Antiplasmodial activity of Ethanolic extract of Cassia spectabilis DC leaf and its inhibition effect in Heme detoxification

Background In previous studies, Cassia spectabilis DC leaf has shown a good antiplasmodial activity. Therefore, this study is a follow-up study of the extract of leaf of C. spectabilis DC on its in vitro and in vivo antiplasmodial activity and mechanism as an antimalarial. Methods The extract was fractionated, sub-fractionated and isolated to obtain the purified compound. In vitro antiplasmodial activity test against Plasmodium falciparum to find out the active compound. In vivo test against P. berghei ANKA-infected mice was conducted to determine prophylactic activity and antiplasmodial activity either alone or in combination with artesunate. The inhibition of heme detoxification test as one of the antimalarial mechanisms was carried out using the Basilico method. Results The results showed that active antimalarial compound isolated from C. spectabilis DC leaf had a structural pattern that was identical to (−)-7-hydroxycassine. Prophylactic test of 90% ethanolic extract of C. spectabilis DC leaf alone against P. berghei ANKA-infected mice obtained the highest percentage inhibition was 68.61%, while positive control (doxycycline 13 mg/kg) was 73.54%. In combination with artesunate, 150 mg/kg three times a day of C. spectabilis DC (D0-D2) + artesunate (D2) was better than the standard combination of amodiaquine + artesunate where the inhibition percentages were 99.18 and 92.88%, respectively. The IC50 of the extract for the inhibitory activity of heme detoxification was 0.375 mg/ml which was better than chloroquine diphosphate (0.682 mg/ml). Conclusion C. spectabilis DC leaf possessed potent antiplasmodial activity and may offer a potential agent for effective and affordable antimalarial phytomedicine.


Background
Research to obtain new antimalarial drugs, both synthetic drugs and those derived from natural materials, especially from plants, is still ongoing. One of Indonesian plants that has been traditionally recognized to treat malaria is Cassia spectabilis DC from the Caesalpiniaceae family. Previous study related to this plant using in vivo test showed that this plant was a potential antimalarial phytomedicine [1]. New antimalarial active compounds have also been obtained from different plant, Cassia siamea from the same genus of C. spectabilis that identified as Cassiarin A alkaloid compound [2,3]. Based on these results, an in vitro test on antiplasmodial activity to find out the active compound isolated from C. spectabilis DC has been conducted.
Prevention of malaria can be done in various ways, one of which is chemoprophylaxis. Chemoprophylaxis is one way to reduce the risk of malaria infection and alleviate clinical symptoms of malaria. Chemoprophylaxis used before traveling to malaria endemic area to avoid infection. In Indonesia, therapeutic choice used for malaria prophylaxis is doxycycline and tetracycline [4]. Both can be used as chemoprophylaxis for malaria but there are many undesirable effects from this drug [5]. Therefore, this research was performed to find out and develop drugs that can be used as an effective antimalarial prophylaxis, safe, have few side effects, cheap, and easy to obtain, especially those from plants, namely C. spectabilis DC leaf.
Furthermore, antimalarial drug development and discovery is expected to provide new drugs which is not only have antiplasmodial activity in vitro and in vivo, but also has a safety mechanism to be applied to human. Research related to the biochemical process that unique of malaria parasites plays an important role in the development of new antimalarial drugs. Malaria parasites consume hemoglobin from erythrocytes during their life cycle, however, parasites are unable to digest ironcontaining heme molecule. Heme is toxic due to the reactivity of iron. Therefore, parasite has developed the mechanism to detoxify it by polymerization of heme to form hemozoin or malaria pigmen [6,7]. The structure of hemozoin through X-ray diffraction and IR spectroscopy has been found to be similar to β-hematin [8] β-hematin is synthetic hemozoin which chemically [8], spectroscopically [9] and crystallographically [10] similar to hemozoin which consists of Ferriprotoporphyrin units linked into a polymer by propionate oxygen-iron bonds [8,11]. The inhibition of heme detoxification has been the target of antimalarial drugs, such as chloroquine and artemisinin [12,13], since inhibit the heme detoxification can kill the parasites. Currently, the effect of ethanol extract of C. spectabilis DC leaf on biochemical activity such as potential inhibition of heme detoxification in the food vacuole of malaria parasite and stage-specific activity against asexual stage of parasite has been tested.
In addition, parasitic resistance to some of the existing antimalarial drugs is the biggest problem in overcoming this disease, especially in malaria endemic areas [14]. The combination therapy with artemisinin derivatives or commonly referred to artemisinin-based combination therapy (ACT) is highly recommended by WHO as the preferred therapy that is able to control the spread of resistance of P. falciparum [15,16]. Previous studies have found an effective dose of 90% ethanolic extract of C. spectabilis DC leaf was 150 mg/kg bodyweight which were given three times daily [1].
Based on this result, both ethanolic extract of C. spectabilis DC leaf alone and in combination with artesunate has been tested to determine the inhibition of growth of parasites in P. berghei ANKA-infected mice in vivo, since the ACT is more effective in reducing parasitemia [17]. The combination therapy will be carried out in extractdrug regimens and an overview of the resulting antimalarial activity will be obtained. A therapeutic effect of an appropriate combination of artesunate and 90% ethanolic extract of C. spectabilis DC leaf is reported herein.

