Antimalarial Activity of Nano Phytomedicine Fraction of Syzygium cumini Fruit in Rodent Malaria

Lilik Maslachah¹*, Neny Purwitasari²

¹Departement of Basic Veterinary Medicine, Veterinary Pharmacy, Faculty of Veterinary Medicine Universitas

Airlangga, Surabaya 60115 Indonesia.

²Departement of Pharmacognosy, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115 Indonesia.

*Corresponding Author E-mail: lilik.maslachah@yahoo.com

ABSTRACT:

The purpose of this study is to prove the antimalarial activity of nanophytomedicine fraction Syzygium cumini fruit in rodent malaria. Preparation of nanoparticle formulations using ionic gelation. This research used 80 mice divided into 8 groups. K: not infected, K-: infected, P1: infected+chloroquine, P2: infected+fraction, P3, P4, P5, were infected + nanoparticles, and P6 were infected + nanoparticles combined with chloroquine. Mice were infected with red blood cells containing 1x10⁶ in 0.2ml P.berghei. Treatment for 4 days and 24hours post-infection. On the 8th day, post-infection blood and organs were collected. The results showed the body weight of mice showed a decrease except for the P1 and P6 groups. Increased % parasitemia and decreased parasite growth inhibition in group K- compared to P1, P6, and P5. The splenic index of the K group was significantly different from the other groups. The hepatic index of the K group was not significantly different from the P1 and P6 groups, but significantly different from the other groups. The hematological changes of hemoglobin and hematocrit in groups K- and P3 showed a decrease. Leukocytes, monocytes, and granulocytes in all groups were in the normal range. The conclusion is Nanoparticles fraction of Syzygium cumini at a dose of 400 mg/kg BW and combination therapy with chloroquine have better potential as an antimalarial seen from the decreased parasitemia, increased inhibition of parasite growth, increased body weight, splenic index, hepatic index and Hematological changes of mice infected with Plasmodium berghei.

KEYWORDS: Hematology, Hepatosplenomegaly, Infectious Disease, Malaria, Mean Survival Time.

INTRODUCTION: 

Malaria is a contagious and infectious disease caused by Plasmodium which occurs especially in tropical countries. Each year, approximately 300-500 million people in the world are infected with malaria, and 1-3 million people die. The greatest number of deaths occurred in Africa and the Sahara with 90% of deaths occurring in children under 5 years old. It is estimated that the average decline in economic growth is around 1.3% in high-risk countries of malaria¹. The development of treatment, prevention, and control of malaria is a major problem in the world. Nowadays, this disease is still dependent on the use of antimalarial drugs.

Currently, there has been a decrease in the efficacy of anti-malarial drugs and many parasites are already resistant to some antimalarial drugs. There is also no effective vaccine to control malaria infection due to the complex life cycle of the Plasmodium parasite².

Due to the spread of multidrug-resistant malaria parasites, it is essential for the development of new antimalarial drugs, finding new drug targets and novel clues in identifying the drug-resistant malarial genes of the parasite using the silico method^3,4. The weaknesses of natural-origin medicine are low stability, solubility, and absorption, thus reducing bioavailability and efficacy. Natural-origin medicine also does not have a specific action on organ targets. The development of new drugs of natural origin with an active compound delivery system with nanoparticle technology will be able to increase stability, solubility, and absorption⁵. In addition, there is a decrease in the efficacy of the antimalarial drugs which is currently in use, so it is very important for the development of formulations of natural origin medicine, namely nanoparticle technology to increase their efficacy and therapeutic effects.

One of the medicinal plants that can be found easily in Indonesia is juwet (Syzygium cumini). The results of the research that Syzygium cumini has radical scavenging activity and strong antioxidants⁶. The use of several parts of the Syzygium cumini antimalaria plant has shown that the leaves of Syzygium cumini as a therapeutic adjuvant had a better inhibition effect on plasmodium than the stem bark⁷. The results of the research by Maslachah et al. (2020) showed that the Syzygium cumini fruit was better than the leaves⁸_. This study developed a nanoparticle formulation from the extract of Syzygium cumini L fruit as an antimalarial. This study used Plasmodium berghei rodent malaria as an in vivo model of malaria by infecting experimental mice⁹.

