parasite growth (IC50) was determined by computer. Parasit amount was monitoring by microscope, and computer by linear program Sigma-plot (IC50) was used for calculated of IC50. The antimalarial activity was expressed as the IC50 value (ppm), which was defined as the concentration of extract required to inhibit 50% parasite growth after 48-h incubation at 38 o C.

TEM (Transmission electron microscope)
Brief, small amount of extract were placed on the grid (formvar carbon coated Cu messed 300), at the acceleration voltage of 80 kV, using Zeiss. Model EM 10c (Jeol Japan). In addition, the apparatus was used for imaging at two different scales 24 and 30 nm. in-situ and after isolation was used for detect hemozoin In TEM, fixed-illumination mode, individual spectra are obtained from whichever area of the sample is illuminated. To control the sampling volume, the microscopist converges the beam to illuminate only the region of interest uses postspecimen apertures to select the divergence angle of electrons reaching the spectrometer. Because the sample and beam are fixed relative to each other, spectra, EDS and/or EELS, can be obtained in TEM mode

TEM imaging-Produccing system.
The specimen grid is conducted in a small holder in amoved specimen stage,. The objective lens is usually of short focsal length 1-5 mm (0.04-0.2 inch) and results a real intermediate image that is furher magnified by the projector lenses. A single projector lens will provide a range of magnification of 5:1. For practical reasons of image stability and brightness, the microscope is operated to give a final magnification of 1,000-250,000 x on the sreen to show the hemozoin crystal. Reduction of hemozoin crystal formation in the present of bioactive fraction had investigated by using TEM (Transmition Electrone Microscope) technich insitu and after isolation. The identification of spesific area of hemozoin an image, or pixels with specified characteristic, To furher characterize the antimalarial activity of Calophyllum, methanol extract (from the late sate of hemozoin formation) in the present sample after 1x24 hr incubation and in present sample after after 2x24 hr incubation were examined by TEM. There existed numerous hemozoin with diameter 1µm after 1x24 incubated, but there are not hemozoin after 2x24 hr incubation).
The transmission electron microscope (TEM) image in Figure 5

Sepration results
The present investigation for antimalarial principles from C. bicolor has led to the finding of three fraction, e.i, hexane (208.29 g), acetone (413.62 g) and methanol 149.8 g.

Antimalarial activity results
According to the previous paper [12] which it's explained that antimalarial activity of methanol fraction of C. bicolor have better than hexane fraction, and hexane fraction have better than acetone fraction while the acetone fraction have not antimalarial activity, these data showed in Figure 7. Antimalarial activity of the metanol fraction best compare hexane and acetone fraction, these methanol fraction are devided further into five sub fraction (e.i sub fraction 9 :1 ; 8 :2 ; 7 :3 ; 6 :4 ; 5 : 5. The data antimalrial activity explaned in

Discussion
The present investigation for antimalarial principal from methanol fraction of C. bicolor has led to the finding of five sub fraction e.i sub fration Percentage of parasite P. falciparum growth versus sample concentration (10 -9 to 10 -1 mg/mL) was observed.
The increasing in sample concentration should decrease the parasite growth percentage.
Unfortunately,In the fact state that the increasing sample concentration from 10 -8 to 10 -7 mg /mL did not cause a decreasing percentage of parasite growth, this illustrates that the percentage decreasing parasite growth does not depend on the sample concentration (see Figure  8).
Signal at δH 3,12 ppm (1H, dd J 5,28 & 11,65) is signal of mehine (CH) at position C-3 of triterpenoid compound, Isolate compound also have one hydroxil group at C-3. Unfortunately isolate compound have not signal at 6-8 ppm, these indicated that isolate compound have not aromatic proton.