Plant material
C. spectabilis DC leaf was obtained from Purwodadi Botanical Garden-Indonesian Institute of Sciences [Lembaga Ilmu Pengetahuan Indonesia, LIPI], Pasuruan District, East Java Province, Indonesia, and the determination of specimen was performed at the above institution. The specimen was then deposited as herbarium in the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga with registration number of 02/W/XI/2016.

Parasite and culture preparation
Plasmodium falciparum 3D7 strain was obtained from Faculty of Pharmacy, Universitas Airlangga, Surabaya. The parasite was cultured in complete RPMI 1640 medium supplemented with 5.96 g HEPES, 0.05 g hypoxanthine, 2.1 g NaHCO 3 , 50 μg/ml gentamycin and completed with 10% human O+ serum under anaerobe condition and incubated in a 37°C incubator [18]. Parasitemia was observed daily prior to antimalarial assay. Plasmodium berghei ANKA strain was originally obtained from Eijkman Institute for Molecular Biology, Jakarta, and maintained at Faculty of Pharmacy, Universitas Airlangga. The P. berghei ANKA was infected into male BALB/c mice and observed the parasitemia level.

Experimental animals
Experimental animal used in this study was BALB/c strain male mice obtained from Faculty of Veterinary At the end of the tests, all animals were followed the euthanasia procedure. The mice were sacrificed by cervical dislocation after anesthesia by intraperitoneal injection of 100 mg/kg bodyweight ketamine [19]. The dead animals were then buried.

Extraction
The extract was made by three times macerating dried C. spectabilis DC leaf powder using 90% ethanol. The macerated extract was then evaporated using a rotavapor.

Isolation of compound from C. spectabilis DC leaf
A thousand grams of dried powder of C. spectabilis DC leaf was macerated with n-hexane, then the pulp powder was extracted again with methanol. The extract was subjected to liquid-liquid partition using ethyl acetate and 3% tartaric acid. The isolation proceess was done by adding 3% tartaric acid until the atmosphere becomes acidic to turn the alkaloids in alkaline form into alkaloids salts. This alkaloid salt was partitioned using ethyl acetate, and the alkaloids were present in the aqueous acidic layer at the bottom. The alkaloid was the isolated from their salt form in aqueous layer, by adding NaCO 3 to increase the pH to 9-10, and the alkaloids returned to alkaline form. Furthermore, the aqueous layer with pH 9-10 was then extracted liquid-liquid using chloroform. The fractions so called chloroform fraction. The isolate pure alkaloid compound from other compounds, the chloroform fraction, was further fractionationed by column chromatography using stationary phase of silica gel 60 with a series of gradient mobile phases was started using 100% chlorofom, chloroform: ethyl acetate (CHCl 3 : EtOAc; 2:1), chloroform:ethyl acetate:methanol (CHCl 3 : EtOAc: MeOH; 2:1:2), and finally 100% methanol. The selection of the mobile phase was based on the results of the orientation by TLC, which gave good separation results. The solvent used for each gradient was 200 ml and the collected fractions were combined based on similarity of the TLC profile. Nine fractions (C.1-C.9) were obtained.
The fractions were then assayed for their antiplasmodial activity. The active fraction was then subjected to silica gel 60 column chromatography and was eluted using CHCl 3 : MeOH (1: 00: 1) to yield sub-fractions (SFC.8.1-SFC.8.4). The sub-fractions were further assayed for their antiplasmodial activity. The antimalarial active sub-fraction was then purified by preparative thinlayer chromatography (PTLC) using CHCl 3 : MeOH (8.5: 1.5) to yield compounds C.8.3.1 and C.8.3.2. Identification of active compound was carried out using the TLCdensitometry, UV-Vis spectrophotometry, FTIR spectroscopy, and NMR. The procedures employed for the preparation of the plant active compounds are illustrated in Fig. 1.