MATERIAL AND METHODS:

Ethical Approval:

This study was conducted after getting approval with certificate number No. 2.KE.039.05.2020 from the Animal Ethics Committees Faculty of Veterinary Medicine Universitas Airlangga Surabaya Indonesia.

Preparation of Nanoparticles from Syzygium cumini Fruit Extract Fraction:

Fruits Syzygium cumini were collected from Lumajang, East Java. Fruits washed and air dried then ground to powder. The powder was extracted by maceration. The dried extract was used for fractionation. The combined results of the ratio of chloroform and methanol fractionation (20:80, 10:90, 0: 100) were made into a nanoparticle formulation by dissolving 1g of the fraction in 5mL of 96% ethanol and sonication for 10 min. 100 mL (0.5%) chitosan was added to tween 80 1mL, stirred using a homogenizer at 1000 rpm for 10 min. Added 1g of fraction, stirred using a homogenizer at 3000 rpm for 30min. After that, add 20mL (0.5%) NaTPP, homogenizer at 4000rpm for 90min. The results were left to stand for 24h and dried by freeze-drying. The nano size was checked with a Scanning Electron Microscope (SEM) and Particle Size analyzer (PSA).

Calculation of the Dose of Plasmodium berghei Infection in Mice:

Mice (Mus musculus) males aged 2.5 months a weight 30 grams were infected with red blood cells containing 1x10⁶ in 0.2mL P.berghei parasites intraperitoneally¹⁰. Calculation of the parasite infection dose is determined by calculating the percentage of parasitemia parasite from a thin blood smear with Giemsa. Calculation of the number of erythrocytes diluted with PBS using an Improved Neubauer Counting Chamber. The total parasite dose is obtained by multiplying the number of erythrocytes times the percentage of parasitemia and the dilution.

In vivo Antimalarial Activity Test:

Eighty male mice were divided into 8 groups. The details of each group are as follows K: not infected P. berghei and given 0.5ml of chitosan-NaTPP polymer, K-: infected and given 0.5mL of chitosan-NaTPP polymer, P1: infected and given 25mg/kg BW chloroquine, P2: infected and given 400mg/kg BW fraction, P3, P4, P5, and P6 were infected and given each 100 mg/kg BW, 200mg/kg BW, 400mg/kg BW nanoparticles and combined with 25mg/kg BW chloroquine, also 400 mg/kg BW nanoparticles. Treatment of infection with 0.2mL of 1x10⁶ P. berghei Intra peritoneally, therapy was given for 4 days 24 h after infection. After 48 h of infection, a thin blood smear with 20% Giemsa was made every day to see the percentage of parasitemia, growth inhibition, and Mean Survival Time. On the 8^th - day post-infection, blood was taken from the heart for hematological examination, and surgery was done to take the organs of mice to check the splenic index and hepatic index.

Calculation of Parasitemia and Growth Inhibition:

The calculation of the percentage of parasitemia and growth inhibition of Plasmodium berghei in mice was carried out on 5^th-day post-infection. A thin blood smear was fixed with methanol, stained with 20% Giemsa.. The percentage of parasitemia and percentage of inhibition of Plamodium berghei were calculated by counting the number of infected erythrocytes in every 5000 erythrocytes under the microscope with 400X magnification. The percentage of parasitemia and growth inhibition was calculated with the formula^11,12,13_.

Examination of the Mean Survival Time:

Mortality from control and treated mice was observed every day starting from day 1 (D1) of Plasmodium berghei infection until the 8^th-day post-infection, calculate the Mean Survival Time calculated using the formula¹⁴_.

MTS = number of mice living in the group (days)/total number of mice in the group

Hematology Examination:

On the 8^th day post-infection, the mice were anesthetized with ketamine (sigma), then a thoracotomy was performed, blood is taken from the heart (1 mL) using a tuberculin syringe put in a vial that has been given an anticoagulant for hematological examination an automated SYSMEX XT 4000i blood analyzer.

