Effect of Combination Songga-Wood-Stem ( Strychnos Ligustrina Blume) And Antimalaria-Act on Il-10 Production of Malaria

Malaria is one of the deadliest infectious diseases in the world. Artemisinin-Based-Combination-Therapy (ACT) an antimalaria recommended by WHO, is starting to experience resistance in Asia. Songga-wood-stem having an immunoprotective effect is traditionally used as antimalaria in a certain community. IL-10 produced during malaria, gives an immunopathological-protection but interfere the ability to control Plasmodium-infection. Whether IL-10-production affected by both Songga-wood-stem and ACT together had not been studied. Objective of this study was to determine the effect of combination of ethanolic-extracted-Songga-wood-stem (EESWS) and ACT on IL-10, a protective-cytokine against malaria-immunopathology. This experimental-study used post-test-only-randomized-controlled-group-design . The thirty- Swiss-webster-mice were grouped into 5 groups. The one-group of healthy-mice (K1), and the four-groups infected with Plasmodium berghei ANKA (PbA) which were untreated-K2-group, ACT-treated-K3-group, EESWS-treated-P1-group, and EESWS-ACT-combination-treated-P2-group. IL-10-level of stimulated-splenocytes-culture was examined by ELISA-method. Data-analysis-used was One-Way-Anova-Welch-test and Post-hoc Games-Howell. P1 and P2-groups had higher IL-10-levels than K1 (p=0.038). Groups of P1 and P2 showed lower IL-10-levels than K3 (p=0.001). IL-10-level of P2-group was not different than P1 (p=0.135). The conclusion is the EEWS or EESWS-ACT-combination-therapy restrains the increase-splenic-IL-10-production above normal value in the healing phase of malaria infection.


INTRODUCTION
Malaria, one of the deadliest infectious diseases in the world, is until now still a major problem on international and national scale. 1,2WHO recommends Artemisinin-Based-Combination-Therapy (ACT) as the treatment of uncomplicated malaria caused by P. falciparum.The ACT-effectiveness, however, is starting to experience resistance in Asia. 3 One solution for antimalarial-treatment is to combine of natural-extractingredients and standard-antimalarial-drugs.This can potentially slow the occurrence of parasite-resistance to standard-antimalarial-drugs. 4 The ethanolic-extract of Songga-wood-stems (EESWS) in mice infected with P.berghei ANKA (PbA) showed strong antimalarial activity with an IC50 of 8,478 mg/Kg weight.This is reinforced by GCMS-analysis which proves that EESWS contains strychnine alkaloid compounds which are very active as antimalarials. 5ongga-wood-plants in addition to having antimalarial activity, also have immunomodulatory-effect during malaria in mice-model.EEWS-adjuvant-administration has protective immunomodulatory-activity against severe-PbA-infections by an increase-splenicimmunoprotective-chemokines-CXCL12-production, without inhibiting the infection-control. 6The variousimmunomodulatory-benefits shown by Songga-woodplants is expected to be immunoprotective in tackling various-malaria-immunopathological-responses.
The immunoprotective-response is successful in controlling the infection without triggering the immunopathologies often associated with malaria, including severe-anemia and cerebral-malaria (CM). 7,8The occurrence of malaria cases is often associated with an imbalance between the overproduction of pro-inflammatory cytokines and the response of anti-inflammatory cytokines. 7IL-10 is an anti-inflammatory and an important-immune-regulatory-cytokine in the host. 6,9It is expected that the EESWS-ACT-combination plays a protective role by inducing sufficient spleen-IL-10production of PbA-infected-Swiss-mice.

