In vitro culture of Plasmodium falciparum
P. falciparum CQ/mefloquine (MQ)-sensitive (3D7) and -resistant (Dd2) strains were originally obtained from Dr. Louis Miller, NIH, USA. The parasites were maintained with 2% hematocrit type O+ red blood cells (RBCs) in RPMI-1640-based complete medium (CM) supplemented with 5% AB+ human serum (prepared from plasma), 0.25% AlbuMax I (Gibco, Waltham, MA), 12.5 µg/mL gentamycin, and 200 mM hypoxanthine at 37°C under mixed gas (5% CO2, 5% O2, and 90% N2) condition basically as described . Japanese Red Cross Society was responsible for supplying RBCs and human plasma (number: 28J0060).
Isolation and culture of primary Adult Mouse Brain cells
Primary Adult Mouse Brain (AMB) cells were isolated and established in NEKKEN Bio-Resource Center, Institute of Tropical Medicine, Nagasaki University as described . The primary cells, which were passaged several times to be adapted to in vitro condition, were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum, penicillin/streptomycin solution (100 units/mL penicillin G, 100 mg/mL streptomycin sulfate) (Wako Pure Chemicals Industrial Ltd, Osaka, Japan) and incubated at 37°C under 5% CO2. The primary cells for cytotoxicity assay were used after three passages.
Kampo crude drug extracts, compounds, and formula
A Kampo library containing 120 herbal extracts (10 mg/mL), 96 Kampo-related active compounds including three compounds (coptisine chloride, berberine chloride, and palmatine chloride), and powder of formulated Orengedokuto (Kampo formula) ) were provided by the Institute of Natural Medicine (WAKANKEN), at the University of Toyama as described  and stored at -80°C. All the herbal extracts were dissolved in ultra-pure water (UPW) generated by Milli-Q (Merck KGaA, Darmstadt, Germany). Compounds were preserved at a concentration of 2 mM dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemicals Industrial Ltd.) solution, the most common solvent for chemicals. For in vivo assay, powder of Orengedokuto and Coptis Rhizome, as well as chloroquine, were dissolved in distilled water (DW) for oral administration.
In vitro antimalarial assay (first screening)
It was done by seeding the P. falciparum cultures (0.75% parasitemia and 2% hematocrit) on 96-well clear flat-bottom plates (Thermo Fisher Scientific, Rochester, NY) and exposed it to Kampo herbal extracts (final concentration of 500 µg/mL). The final UPW solution was less than or equal to 5% of the culture volume, which had no inhibitory effect on parasite growth. CQ (Sigma-Aldrich, St. Louise, MO) and artesunate (Shin Poong Pharm Co, Seoul, South Korea) were used as positive controls (10 µM – 0.508 nM), while 5% UPW was used as negative control. The culture plates were kept at 37°C under mixed gas (90% nitrogen, 5% oxygen and 5% CO2) condition for 48 hrs. Each in vitro experiment was performed in duplicated wells and repeated twice. The inhibition was obtained by dividing the parasitemia of test samples by the average of the negative controls.
In vitro dose response assay
The dose-response assay was performed for samples that showed more than 50% inhibition in the first screening to obtain the 50% inhibitory concentration (IC50; 10(LOG(A/B) × (50 ‒ C)/(D ‒ C) + LOG(B), where A represented the lowest concentration value at which the percentage inhibition showed greater than 50%, B was the highest concentration value at which the percentage inhibition showed less than 50%, C was the percentage inhibition value of the sample at concentration B, and D was the percentage inhibition value of the sample at concentration A). For the herbal extracts/Kampo formula, and for the compounds in the library, the anti-malarial activity was analyzed using a serial dilution of test samples at 500 µg/mL – 25.4 ng/mL or at 20 µM – 0.619 nM respectively. Artesunate (10 µM – 0.508 nM) for 3D7 and CQ (10 µM – 0.508 nM) for Dd2 were served as positive controls, while UPW (final 5%) or DMSO (final 0.5%) were assigned as negative controls. The final concentration of DMSO for all tested compounds, negative and positive controls were adjusted to 0.5%.