Examination of the Splenic Index and Hepatic Index:

The spleen and liver were taken and weighed using analytical scales, then measured the length and width of the spleen and liver using a caliper with an mm scale. The value of the splenic index and hepatic index were calculated from the organ weight divided by the body weight of the mice.

Processing and Analyzing Data:

Data from the observation of hematology, splenic index, hepatic index and Mean Survival Time were processed using Analysis of Variance using SPSS System 24.0 then followed by the Duncan Multiple Range Test with a level of 5% to determine differences in the treatment given. For % parasitemia and % growth inhibition were analyzed by linear regression analysis.

RESULTS:

Nanoparticles Characterization of Syzygium cumini Fruit Fraction:

Results of characterization of the fruit fraction of Syzygium cumini nanoparticles using Particle Size Analysis (PSA). The size distribution of the nanoparticles was 7.035nm - 990nm. Polydispersity Index Value is 0.472. Zeta Potential Value is +27.6mV.

Weight Loss:

Data of the mean weight loss in the control group and treatment group can be seen in figure 2. Data of body weight of mice after 5 days of infection in the chloroquine-treated group (P1) and the fraction-treated group (P2) showed an increase in body weight, while the other groups showed a decrease. Whereas at 8^th days post-infection, there was a decrease in body weight in all treatment groups except for the group that was treated with chloroquine (P1) and the group that was treated with the combination of chloroquine and nanoparticle fraction of Syzygium cumini at a dose of 400 mg/kg BW (P6), the results are as shown in Figure 1

Fig 1: Graph of the Average Weight Loss of Mice in the Control Group and Treatment Groups, 5^th and 8^th Days Post Infection

Percentage of Parasitemia, Growth Inhibition, and Mean Survival Time:

The percentage of parasitemia and growth inhibition of P. berghei in the control and treatment groups after 7 days post-infection can be seen in Table 1. The table shows that % parasitemia in group K- shows a significant difference with all treatment groups with p≤ 0.05. Group P1 was not significantly different from the P6 group at p≥0.05 but significantly different from the K-, P2, P3, P2, and P3 groups (p≤0.05). Group P2 and P3, P4 did not show significant differences but were significantly different from groups K-, P1, P5, and P6, while group K- showed a difference with all treatment groups p ≤ 0.05.

The percentage of growth inhibition in the K-group showed a significant difference with all treatment groups with p ≤ 0.05. Group P1 was not significantly different from group P6 (p≥0.05) but significantly different from groups K-, P2, P3, P4, and P5 (p≤0.05). Groups P2 and P3, P4, and P5 did not show significant differences (p≥0.05) but were significantly different from groups K-, P1, and P6 (p≤0.05).

The percentage of live mice in groups K, P1, P5, and P6, 100% of all mice still survive until the 8^th-day post-infection, while the percentage of live mice in K- group is only 37.5% followed by group P3 50%, P4 75% and P2 87.5%. While the mean survival time for groups K, P1, and P6 in 8^th days after infection, mice were still alive.

Table 1: Results of Percentage of Parasitemia, Growth Inhibition and Mean Survival Time of P. berghei in the Control group and the Treatment Group After 7 days Post Infection

Group	% Parasitemia (Mean ± sd)	% Growth Inhibition (Mean ± sd)	Percentage of live mice	Mean Survival Time
K	-	-	100 % (8/8)	8.00
K -	3.58^a ± 0.32	0.00^a ± 0.00	37.5% (3/8)	7,21
P1	0.73^d ± 0.21	70.84^c±14.79	100% (8/8)	8,00
P2	2.21^b ± 0.18	47.66^b± 13.65	87.5%(7/8)	7,89
P3	2.39^b ± 0,28	41.69^b±13.14	50.0% (4/8)	7,50
P4	2.41^b ± 0.14	49.57^b±17.35	75.0% (6/8)	7,75
P5	1.58^c ± 0.25	53.99^b±8.27	100 % (8/8)	8,00
P6	0.77^d ± 0.43	70.57^c±14.40	100% (8/8)	8,00