In vitro antiplasmodial activity test
The extract was dissolved in DMSO (the final DMSO concentration in a well culture plate not more than 0.5%) and diluted with complete RPMI medium containing RPMI 1640, 10% human plasma, 25 mM HEPES, and 25 mM NaHCO 3 to make the final concentrations of 10, 1, 0.1, 0.01, and 0.001 μg/ml. Stock of parasite cultures were further diluted with uninfected type O+ human erythrocytes and culture medium to make initial parasitemia of 1% and a hematocrit of 2%. This final parasite culture was immediately used for antiplasmodial assay.
The test was carried out in duplicate. The plates containing parasite cultures and extracts were then incubated in a 37°C incubator in a candle jar for 48 h. Observation of stage-specific antiplasmodial activity in this in vitro test was performed by sampling the blood films from each well at 6, 12, 24, and 48 h. At the end of test thin films were prepared from each well and stained with 10% Giemsa solution prior to counting parasitemia [20]. The 50% inhibitory concentration (IC 50 ) value was determined using probit analysis based on the relation of log concentration of test compound and % inhibition of parasites growth.

In vivo antimalarial prophylactic activity test
In vivo test for antimalarial prophylactic activity of 90% ethanolic extract of C. spectabilis DC leaf used Peters method with slight modification [21]. Forty two male adult BALB/c mice were randomly divided into six groups [22]. Group 1 as a negative control group was given a 0.5% Na CMC suspension solution. Groups 2, 3, 4, and 5 were given extract at the doses of 100, 200, 400, and 800 mg/kg, respectively. Group 6 as a positive control group was given a doxycycline of 13 mg/kg suspension solution. Each treatment was given orally once a day for 4 days before parasite infection. On the fourth day the mice were infected with P. berghei ANKA. Each mouse in each group was infected with 1 × 10 6 infected erythrocytes. Thin blood films from each mouse were made at 72 h post infection. Determination of parasitemia, percentage of the parasites' growth, percentage inhibition of parasites' growth, and effective dose 50 (ED 50 ) were based on Ekasari et al. [20].
Suppressive effect of ethanolic extract of C. spectabilis DC leaf combined with artesunate The purpose of this test was to increase the effectiveness of the extract. The suppressive effect of ethanolic extract of C. spectabilis DC leaf in combination with artesunate, respectively against P. berghei ANKA infection in mice was determined using Peters' 4-day suppression test procedure [21]. A donor mouse densely infected with parasites was anaesthetized with chloroform and the blood was collected through cardiac puncture. The presence of parasitemia was established by microscopic examination of a thin blood film. The blood was diluted with phosphate buffered saline (PBS) so that each 0.2 ml of blood contained 1 × 10 6 P. berghei ANKA infected with erythrocytes. A total of 0.2 ml of diluted blood was injected intraperitoneally into 36 healthy mice. The infected animals were randomly divided into six groups those were Group A-F. The animals were treated shortly after inoculation on day zero (D 0 Determination of parasitemia level, percentage parasites' growth, percentage inhibition of parasites' growth, and the ED 50 were as described on Ekasari et al. [20].

Heme polymerization inhibition test
The inhibition of heme polymerization test was performed based on the Basilico method [23] with slight modification in the concentration of hematin solution and the sample used. The Basilico method is an in vitro spectrophotometric microassay of heme polymerization. A 96-well Ubottomed microplates was used in this assay. The relative amounts of polymerized and unpolymerized hematin were determined using an ELISA reader. The final concentration of the extract samples ranged from 2 to 0.01 mg/ml.

In vitro antiplasmodial activity test
All the extracts, fractions, sub-fractions, and isolated compounds from C. spectabilis DC leaf were continuously screened in vitro for their antiplasmodial activity against chloroquine-sensitive P. falciparum (3D7 strain), using microtechnique which demonstrated on the previous study [24]. The samples for these tests were hexane extract, methanolic extract, ethyl acetate fraction and chloroform fraction. The results are shown in Table 1.
Identification using 1 H-NMR spectroscopy showed a characteristic signal of two hydroxyl protons at δ 3.  Effect of C. spectabilis on parasitic stage development Stage-specific activity test of 90% ethanolic extract of C. spectabilis DC leaf against P. falciparum 3D7 strain was performed at different incubation periods of 0, 6, 12, 24, and 48 h to find out the effect of the extract to the growth of parasites at each stage of development. The extract concentration used was 100 μg/ml. The results of this experiment are shown in Table 5 and Fig. 4.
No sharp difference was observed on the growth of each parasitic stage during incubation period of 0-12 h. However, opposite direction of percentage parasitemia was seen at 12 to 48 h post incubation compared with  control which moved upward but the tests extract went to 0. At 12-24 h of incubation period, parasite growth decreased but not significantly compared to negative control. Whereas at 48 h of incubation, parasite growth decreased significantly (p = 0.005) compared to negative control, with an inhibition percentage of 100% (Fig. 5).

