Gymnema inodorum and preparation of extract
Gymnema inodorum leaves were obtained from the Chiangda Organic Company Garden, Chiang Mai, Thailand. A plant biologist authenticated this plant at Chiang Mai University and the voucher specimen (NRU64/036-001) was subsequently deposited at the Research Excellence Center for Innovation and Health Products, Walailak University. The plant materials were dried in a hot-air oven at 50oC, and dried powdered material was then prepared using the electric blender. To prepare of aqueous crude extract, 250 g of the dried powdered G. inodorum was soaked in 750 ml of distilled water at room temperature for seven days with occasional stirring. Filtration was then performed with Whatman no. 1 filter paper and collect the filtrate. Lyophilization was carried out to obtain the dried powdered form of aqueous crude extract of G. inodorum (GIE) and stored at –20oC until further use (Ounjaijean et al. 2021a). Before experiments, GIE at the chosen doses was freshly prepared in 20% Tween-80 according to the weight of the mice.
Preparation of standard antimalarial drug
Dihydroartemisinin (DHA) was obtained from Sigma-Aldrich Co. (St Louis, MO, U.S.A.) and stored at -20oC. For experiments, DHA at the chosen doses was dissolved in 20% Tween-80 according to the mice’ body weight for administering orally.
Healthy Balb/c male mice, 4-6 weeks old, weighing 20-25 g at the time of primary infection obtained from Nomura Siam International Co., Ltd., were used throughout the study. The mice were kept in a room with temperature control between 22-25oC and 12 h light/12 h dark cycle. They were fed on a commercial pellet diet 082G and clean tap water ad libitum.
Rodent malaria parasite
Plasmodium berghei strain ANKA (PbANKA) obtained from MR4 (Malaria Research and Reference Reagent Resource Center, https://www.beiresources.org/About/MR4.aspx) was used in this study. Cryopreservative stock of PbANKA was thawed in a water bath at 37oC, and 200 ml of the suspension was inoculated by intraperitoneal (IP) injection into naïve Balb/c mice. Parasitemia was monitored daily by microscopic examination of Giemsa-stained blood film, and serial passage was then performed when parasitemia reached about 10-20%. Blood was collected by cardiac puncture and diluted with normal saline to obtain 1x107 parasitized erythrocytes for IP injection.
Determination of parasitemia
Tail blood from PbANKA infected mice was smeared on a microscopic slide. After air-dried, smeared slide was then fixed with absolute methanol and stained with 10% Giemsa solution for 15 min at room temperature. Parasitized erythrocytes were counted under a light microscope with a 100x oil immersion lens, and parasitemia was subsequently calculated using the following formula.
The antimalarial assay was first carried out to determine the effective dose (ED50) of the individual substances (GIE and DHA) according to standard 4-day suppressive test as previously described (Peters 1975). Groups of mice (5 mice/group) were inoculated with 1x107 parasitized erythrocytes of PbANKA by IP injection. Two hours after infection, mice were administered orally by gavage with GIE (1, 10, 50, 100, and 200 mg/kg) and DHA (0.1, 1, 5, 10, 20 mg/kg) once a day for 4-consecutive days (day 0-3). The untreated control was given 10 ml/kg of 2% Tween-80. On day 4, parasitemia was determined by microscopic examination of Giemsa-stained blood film, and the percentage of inhibition was subsequently calculated using the following formula.
The obtained ED50 values of both GIE and DHA were used for combination treatment. The combination was prepared in fixed ratios of 100/0, 80/20, 60/40, 40/60, 20/80, and 0/100 of GIE/DHA according to the fixed ratio method as previously described (Nateghpour et al. 2012). The standard 4-day suppressive test was used to test the combination treatment in this step. On day 4, parasitemia was estimated, and % inhibition was then calculated. Interaction between DHA and GIE against the PbANKA was interpreted as lying the points above the joint line indicated synergism and either around the line or below indicated additive or antagonism, respectively. A combination index was also calculated to interpret the interaction between GIE and DHA against PbANKA. Moreover, body weight, packed cell volume, and mean survival time for each group were recorded.
Determination of body weight and packed cell volume
Each mouse’s body weight (BW) in all groups was measured and recorded using sensitive electronic balance before infection on day 0 and on day 4 post-infection. To determine packed cell volume (PCV), blood was collected from the tail vein of each mouse in heparinized capillary tubes by filling ¾ of its volume. The tubes were sealed and centrifuged at 12,000 rpm for 15 min using a microhematocrit centrifuge. PCV was then calculated using the following formula before infection on day 0 and day 4 post-infection.
Mean survival time
The mortality of each mouse was monitored and recorded from the time of infection until death throughout the follow-up period (30 days). Mean survival time (MST) was determined using the following formula.
GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA) was used to analyze the results of this study. Data were presented as the mean + standard error of the mean (SEM). The non-linear regression function for the sigmoidal dose-response variable slope was conducted to obtain the best-fit ED50 value. The comparison between the mean of control and treatment groups was tested with one-way analysis of variance (ANOVA) and Tukey's post-test. The 95% confidence, p < 0.05 was considered as statistical significances. Moreover, combination index (CI) was automatically simulated by CompuSyn software (ComboSyn, Inc., USA), which defines synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1).