Note: Different superscripts in the same column show significant differences p≤0.05

Splenic Index and Hepatic Index:

Table 2 shows an increase in Spleen weight and spleen size which is longer and wider in all K-, P1, P2, P3, P4, and P5 groups when compared to the K group. The color of the spleen becomes blackish in K-, P1, P2, P3, P4, and P5 groups. The splenic index of group K was significantly different from K-, P1, P2, P3, P4, P4, and P5. Group K was not significantly different from groups P2, P3, P4, and P5 but significantly different from K, P1, and P6. While P1 was not significantly different from P6 but significantly different from groups K-, P1, P2, P3, P4, and P5.

Table 3 shows that the average liver weight and average live length were longer in the K group. The group K hepatic index was not significantly different from P1 and P6 but significantly different from groups K-, P2, P3, P4, and P5.

Table 2: Results of Splenic Index in Control and Treatment Group

Group	Mean of body weight	Mean of Spleen weight	Spleen size		Spleen Color	Splenic Index Mean ± SD
			Length mean	Width mean
K	25.18	0.21	1.86	0.78	Fresh red	0.86^a± 0.43
K-	20.46	0.53	2.65	0.91	Blackish brown	2.59^d± 0.13
P1	27.00	0.38	2.39	0.76	Fresh red	1.39^ab± 0.25
P2	20.81	0.44	2.53	1.06	Blackish brown	2.13^cd± 0.31
P3	17.39	0.39	2.57	0.69	Blackish brown	2.29^d± 0.60
P4	18.54	0.46	2.33	1.02	Blackish brown	2.45^d± 0.55
P5	23.20	0.62	2.97	0.95	Blackish brown	2.63^d± 0.69
P6	23.31	0.36	2.47	1.15	Fresh red	1.57^bc± 0.27

Note: Different superscripts in the same column show significant differences p≤0.05

Table 3: Results of Hepatic Index in Control and Treatment Group

Group	Mean of body weight	Mean of liver weight	liver size		Liver Color	Hepatic Index Mean ± SD
			Length mean	Width mean
K	25.18	1.36	1.88	2,33	Fresh red	5.39^a± 0.49
K-	20.46	1.55	2.36	2.15	Blackish brown	7.69^b± 1.67
P1	27.00	1.57	2.34	2.07	Fresh red	5.82^a± 1.00
P2	20.81	1.80	2.40	2.26	Blackish brown	8.69^bc±0.96
P3	17.39	1.46	2.12	2.07	Blackish brown	8.39^bc±1.11
P4	18.54	1,78	2.16	2.41	Blackish brown	9.61^c± 1.22
P5	23.20	1.84	2.25	2.60	Blackish brown	7.95^b± 1.52
P6	23.31	1.24	2.26	2.24	Fresh red	5.39^a± 0.91

Note: Different superscripts in the same column show significant differences p≤0.05

Hematology Changes:

The hematological changes show that the leukocyte levels of all groups are still in the normal range, namely 5.1-11.6 x10³/mm³. Erythrocytes in all groups still showed a normal value range, which is 6.8-11.7 x10⁶/ mm³. Hemoglobin groups K- and P3 showed a decrease in hemoglobin levels, while the other groups were still within the normal range which is 12.4-14.6g/dL. Hematocrit also showed a decrease in the K- and P3 groups, while the other groups were still within the normal range, which is 38.5-45.1%. Platelet levels in all groups were still within the normal range (150-350 x10³ /mm³). The results of the leukocytes, monocyte, and granulocyte count in all treatment groups still showed values within the normal range, which is 25-33% for leucocytes, 1-6% for monocytes, and 12-68% for granulocytes (Table 4 and 5).

