2.1 Plant materials and extraction
Ensete glaucum seeds taken from ripe fruits were collected from Bac Ai District, Ninh Thuan Province, Vietnam in September 2020. The samples were identified and authenticated by MSc. Le Duc Thanh (Research Center of Ginseng and Medicinal Materials Ho Chi Minh City, Vietnam) and a voucher specimen (TNDL-EGS-2020) were deposited for Ensete glaucum (Roxb.) Cheesman. Bad seeds were removed, and then the sample was washed with tap water and distilled water. This was followed by drying and grinding into powder for study.
The dried powdered material was extracted with distilled water by the decoction method for 60 minutes (total ratio is 1: 20 w/v) to obtain liquid extracts. The liquid extracts were collected by filtering and then concentrated using a rotary evaporator at 80 °C under reduced pressure to obtain crude extracts. The crude extract was stored at -15 °C and dissolved in a suitable solvent to yield a stock solution.
2.2 Experimental animals
All healthy Swiss albino mice of both genders weighing from 20 ± 2 g (from the Institute of Vaccines and Medical Biologicals in Nha Trang City, Vietnam) were stabilized for 7 days before testing, and raised in a room at a temperature of 25 0C, humidity at 50-65%, and on a 12 light/12 dark cycle. They were raised in PP-plastic cages (33 × 21 × 15 cm) with adequate food and water. The oral administration volume was 10 mL/kg body weight, and the oral administration time in the experiment was between 8 and 9 am. Experimental studies on mice followed the guidelines of the Guidelines for the Care and Use of Laboratory Animals and Ministry of Health-Vietnam (No. 141/QĐ-K2ĐT).
2.3 Acute oral toxicity test
Acute oral toxicity of the polyextract was investigated according to the Organization for Economic Cooperation and Development (OECD) guideline number 423 and the Vietnam Ministry of Health [10, 11]. The test was conducted in two phases (mice were starved for 12 hours before testing): Phase 1 (primary), 6 mice (3 males, 3 females) were given the sample at the highest concentration that could be injected via a needle (50 mL/kg volume), general movements, behavioral manifestations, hair state, feeding, urination and number of dead mice were monitored and recorded in 4-72 hours. After 72 hours, the mice showed no signs of abnormality or death, and continued monitoring for 7 days and 14 days. In the case of dead mice, the dose was lowered to find the LD50 (lethal dose, 50%). Mice were dissected to observe the macroscopic and microscopic organs (if there was death).
2.4 Antinociceptive tests
2.4.1 Acetic acid-induced writhing test
The writhing test was performed as described previously with some slight modifications [12]. Male mice were divided randomly into 6 groups (n = 9): (I) Negative control group (distilled water, per os, p. o.); (II) Positive control group (aspirin pH 8 100 mg/kg, p. o.); (III-VI) Experimental groups with 4 doses of the extract (50, 100, 200, 400 mg/kg, p. o.). Two hours after oral administration of the drug and extract, mice were intraperitoneally injected with 0.6% (v/v) acetic acid (Merck Co., Darmstadt, Germany) to induce pain. The pain manifestation was measured after 10 minutes of injection over a period of 30 min. Analgesic activity was determined based on the reduction in the number of abdominal writhing frequencies.
2.4.2 Hot plate test
The method was described previously with some minor modifications [13]. Male mice were divided randomly into 4 groups (n = 8): (I) Negative control group (distilled water, p. o.); (II) Positive control group (tatanol codeine 177 mg/kg, p. o.); (III-IV) Experimental groups with 2 doses of the extract (100 mg/kg and 200 mg/kg, p. o.). The drug and extract were administered via the oral route. The hot-plate temperature was set at 55 ± 0.5 0C. Mice showing an initial nociceptive response between 8 and 30 seconds were selected for the experiment. To avoid tissue damage, the cut-off time was set at 60 seconds. The latency time recorded was from the moment the animal was placed on the hot plate until it licked the hind paw or jumped. The estimation was determined before administration, and then 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, and 240 min after extract/drug administration.
