Collection and drying of T. cacao husk powder
The T. cacao plant was authenticated at the Ateneo de Davao University (AdDU) Biological Collections, Davao City, Philippines. The voucher specimen (AdDU2019.01) was kept in the AdDU Herbarium for future reference. T. cacao husks (1 kg) were collected from Calinan, Davao City, Philippines. They were washed with distilled water three times to remove contaminants and blot-dried with paper towels. The husks were peeled using a stainless peeler, placed in plastic trays, and air-dried at room temperature for four weeks. The peelings were transferred in different, clean trays every day for four weeks to avoid contamination. At the end of the fourth week, the peelings were oven-dried (BIOBASE BOV-T105F, Metro Manila, Philippines) for 24 h at 37°C. The dried peelings were milled using an electric blender (AVINAS AV-126, Bulacan, Philippines) and sifted using 0.75 mm sieves (MonotaRO, Japan). The powder (20 mg) was placed in a wide-mouthed amber glass bottle and kept at room temperature until further use.
Preparation of T. cacao husk aqueous extract
T. cacao powder (1 g) was suspended to 100 ml of deionized water and heated in a 60°C water bath (Thermo Fisher Scientific, Waltham, USA) for 1 h. The water bath temperature was checked every 5 min to ascertain that it is within the temperature range indicated. After heating, the suspension was filtered using a cheesecloth to a beaker. The filtrate was centrifuged (Fisher Scientific, Waltham, USA) at 3,000 rpm for 5 min, and the supernatant (20 ml) was placed in an amber glass bottle.
Preparation of T. cacao husk extract concentrations and positive controls
TCE was prepared by diluting different amounts of aqueous T. cacao extract to varying amounts of deionized water to make 10%, 7%, 5%, 3%, and 1% concentrations. Metformin and Diclofenac were the positive controls used for the antidiabetic assays and anti-inflammatory assay, respectively. A 5 mg Metformin tablet was dissolved in 100 ml deionized water to make a 5% concentration. The same procedure was used in preparing a 5% concentration of Diclofenac. TCE concentrations for the brine shrimp lethality assay were made by diluting different amounts of aqueous cacao extract to varying quantities of artificial seawater to make 10%, 7%, 5%, 3%, and 1% concentrations. The artificial seawater was prepared by dissolving 3.8 g of non-iodized rock salt in 100 ml deionized water. All solutions were stored in amber glass bottles and kept in a refrigerator (4°C) until further use.
Glucose diffusion assay
The protocol used in the study was based on Ahmed et al. [15]. This assay was conducted to determine if the TCE samples can inhibit the movement of glucose across a dialysis membrane, indicating a potential to reduce sugar levels in the bloodstream. Reaction mixtures consisting of 1 ml of 25 mmol/L glucose solution, 1 ml of 0.15 mol NaCl solution, and 1 ml of the different TCE samples and 5% Metformin were prepared in separate dialysis bags (Thermo Fisher Scientific, Waltham, USA). The dialysis bags were tied and immersed in separate beakers containing 10 ml of deionized water and 40 ml 0.15 mol NaCl solution. The reaction mixtures were incubated at room temperature. An aliquot (2 ml) of dialysate from every mixture was transferred using a micro pipettor in different test tubes after 30, 60, 120, and 180 min of incubation. Benedict's reagent (Chem Vest Commercial Trading, Davao, Philippines) (2 ml), which served as an indicator of glucose content, was added to every test tube, shaken carefully for 1 min, and heated in a water bath at 70°C for 5 min. An aliquot (500 μl) of the mixture from each test tube was then transferred in a microwell, and the absorbance was read at 540 nm using Epoch Microplate Spectrophotometer (Biotek, Winooski, USA). The experiment was conducted in triplicates and three independent trials. Glucose dialysis retardation index (GDRI) was calculated using the formula:
$$ GDRI\ \left(\%\right)=1-\frac{Glucose\ content\ \left( Samples\frac{mg}{mL}\right)}{Glucose\ content\ \left( Without\ Sample\frac{mg}{mL}\right)}\times 100 $$
where: samples = glucose content of the samples and controls.