Table 4: Results of Hematological Features of the Control Group and the Treatment Group

Group	Hematological changes
	Leukocytes (10^3/mm^3)	Erythrocytes (10^6/mm^3)	Hemoglobin (g/dL)	Hematocrit (%)	Platelets (10^3/ mm^3)
K	6.24^b±0.27	6.86^ab± 0.05	12.72^bcd±0.27	39.28^cd±0.39	244.6^b± 7.60
K-	5.46^a±0.18	6.08^a±0.08	10.6^a± 0.14	36.08^a± 0.16	200.2^a± 0.44
P1	6.42^b±0.38	7.30^abc±0.71	12.96^cd±0.64	38.90^bc±0.53	272.2^c± 3.34
P2	5.7^a± 0.49	6.68^ab± 0.22	12.2^bc±0.49	38.14^b± 0.25	214.2^a± 9.41
P3	5.56^a±0.05	6.45^a± 0.29	11.94^b±0.49	37.00^a± 0.14	206.0^a± 1.73
P4	6.86^c±0.44	8.86^d± 0.85	13.86^c±0.29	39.66^cd±0.18	301.8^d± 3.49
P5	6.36^bc±0.40	8.08^bcd±1.49	13.46^dc±0.97	40.12^d± 1.15	300.0^d± 1.91
P6	6.66^bc±0.65	8.48^cd± 2.17	13.38^dc±0.92	39.82^cd±1.84	285.4^cd±4.97

Note: Different superscripts in the same column show significant differences p≤0.05

Table 5: Results of Calculation the Type of Leukocytes in the Control Group and the Treatment Group

Group	Type of leukocytes
	Lymphocytes (%)	Monocytes (%)	Granulocytes (%)
K	30.0^ab ± 0.70	4.6^a ± 0.89	65.4^bc ±0.54
K-	29.0^a ± 0.00	5.0^a ± 0.70	66.0^d ±0.70
P1	32.2^d± 1.09	4.2^a ± 1.92	63.6^ab ±2.70
P2	29.4^ab± 1.34	5.6^a ± 0.54	65.0^bc ±1.22
P3	30.6^b± 0.54	4.2^a ± 0.44	65.4^bc ±0.54
P4	29.0^a ± 1,00	4.6^a± 1.14	66.4^d ±0.89
P5	30.4^abc ± 0.54	4.0^a ± 1.58	65.6^bc ±1.81
P6	31.6^cd ± 1.63	5.0^a ± 0.70	63.4^a ±1.67

Note: Different superscripts in the same column show significant differences p≤0.05

DISCUSSION:

Nanoparticles are solid colloid particles with a diameter of 1-1000nm, fraction nanoparticles chloroform: methanol (20:80, 10:90, 0: 100) Syzygium cumini fruit using the ionic gelation method using chitosan and NaTPP with a ratio of 5:1. Nanoparticles consist of macromolecular materials used as drug carriers by dissolving, trapping, encapsulating, absorbing or chemically attaching active ingredients. Chitosan is one of the polymers used to form nanoparticles. Chitosan is ideal, biocompatible, biodegradable, non-toxic and inexpensive¹⁵. Characterization of the Syzygium cumini fraction of nanoparticles by Particle Size Analysis (PSA) showed the smallest particle size distribution was 7.035 nm and the largest particle size was 990 nm. Based on these results, the size of the Syzygium cumini fraction of nanoparticles has fulfilled the shape of the nanoparticles. The particle size in this nanocarrier is very important because it affects stability, encapsulation efficiency, drug release profile, biodistribution, mucoadhesion, and cellular uptake. The size of the drug delivery system affects the pharmacokinetics, tissue distribution, and clearance¹⁶. The polydispersity index value is 0.472, this result indicates a relatively homogeneous or uniform size of dispersion because it is smaller than 0.7. Zeta potential value was +27.6mV and it showed that the nanoparticles of the Syzygium cumini fraction were stable.

The body weight of mice 8 days post-infection showed weight loss in all treatments except those treated with chloroquine and those treated with a combination of chloroquine and nanoparticle fraction of Syzygium cumini at a dose of 400mg/kg BW. Malaria is a complex disease that can cause a variety of pathological conditions. Clinical conditions in the early stages of malaria infection are characterized by periodic fever, chills, headaches, dizziness, malaise, abdominal discomfort, nausea, muscle, and joint aches¹⁷. This results in decreased appetite, leading to weight loss. If the infection develops and the number of parasites increases, the pathogenic process will take place and cause severe anemia, blood acidosis, splenomegaly, hepatomegaly, acute respiratory distress syndrome and other clinical conditions, sequestration of parasites in the brain, lung, liver, and intestinal organs which can cause death^18.19.