A SYBR Green based microfluorometric method was used to quantify parasite level as previously described . Briefly, after 48 hrs of incubation with herbal extracts or compounds, we added 100 µL of lysis buffer to RBCs by using 20 mM Tris, 10 mM EDTA, 0.01% saponin (wt/vol), and then we added 0.1% Triton X-100 (vol/vol), in pH 7.5 as well as 1× the final concentration of SYBR Green - I (Lonza, Rockland, ME) into each well. The plates were then incubated at room temperature for one hr with gentle agitation. Finally, the relative fluorescence unit (RFU) per well was detected using a multilabel plate reader (ARVO 1430; Perkin Elmer, Waltham, MA) with 485 - 515 nm for 0.1 seconds per exposure.
Cytotoxicity was initially screened at 500 µg/mL for herbal extracts and 20 µM for compounds. AMB cells (1×104 cell) were seeded in a 96-well plate (black plate with clear bottom) and incubated at 37°C in a CO2 incubator for 48 hrs. Then, herbal extracts, compounds, or their negative controls were added, and the cells were further incubated for 48 hrs. To examine the cell viability (%), 10 µL of Alamar Blue solution (10%, Funakoshi Co., Tokyo, Japan) were added into each well and the cells were incubated for 2 hrs. Then we measured the fluorescence intensity of each well using a multilabel plate reader at 590 nm for 0.1 seconds per exposure. The concentration of drug required to reduce cell viability by 50% (CC50; 10(LOG(A/B) × (50 ‒ C)/(D ‒ C) + LOG(B), where A represented the lowest concentration value at which the percentage viable cell showed greater than 50%, B was the highest concentration value at which the percentage viable cell showed less than 50%, C was the percentage viable cell value of the sample at concentration B, and D was the percentage viable cell value of the sample at concentration A) was determined for samples that showed less than 50% viability in the initial screening. The assays of duplicated well were performed twice independently. IC50 and CC50 values were used as an indicator of in vitro antimalarial activity and an indicator of cytotoxicity in AMB cells. The curve was plotted using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA). Selectivity index (SI) was calculated by dividing CC50 value by IC50 value.
Assessment of antimalarial activity in mouse model
A Kampo herbal extract (Coptis Rhizome) and formula (Orengedokuto) exhibited in vitro antimalarial activity were tested for in vivo activity against P. yoelii strain 17X in a mouse model. Female of 6-7 weeks C57BL/6N mice (SLC Japan), weighing 18–20 g, were used. The mice were kept in a cleanroom under conventional conditions then were acclimatized for one week before the experiments.
The P. yoelii 17X strain was provided by Dr. Tetsuo Yanagi, of National Bio-Resource Center (NBRC), NEKKEN, Nagasaki University, Nagasaki, Japan. and maintained by successive serial passage in mice of study. The parasite was maintained frozen at -80°C. For each individual assay, an aliquot was injected intraperitoneally (IP) in a mice, and infected donor mice were produced after three in vivo passage. A female C57BL/6N mouse previously infected with P. yoelii and having parasitemia levels of 20 to 30% were used as a parasite donor. At day 0, mice were injected IP with 0.2 mL of infected blood suspension containing 1×104 P. yoelii parasitized red blood cells obtained from the tail vein of P. yoelii infected donor mouse. The P. yoelii infected blood was diluted in physiological saline and injected via syringes.
To evaluate the antimalarial effect of both Coptis Rhizome and Orengedokuto, we randomly distributed infected mice into four groups of five individuals per cage. Tested drug and CQ were dissolved in DW. Each groups received the drugs 2 hrs after infection with P. yoelii on day 0 and continued daily for 7 days. Animals in test groups were treated twice a day with 365mg/kg /day of Orengedokuto (Kampo formula) and 122mg/kg/day of Coptis Rhizome (Herbal extract) in 0.2 mL solution by oral administration. CQ groups, served as a positive control, received a dose of 10 mg/kg/day and DW groups as a negative control, received 0.2 mL. Amount of dosage is calculated according to the normal usage for humans. Moreover, blood was collected after 1hrs from mice treated with Coptis Rhizome and Orengedokuto to detect berberine, palmatine, and coptisine.