2.4.3 Formalin-induced licking test
The formalin-induced pain method was carried out previously described with minor modifications [14]. Male mice were divided randomly into 4 groups (n = 8): (I) Negative control group (distilled water, p. o.); (II) Positive control group (Tatanol codeine 177 mg/kg, p. o.); (III-IV) Experimental groups with 2 doses of the extract (200 mg/kg and 400 mg/kg, p. o.). Two hours after oral administration of the drug and extract, mice received an intraplanter injection with 20 µL of 2.5% (v/v) formalin (Merck Co., Darmstadt, Germany) into the left hind paw. Then, the animal was placed individually in the flat, translucent chamber (15 × 14 × 20 cm) and observed for 30 minutes. After the injection, two distinct periods or phases of licking/biting behaviour occur in phase I (0-5 min) and phase II (15-30 min). The analgesic activity was evaluated based on the total duration of paw licking in 2 phases, and on scoring the behavior in phase II. The rodent stood and walked firmly on the injected paw equal to 0, the injected paw was not fully lifted equal to 1; the injected paw completely lifted off the floor equal to 2; the injected paw was licked or chewed – which was the expression of the most painful sensation, equal to 3.
2.5 Experimental design of sodium glyoxylate-induced urolithiasis in mice
The animal model was carried out within 14 days. After being housed for one week, 56 mice were equally divided into 7 groups of 8 mice. To induce renal calculi CaOx, sodium glyoxylate (Sigma Co., St. Louis, MO, USA) at a dose of 100 mg/kg was administered by successive intraperitoneal injections for 7 consecutive days (from day 1 to day 7). In the normal group, 0.9% saline NaCl was intraperitoneally injected from day 1 to day 7. Groups that received the treatment with the extract would drink the extract for 14 days, while the cystone-treated group only drank from day 7 to day 14 [15].
2.5.1 Serum analysis
On the 7th and 14th days, blood from mouse tails was collected, centrifuged to obtain the serum, and analysed for the content of creatinine, urea nitrogen, uric acid, calcium, and phosphate by biochemical kits.
2.5.2 Urine analysis
On the 7th and 14th days, animals were kept in individual metabolic cages and 24 h urine samples were collected. Animals had free access to drinking water during the urine collection period. The urine samples were analysed for the levels of phosphate, calcium, and magnesium with the help of diagnostic biochemical kits.
2.5.3 Histopathological examination
Kidney tissues were collected and fixed in 10% Neutral Buffered Formalin, processed routinely, and embedded in paraffin. Three-micron-thick sections were prepared and stained with hematoxylin and eosin dye for microscopic investigation. The stained sections were examined and photographed under a light microscope (Nikon Eclipse Ts2R-FL).
2.6 Nucleation and aggregation assays
2.6.1 Nucleation assay
First, the solution of 10 mM calcium chloride and 1 mM sodium oxalate was prepared in crystallization buffer containing 10 mM Tris–HCl and 90 mM NaCl (pH 7.4). The experiment was performed in triplicate to a 24-well plate. As described briefly, 190 µL of 10 mM CaCl2 was added into 24 well-plate before adding 20 µL of the tested extract at various concentrations. Cystone was used as the positive control group, and it was dissolved in distilled water to give tested concentrations. In addition, for the control of each time, an equal volume of the basic buffer was added into the well. To induce the crystallization reaction, 190 µL of 1 mM Na2C2O4 was added into each well. Thereafter, the mixture was incubated at 37 °C in a water bath for 30 minutes [16]. Crystal images were captured randomly from 9 high-power fields (HPFs) with 400× magnification under Nikon inverted phase-contrast light microscope ECLIPSE Ts2 (Nikon Eclipse Ts2R-FL). Crystal sizes and the number of crystals were measured using NIS Element D software (Nikon), whereas crystal mass was calculated from total crystal areas of 9 HPFs per well using the following equation:
2.6.2 Aggregation
First, individual COM (calcium oxalate monohydrate) crystals were prepared by mixing 10 mM calcium chloride and1 mM sodium oxalate in buffer at a ratio of 1: 1 (v/v). Then the solution was incubated at 25 °C for at least one hour. The supernatant was discarded by centrifugation, whereas COM crystals were harvested and washed three times with methanol. After another centrifugation, methanol was discarded, and the crystals were air-dried by evaporation overnight at room temperature. The COM crystals were resuspended in Tris-buffer saline at a concentration of 800 µg/mL. Briefly, 150 µL of COM crystal solution was added to 50 µL of the extract at different concentrations and as the same procedure for positive control cystone. Besides, for the control of each time, an equal volume of the basic buffer was added into the well. Then the plate was continuously shaken on a rotary shaking machine at 25 °C for one hour [16, 17]. Thereafter, images of the formation of CaOx crystal aggregates were observed under the Nikon inverted phase-contrast light microscope ECLIPSE Ts2. Several CaOx crystal aggregates were counted from 3 randomized HPFs per well. The experiment was performed in triplicate in 96-well plate.
2.7 Anti-inflammatory assays
The anti-inflammatory activity of the aqueous extract of E. glaucum seeds was examined by protein denaturation, heat-induced hemolysis, and hypotonicity-induced hemolysis assays according to the protocol previously described [18].