In vivo antiplasmodial prophylactic activity test
The results of in vivo antiplasmodial prophylactic activity test of 90% ethanolic extract of C. spectabilis DC leaf against P. berghei ANKA infection in mice is shown in Table 6. The dose of extract of 800 mg/kg provided the greatest inhibitory effect (68.61%) compared to other doses. Probit analysis resulted in ED 50 value was 161.20 mg/kg. Table 7 shows the results of suppressive activity tests by C. spectabilis DC leaf extract combined with artesunate. Suppressive effects produced by the three extractartesunate combinations were higher than artesunate alone. Moreover, the suppressive effects of group D (ethanolic extract of C. spectabilis DC leaf at 150 mg/kg (three times a day) on D 0 -D 2 and artesunate at 36.4 mg/ kg on D 2 ) was higher (99.18%) than those showed by artesunate alone (82.60%) and artesunate-amodiaquine combination (92.88%).

Heme polymerization inhibition test
The IC 50 value of the heme inhibition test by 90% ethanolic extract of C. spectabilis DC leaf was 0.375 mg/ml, while chloroquine as an antimalarial standard compound was 0.682 mg/ml (Table 8).

Discussion
Anti-malarial activities of C. spectabilis DC leaf was conducted to extract, fraction and pure isolate. The active compounds were identified by TLC-densitometry, UV-Vis spectrophotometry, FTIR spectroscopy, and NMR.
The greatest prophylactic inhibitory effect in vivo of 90% ethanolic extract of C. spectabilis DC leaf against P. berghei ANKA was 68.61% provided by the mice treated with 800 mg/kg of the extract. This result was lower than that of mice treated with doxycycline (73.54%). The highest dose that can be used in mice is 1000 mg/kg of bodyweight [27].
The effect of 90% ethanolic extract of C. spectabilis DC leaf to inhibit heme detoxification process showed the IC 50 value of 0.375 mg/ml which was higher than that of chloroquine as a standard antimalarial drug which was 0.682 mg/ml.
The potential for 90% ethanolic extract of C. spectabilis DC leaf in the inhibition of hemozoin formation caused morphological and growth disturbances of malaria parasites due to membrane damage and disruption of the activity of several enzymes [28] as seen during the 12-h incubation period where the inhibitory activity 70% increase compared to controls. Furthermore, after 24 h incubation, the growth of parasites was 100% inhibited compared to the control ( Table 5).
The advantage of the combination therapy was to increase the effectiveness of extract to prevent or slow the onset of resistance to a single antimalarial drug [29]. Selection of 90% ethanolic extract of C. spectabilis DC leaf combination with artesunate referred to the basis of malaria treatment which is a standard antimalarial drug recommended by WHO [30].
The dose of 90% ethanolic extract of C. spectabilis DC leaf used was 150 mg/kg given three times [1]. Artesunate is the artemisinin derivative is a schizonticidal with fast onset of action and gametocytocidal which can reduce malaria transmission in endemic areas [31]. Artesunate at a dose of 36.4 mg/kg was converted dose from human to mouse. The three times a day of artesunate therapy was compared to single dose therapy to prevent the immediate emerging of parasites resistance to this antimalarial drug. The control used in this study was a single dose of artesunate alone at the dose of 36.4 mg/kg and amodiaquine alone at the dose of 72.8 mg/kg which were given for 3 days. As recommended by WHO that ACT administration that given for 3 days has been able to effectively inhibit the growth of parasites, beside antimalarial drug should be effective, safe, and used in a short time. The Na CMC was used as negative control since this solvent was used to dissolve the extract in this test.
The interesting result was seen in the combination therapy using three times a day of 90% ethanolic extract of C. spectabilis DC leaf at dose of 150 mg/kg for 3 days with a single dose of artesunate at the dose of 36.4 mg/ kg given on the third day. This combination therapy showed a higher inhibitory activity (99.18%) compared to that of combination artesunate and amodiaquine for 3 days (92.88%). This combination therapy may overcome  the resistance of parasites to artesunate because shorten the duration of treatment period. Thus, the combination of 90% ethanolic extract of C. spectabilis DC leaf with artesunate can be expected as a new antimalarial combination drug which may replace the artesunate-amodiaquine combination drug that has been used so far. The mixture of artesunate-amodiaquine caused the drug preparations will become unstable [32]. Concurrent drug administration in a separate formula have the disadvantage of reducing patient compliance to take the drug, but the combination therapy can shorten the duration of treatment therapy [30].

Conclusion
The results show 90% ethanolic extract of C.spectabilis DC leaf has a very good antiplasmodial activities both in vitro and in vivo study and revealed potentiated effect in combination with artesunate. The 90% ethanol extract of C. spectabilis DC leaf also found to be active in inhibiting the heme detoxification process. Compound of (−)-7-hydroxycassine may play as important role in antimalarial activities. Therefore, this plant can be potentially used as a new source for the development of new plant-based antimalarial agent.