Increased % parasitemia and decreased parasite growth inhibition in the Plasmodium-infected and untreated group compared to those treated with chloroquine. Combination of chloroquine and Syzygium cumini fraction nanoparticles and the group treated with 400 mg/kg BW of the Syzygium cumini fraction nanoparticles. Chloroquine is a weak base antimalarial. The accumulation of chloroquine in the digestive vacuole Plasmodium causes an increase in pH in the digestive vacuole which will bind to heme and inhibit the formation of hemozoin. Heme is toxic to parasites so the parasite dies. Degradation of hemoglobin by protease enzymes in the digestive vacoula under acidic conditions is needed by parasites to live like amino acids, while free heme (Fe2 + protophorphyrin IX) will be detoxified and converted to hemozoin crystals (Fe3 + protophorphyrin IX)²⁰. Chloroquine combination therapy and Syzygium cumini fraction nanoparticles as adjuvant therapy to increase efficacy and reduce disease complications. The antioxidant potential of flavonoid compounds, phenolics in the nanoparticles fraction Syzygium cumini works as radical scavenging, inhibits the kinase enzyme, causing the formation of hemozoin and upregulation of MMP-9, TNFα and inhibit              IL-1β^21,22,23. For inhibiting parasite growth, the antimalarial activity of the nanoparticles from the Syzygium cumini fraction was dependent on the dose. The content of flavonoid compounds contained in Syzygium cumini also works to inhibit fatty acid biosynthesis (FAS II) from malaria parasites²⁴. Inhibit the influx of L-glutamine and myoinositol into the infected erythrocytes and directly interacting with the functional structures like DNA, enzymes, proteins of the parasites²⁵. Apigenin is a natural compound that is belonging to the flavone subclass of flavonoid, Antimalarial mechanisms of apigenin are confirmed to induce ABCC1 transporters, inhibit protein kinase and act as an antioxidant²⁶. The bioactive conformation was explored and explained by docking of compounds the active binding site²⁷.

The percentage of live mice in normal group, the group that was treated with chloroquine, 400mg/kg BW Syzygium cumini fraction nanoparticles, and combination therapy of chloroquine and nanoparticles, 100% of all mice still survive until the 8^th day post-infection, while the percentage of live mice in the group that infected but not treated was only 37.5% followed by a group that infected and treated with 100mg/kg BW Syzygium cumini nanoparticles which are 50%, 200mg/kg BW Syzygium cumini nanoparticles which is 75% and fraction 400mg/kg BW Syzygium cumini nanoparticles which is 87.5%. While the mean survival time for normal groups, a group treated with chloroquine, a group that was treated with 400mg/kg BW Syzygium cumini fraction nanoparticles, and a group that was given a combination therapy of nanoparticles and chloroquine for 8 days, mice were still alive. Administration of chloroquine, nanoparticle fraction of Syzygium cumini, was able to reduce the pathological effect of plasmodium parasite infection. The higher the dose of nanoparticles given indicates an increase in the survival of the host, this indicates that the antimalarial activity or the ability to kill plasmodium from the Syzygium cumini fraction of nanoparticles depends on the dose given. This could also be attributed to the results of decreased parasitemia and increased growth inhibition with increasing doses¹⁴. In the present study nanoparticles extract of Syzygium cumini leaf and fruit as an adjuvant therapy can reduce liver, kidney, lung and brain damage of mice infected with Plasmodium berghei²⁸.