2.8 Antioxidant assays
The antioxidant activity of the extract was evaluated through the DPPH radical scavenging assay, ABTS radical cation decolorization assay, and reducing power assay [19].
In the DPPH assay, 25 µL of the extract at different concentrations was incubated with 25 µL of 0.6 mM DPPH solution (Sigma Co., St. Louis, MO, USA) in methanol to reach a total volume of 200 µL in total volume. The mixture was kept in the dark for 30 min at room temperature. The absorbance was measured at 515 nm.
In the ABTS assay, 5 μL of the extract at different concentrations was mixed with 145 μL of ABTS (Sigma Co., St. Louis, MO, USA) solution, the reactions were incubated for 6 min at room temperature and the absorbance was measured at 734 nm.
In the reducing power assay, the reactive mixture, 40 µL of the extract at various concentrations, 100 µL of 200 mM sodium phosphate buffer (pH 6.6), and 100 µL potassium ferricyanide 1%, were incubated at 50 °C for 30 min. Then 100 µL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 3000 rpm for 10 min. Next, 100 µL of the supernatant was mixed with an equal volume of double-distilled water and 20 µL of 0.1% ferric chloride. The absorbance was measured at 700 nm. The reducing power activity was subsequently analysed via optical density and EC50 values. The lower the optical density value, the weaker the reduction activity of the sample. The EC50 value is the concentration of effective antioxidants for the absorbance to reach 0.5, which was calculated through the equation illustrating the correlation between the concentration of the sample and its optical density.
Ascorbic acid (Sigma Co., St. Louis, MO, USA) was used as a positive control. All measurements were performed in triplicate using a plate reader (Biotek, USA), and an average of each sample was calculated. The percent DPPH/ABTS inhibition was calculated by the equation: where Ac, A0c, At and A0t are the absorbance of the control (with DPPH/ABTS solution and without test extract), the blank (without DPPH/ABTS solution and test extract), the sample (with DPPH/ABTS solution and test extract), and the control sample (without DPPH/ABTS solution and with test extract), respectively.
2.9 Phytochemical screening
Preliminary qualitative phytochemical analysis was carried out to identify the secondary metabolites present in E. glaucum seeds. Screening was performed according to the method of Cuilei [20] with alkaloids, flavonoids, tannins, triterpenoids, saponins, coumarins, anthraquinones, anthocyanosides, proanthocyanidins, lipids, volatile oils, carotenoids, reducing agents, and organic acids. The qualitative results are expressed as (+) for the presence and (−) for the absence of phytochemicals.
2.10 Determination of total polyphenol and flavonoid contents
The total polyphenol content was estimated by Folin-Ciocalteu’s method. Briefly, a 5 μL test sample was mixed with 10 μL of Folin-Ciocalteu’s reagent (Merck Co., Darmstadt, Germany) and 165 μL of double-distilled water. Then 30 μL of Na2CO3 (20% w/v) was poured into this mixture immediately 5 min. The reaction was kept in less-exposure-to light condition for 2 hours at room temperature. The absorbance was measured at 758 nm using a plate reader (Biotek, USA) and all determinations were made in triplicate. The total polyphenol content was calculated from the calibration plot of the gallic acid standard (Sigma Co., St. Louis, MO, USA) and expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g d. w.) [19].
The flavonoid content was estimated based on the aluminum chloride colorimetric method. Briefly, 20 μL of 2% w/v AlCl3 (Merck Co., Darmstadt, Germany) was added to 30 mL diluted extract, and 150 μL methanol was then added to the mixture. After that, the solution was mixed and incubated for 15 min at room temperature. The absorbance of the reaction mixtures was calibrated at 433 nm by a plate reader (Biotek, USA). The tests were carried out in triplicate. The total flavonoid content was estimated from the calibration plot of quercetin standard (Sigma Co., St. Louis, MO, USA) and expressed as milligrams of quercetin equivalents per gram of dry weight (mg QE/g d. w.) [19].
2.11 Statistical analysis
The results are expressed as mean ± standard error of mean (mean ± SEM). Data analysis was performed using R software, version 4.2.2. Analysis of variance with ANOVA and subsequent multiple comparisons of means according to Tukey’s test were performed. Shapiro Wilk’s test was used to test the normal distribution of data. If the normal distribution was not given, a Kruskal-Wallis test on ranks was performed. Differences were considered statistically significant if the P values were less than 0.05. For regression analysis, dose-response relationships and IC50 were calculated using R-package “drc” (Analysis of Dose-Response Curves).