Glucose uptake by yeast cells assay
The method used in the study was based on Tarnam et al. [16]. An antidiabetic substance can increase glucose uptake in different cells and tissues, reducing its amount in the bloodstream. This assay was conducted to test if the TCE samples are potentially antidiabetic by determining if they can increase glucose uptake using yeast cells as model specimens. Commercial baker’s yeast was dissolved in distilled water and centrifuged (Fisher Scientific, Waltham, USA) at 3000 rpm for 5 min. The supernatant (1 ml) was added to 9 ml of distilled water to make a 10% (v/v) yeast suspension in a beaker. In different test tubes, 1 ml of the TCE samples and 5% Metformin were added separately using a micro pipettor to 1 ml of varying glucose solutions (5 mmol/L, 10 mmol/L, and 25 mmol/L) and incubated for 10 min at 37°C. To each of the mixtures, 100 μl of yeast suspension was added to start the reaction. The mixtures were vortexed for 1 minute and incubated at 37°C for 60 min. The mixtures were added with 2 ml of Benedict's reagent and heated at 60°C in a water bath for 5 min. An aliquot (300 μl) from every mixture was transferred in a microwell, and absorbance was read at 540 nm using Epoch Microplate Spectrophotometer (Biotek, Winooski, USA). The experiment was conducted in triplicates and three independent trials. The percentage glucose uptake by yeast cells was calculated using the formula:
$$ Percent\ Glucose\ Uptake\ by\ Yeast\ Cells=\frac{Abs\ Sample- Abs\ Blank}{Abs\ Untreated\ Control- Abs\ Blank}\times 100 $$
Egg albumin denaturation assay
The protocol used in the study was based on Ullah et al. [17]. This assay determined if the TCE samples can inhibit protein denaturation, an event that activates inflammatory responses in the body. The assay was conducted by mixing 200 μl of egg albumin (from fresh hen’s egg), 2.8 ml of phosphate-buffered saline (PBS) (pH 6.4), and 2 ml of the samples or control. The mixtures were incubated at 37°C for 15 min and heated in a water bath at 70 °C for 5 min. The mixtures were cooled for 5 min, and 500 μl of every mixture was transferred in a microwell. The absorbance of every sample was read at 660 nm using Epoch Microplate Spectrophotometer (Biotek, Winooski, USA). The percentage inhibition of protein denaturation was calculated using the formula:
$$ Percent\ Inhibition\ of\ Protein\ Denaturation=1-\frac{Abs\ Sample- Abs\ Blank}{Abs\ Untreated\ Control- Abs\ Blank}\times 100 $$
Brine shrimp lethality assay
The protocol used in the study was based on Meyer et al. [18] and Guevarra [19]. This assay was conducted to determine the cytotoxicity of the TCE samples. Artemia salina eggs (5 mg) were hatched in a shallow rectangular dish (approximately 1 inch deep) filled with artificial seawater. The rectangular dish is clamped with a plastic divider punched with 2 mm holes to make two unequal compartments. The eggs were sprinkled into the larger chamber and illuminated for 48 h. The hatched brownish nauplii that moved to the illuminated smaller compartment were collected with a Pasteur pipette and used for the assay. An aliquot (3 ml) from every TCE sample was placed in a microwell, and ten brine shrimps were transferred in every well using pipettes. The number of alive and dead brine shrimps was counted after 24 h of exposure to the TCE samples. Brine shrimps were considered dead if they did not exhibit any movement during several seconds of observation. The experiment was conducted in five replicates and three independent trials. Percentage mortality was calculated using the formula: % Mortality of Brine Shrimps=(No.of Dead Brine Shrimp/s)/( Total No.of Brine Shrimps) ×100.
Statistical analysis
All values from the antidiabetic and anti-inflammatory bioassays were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test. The values of P ≤ 0.05 are considered statistically significant. This test determined if the results of the antidiabetic and anti-inflammatory assays of the extracts have significant differences compared to the results of the positive controls.