The spleen and liver weight increased and the size of the spleen and liver was longer and wider and the color of the spleen and liver became black in all groups infected with Plasmodium berghei when compared to the group that was not infected (normal). Splenic index in the normal group was not infected significantly different from all treatment groups. The hepatic index of the normal group was not significantly different from the group treated with chloroquine and the group treated with the combination of chloroquine and nanoparticles but significantly different from the other groups. Splenomegaly was used as a marker index for plasmodium endemicity. The spleen is an important organ for cleaning infected red blood cells. Malaria infection can cause splenomegaly and rupture of the spleen. Activation of the innate immune system, expansion of monocytes, and clearance of infected red blood cells play a role in the damage of microarchitecture and splenomegaly in malaria infection. This can be due to increased intra-splenic tension due to hyperplasia and enlargement, vascular occlusion due to endothelial reticulo hyperplasia which causes thrombosis and infarction^29,30. In the present study thrombocytopenia has emerged as the strongest predictor of severity of falciparum malaria³¹. Hepatosplenomegaly in malaria infection is caused by an increase in pro-inflammatory mediators that are higher than the levels of regulatory mediators and tissue repair cytokines so that they are unable to control the inflammatory response that occurs. An increase in IL12, IL10, and TNF levels in malaria infection can induce an inflammatory response and cause             hepatosplenomegaly ^32,11.

The hematological changes show that the leukocyte levels of all groups are still in the normal value range, which is 5.1-11.6 x10³/mm³, and erythrocyte in all groups still shows a normal value range, which is 6.8-11.7 x10⁶/mm³, hemoglobin and hematocrit in the infected group and untreated and those treated with 100mg/kg BW nanoparticles showed a decrease in hemoglobin levels while the other groups were still within the normal range, which is 12.4-14.6 g/dL. and 38.5-45.1%. Platelet levels in all groups were still within the normal range, which is 150-350 x x10³/mm³. The results of the leukocyte, monocyte, and granulocyte count in all treatment groups still showed values within the normal range, which is leukocytes 25-33%, monocytes 1-6%, and granulocytes 12-68%. Multiplication of parasites causes a decrease in hematocrit levels because red blood cells will lysis when releasing mature parasites. During acute infection, hemolytic occurs in more than 90% of erythrocytes. This causes a decrease in hemoglobin levels. Loss of complement regulatory proteins including complement receptor type 1 (CR1) and CD55 from the surface of red blood cells leads to increased erythrocyte lysis³³.

Increasing hemozoin levels during malaria infection are associated with anemia and reticulocyte suppression. Hemozoin can induce apoptosis of erythroid precursors, induces the release of migration inhibitory factor (MIF) macrophages from macrophages, inhibits erythroid and the formation of the progenitor-derived colony can also inhibit proliferation, differentiation, and maturation of blood cell precursors. Malaria toxin can also stimulate macrophages to produce TNF α. TNF α together with IFN g and IL12 pro-inflammatory cytokines can inhibit the growth of red blood cell precursors and inhibit erythropoiesis which can reduce the life span of red blood cells, causing increased erythrophagocytosis and inducing apoptosis of erythroid precursors^34,35. In malaria infection, malaria toxins such as glycosylphosphatidylinositol act directly on monocytes and macrophages and trigger the release of pro-inflammatory cytokines, causing suppression of hemopoiesis and dyshemopoiesis. Malaria pigments that are released during schizogony and reactive oxygen species can inhibit macrophage and dendritic cell function and can suppress erythropoiesis³⁰. The conclusions of the study Nanoparticles of Syzygium cumini fraction using ionic chitosan gelation method and 5:1 ratio of NaTPP were able to produce homogeneous and stable nanoparticles. Nanoparticles of Syzygium cumini fraction at dose of 400 mg/kg BW and combination therapy 400 mg/kg BW nanoparticles of Syzygium cumini fraction with 25 mg/kg BWchloroquine has potential as an antimalarial when seen from the results of decreased parasitemia, increased inhibition of parasite growth, increased body weight, splenic index, hepatic index and Hematological changes of mice infected with Plasmodium berghei.

ACKNOWLEDGEMENTS:

The author wishes to thank Kemenristekdikti that has been given PUPT funding for this research.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest

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Received on 10.09.2022            Modified on 05.12.2022

Accepted on 06.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(9):4288-4294.

DOI: 10.52711/0974-360X.2023.00702