MATERIALS AND METHODS
A true-experimental-study with post-test-onlyrandomized-controlled-group-design was approved by Health Research Ethical Committee Faculty of Medicine Universitas Diponegoro (Ethical Clearance No. 43/EC/FK-UNDIP/IV/2021).The research was carried out at the Experimental-Animal-Laboratory of Sultan-Agung-University (Unissula), the Integrated-Biomedical-Laboratory-Unissula and the GAKY-laboratory of the Faculty of Medicine, Diponegoro-University.This research used thirtyfemale-Swiss-mice, and detailed research-interventionprotocols were mentioned elsewhere. 6Control-groups were K1, K2 and K3-groups; treatment-groups were P1 and P2-groups.The K1-group consisted of healthymice, and those of K2, K3, P1 and P2-groups were inoculated with PbA intraperitoneally.K2-group was without any treatment, and K3-group was given ACT.P1 and P2-groups were given EESWS, obtained from Maluku Province, and extracted using ethanol in UNISSULA-biomedical-laboratory, and ACT-EESWS-combination, respectively.The culturesupernatant of lipopolysaccharide (LPS)-stimulated splenic-cells was collected and measured for the IL-10levels using IL-10-Enzym-Linked-Immuno-Assay (ELISA)-kit (Legend Max TM , Biolegend Inc, USA). 10 Data analysis was used statistical software on a computer.Each data was tested for normality using Saphiro-Wilk-test.The One-Way-Annova-test was carried out to see a different mean between the fiveresearch-groups.The magnitude of the difference in the mean between two-groups was further analyzed using the Games-Howell Post Hoc Test for IL-10.The significant difference was indicated by p < 0.05.

DISCUSSION
The splenic-IL-10-production in the EESWS-treated-P1 and EESWS-ACT-treated-P2-groups was significantly higher than the healthy-control-K1-group (Table ).The culture-supernatants of this study were obtained from a previous-study showed the no difference of parasitemia-levels among groups of mice treated with EESWS-treated-P1, EESWS-ACT-treated-P2 and ACTtreated-K3-groups, and these three-groups showed significantly-lower-parasitemia-levels than the PbAinfection-control-K2-group which did not receive any therapy. 6These together indicate that the EESWS and the EESWS-ACT-combination are associated with an increase-IL-10-production above normal during the PbAinfection-recovery-phase.The EESWS and the EESWS-ACT-combination are associated with normal-spleenproduction of CXCL12, a chemokine that increases IL-10production, in the PbA-infection-recovery-phase. 6Other mediators, therefore may involve in the increase in IL-10-production.It worthy of note was that the highest IL-10-production among the groups was observed in the PbA-infected-ACT-treated-K3-group, and the differences were significant (Table ).A significantly lower IL-10-production in the P1 and P2-groups than K3group indeed was noticed.This indicates that either EESWS or EESWS-ACT-combination-treatment associates with a restricted-IL-10-elevation in the malaria-recovery-phase.The PbA-infection prevents the increase-anti-inflammatory-cytokine-production.The PbA-infected-control-K2-group and the healthy-control-K1-group showed no different IL-10-production (p = 0.619; Table ).This was in accordance with the finding that the spleen-CXCL12-production in the K2 and K1groups was not different. 6These indicate that the day7-PbA-infection associates with inhibition of a significant increase in spleen-IL-10 and CXCL12-production.ACT was associated with the increase-splenic-IL-10production in the recovery-phase of PbA-infected-Swiss-mice (Table ).The inhibition-IL-10-production of ACT on PbA-infection is thus not proven.ACT reduces the proportion of IL-10-producing-Th2-cells in autoimmune-experimental-animals. 11ACT therefore might have different-effect on the different-diseases.Interestingly, IL-10 protects the severity of the immunopathology of Plasmodium-infection, but IL-10 inhibits the control of Plasmodium-infection and the recurrence of parasitemia.Research is needed to prove whether the recurrence of parasitemia can be protected by EESWS treatment or the EESWS-ACT-combination.

CONCLUSION
The EESWS-ACT-combination or EESWS alone might constrain the increase-splenic-IL-10-production above normal in Swiss mice in the recovery phase of PbA-infection.