For the in vivo antimalarial evaluation of the coptisine chloride (Toronto Research Chemicals (TRC), North York, Canada), coptisine chloride, and CQ were dissolved in DW. Three groups of mice were injected intraperitoneally with 0.2ml of the test sample (30mg/kg /day Coptisine chloride), positive control (10 mg/kg/day CQ), and negative control (DW) 2hrs after infection with P. yoelii. The same dose of injection was performed once a day until day 6. The IP route of administration was used since the previous study revealed that coptisine has low oral bioavailability and poorly absorbed through gastrointestinal tracts [38, 39].
On day 3 (72 hrs post-infection), the parasitemia level were determined by Giemsa-staining of the tail vein blood smears that was characterized by random counting of the number of parasitized erythrocytes on randomly selected fields of the slide under microscopy of 2,000–4,000 erythrocytes when parasitemia was low (≤10 %) or up to 1000 erythrocytes when parasitemia was higher.
Results of the in vivo antimalarial activity were expressed as a mean ± standard deviation (SD) and the comparison of parasitemia was determined by using a Student's t-test in Microsoft Excel 2016 (Microsoft, USA). The Statistical significance level was set at P < .05 for all tests. The different field on each slide was examined to calculate the average parasitemia as shown below.
Percentage parasitemia = (Number of parasitized RBC/ Total number of parasites) x100
The average percentage of parasite growth suppression was calculated by comparing percentage parasitemia suppression of the test group with respect to the control according to the equation:
Percentage suppression = (Mean parasitemia of negative control – Mean parasitemia of treated group)/ (Mean parasitemia of negative control group) x100
Preparation of plasma samples
Healthy 6 weeks old female mice that were subjected to overnight fasting were used for this study. To analyze berberine, coptisine, and palmatine after oral administration of Coptis Rhizome and Orengedokuto, doses of 122mg/kg and 365mg/kg were used for each group, respectively. Five mice per cage were used for each tested drugs. One hour after administration, the blood samples were collected from the tail vein with heparin and centrifuged at 1000xg for 20 min to yield plasma sample. Plasma samples were stored at -80°C. Before analysis, thawed plasma samples were mixed with methanol with 0.05% (vol/vol) formic acid for 15 min and centrifuged at 14000 rpm for 15 min. The supernatant was transferred into an Amicon Ultra filter (molecular weight cutoff of 10 kDa, Millipore Corporation), and centrifuged at 14000 rpm for 60 min at 4°C. The filtrate was evaporated and redissolved with 50 µL of 30% (vol/vol) MeOH in water to prepare LC-MS sample. LC-MS analyses were conducted with ODS Atlantis T3 (3 μm, 2.1×150 mm) column and Shimadzu LCMS system (Shimadzu, Tokyo, Japan) consisting DGU-20A5 on-line degasser, LC-20AD pumps (2 units), SIL-20A autosampler, CTO-20A column oven, SPD-M20A PDA detector, and hybrid ion trap time-of-flight (IT-TOF) mass spectrometer equipped with an ESI (electrospray ionization) interface and chromatogram data were acquired and processed by LCMS Solution (ver. 3.81, Shimadzu). Gradient elution of two solvent mixture consisting of 0.1% (vol/vol) formic acid in water (mobile phase A) and 0.1% (vol/vol) formic acid in acetonitrile (mobile phase B) was run at a flow rate of 0.2 mL/min under the following gradient program: 10% B (0-2min), 10-100% B (2-20 min), 100% B (20-25 min), 100-10% B (25-26 min), and 10% B (26-36 min). TOF analyzer was calibrated by sodium trifluoroacetate solution. Data was acquired using the following parameters: detector voltage, 1.80 kV; probe voltage, +4.5 kV (positive mode) or -3.5 kV (negative mode); nebulizing gas flow, 1.5 L/min.; drying gas pressure, 100 kPa; temperature for CDL (curved desolvation line) and heat block, 200°C; ion accumulation time, 30 msec.; scanning range, m/z 100-2000. The temperature of the column oven was set at 40°C and the injection volume was 5 μL
Human RBCs and plasma were obtained and used after the approval (number: 15 12 03 146-2) by the institutional ethical review board of Institute of Tropical Medicine, Nagasaki University. The animals in this study were handled according to the international guidelines and institutional guideline of Nagasaki University for the use and maintenance of experimental animals and used after approval (number 1710061412) by the institutional ethical review board of Institute of Tropical Medicine, Nagasaki University.