Pepper cultivation, harvesting and extraction
The seeds of the “Bang Chang” cultivar chili pepper (
C.
annuum var.
acuminatum) were obtained from The Tropical Vegetable Research Center, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand and planted in Samut Songkhram Campus, Suan Sunandha Rajabhat University, Thailand, as the original area from November 2022 to February 2023. Plant identification was confirmed according to a previous study
(Kaewdoungdee and Tanee, 2013;
Sudjaroen, 2014). Red peppers or ripe fruits were harvested and preserved as sun-dried chili peppers. Sun-dried chili peppers were qualified by monitoring their moisture content, which was lower than 1%. The pedicels were removed and separated from the fruits and the remaining part was ground to a powder form. Five hundred grams of chili pepper powder was ground and extracted with one liter of RBO by maceration within three days. The Capsicum extract in RBO was filtered to remove waste matter, transferred into an amber glass bottle and kept at 4°C.
Phytochemical analysis
Capsaicin
Capsicum extract and dried chili pepper were prepared with acetone and ethanol according to the AOAC official method. The capsaicin content was determined using an Agilent HPLC system equipped with ZORBAX C18 (4.6 x 250 mm, 5 mm). A sample (10 µl) was injected through methanol/water (70:30 v/v) and the flow rate was 1.0 ml/min for 15 min. The photodiode array detector and mass spectrophotometer represented the signal as a chromatogram and mass spectrum, respectively, which corresponded to standard capsaicin. The retention time of capsaicin was around 8.5 min at UV wavelength (l = 228 and 280 nm). Capsaicin in each sample was quantified by comparison with standard capsaicin (Sigma, USA) and reported as mg/100 g or mg%. The pungency level in Scoville Heat Units (SHU) was approximated using the capsaicin content in ppm x 15.
β-carotene
A sample (100 mg) was homogenized in 3.0 ml of 95% hexane/ethanol/acetone (2:1:1 v/v) and 5 ml of distilled water was added. The mixture was centrifuged at 5,000 rpm for 10 min. The supernatant (5 ml) was adjusted with 95% hexane (5 ml). β-carotene was determined using an Agilent HPLC system equipped with ZORBAX C18 (4.6 x 250 mm, 5 mm). A sample (20 µl) was injected through hexane/water (95:5 v/v) and the flow rate was 1.0 ml/min for 10 min. The retention time of β-carotene was around 4.0 min at lmax = 470 nm. β-carotene in each sample was quantified by comparison with standard capsaicin (Sigma, USA) and reported as µg/100 g or µg%.
g-oryzanol and α-tocopherol
g-oryzanol and α-tocopherol analysis were carried out by the Central Laboratory Co., Ltd., Bangkok, Thailand. g-oryzanol was detected and quantified by an HPLC system equipped with ZORBAX C18 (4.6 x 250 mm, 5 mm) and a PDA detector at 325 nm. The mobile phase consisted of acetonitrile/methanol/isopropanol (50:45:5 v/v). While the mobile phase of α-tocopherol was methanol/water (94:6) and the fluorescence detector was excited at 290 nm and emitted at 330 nm.
Total phenolic content
The Capsicum extract was measured for total phenolic content (TPC) using the Folin-Ciocalteu method and equipped with a spectrophotometer at 760 nm. TPC was represented as mg of gallic acid equivalent (GAE) per g.
In vitro biological assays
Antioxidant assay
DPPH scavenging activity: DPPH radicals in the presence of the extract (0.01-1 mg/ml) were scavenged and their absorbance was monitored. DPPH radicals (6 x 10-5 M) and ascorbic acid were used as negative and positive controls, respectively.
Nitric oxide scavenging activity
NO radicals in the presence of the extract (0.01-1 mg/ml) were scavenged and the absorbance of the reaction from the Griess reagent was monitored. Ascorbic acid was used as a positive control.
Anti-lipase assay
Capsicum extract (0.001-10 mg/ml) was dissolved in 10% (v/v) DMSO. Orlistat (0.0005-5.0 mg/ml) as a positive control was also adjusted with 10% (v/v) DMSO. Enzymatic inhibitory activity was evaluated by monitoring the absorbance (lmax = 415 nm) of p-nitrophenol, the product from
p-nitrophenyl butyrate catalyzed by pancreatic lipase.
Anti-α-glucosidase assay
Capsicum extract (0.001-10 mg/ml) was dissolved in 10% (v/v) DMSO, while acarbose (0.0005-5 mg/ml) as a positive control was dissolved in phosphate buffer. Enzymatic inhibitory activity was evaluated by monitoring the absorbance (lmax = 415 nm) of p-nitrophenol, the product from
p-nitrophenyl-α-D-glucopyranose catalyzed by α-glucosidase.
Anti-α-amylase assay
Capsicum extract and the positive control were similar to the anti-α-glucosidase assay. While the enzymatic inhibitory activity was evaluated by monitoring the absorbance (lmax = 450 nm) of reducing sugar, the product from starch catalyzed by α-amylase.
Cytotoxicity test
The Capsicum extract was dissolved in DMEM, containing DMSO (10%), FBS (10%), penicillin/streptomycin (1%), filtered with a 0.2 µM membrane and adjusted to the concentration within each well of the microtiter plate by sterile culture medium (0.0001, 0.001, 0.01, 0.1 and 1.0 mg/ml). Each sample or control well was suspended in human skin fibroblasts (2.2-3.3 x 104 cells/ml) and incubated for 48 h. Cell cytotoxicity was determined by sulforhodamine B staining with viable skin cells. Cell viability of human skin fibroblast cells against the extract was explained as cell viability (%), which was calculated from four-time repeated experiments. The positive control (cytotoxic) was sodium lauryl sulfate and the negative control (non-toxic) was DMEM.
Anti-inflammation assay
Albumin degradation test
The capsicum extract was dissolved with 20% Tween 20 and centrifuged at 150 rpm for 5 min. The supernatant was adjusted to 0.01, 0.1, 1, 10 and 100 mg/ml. Each sample was incubated with albumin solution at 70±2°C for 5 min. The absorbance of albumin was monitored and diclofenac diethyl ammonium was used as a positive control. Anti-inflammatory activity was represented as a reduction of albumin degradation.
Reduction of NO produced from lipopolysaccharide (LPS)-induced macrophage cells
Mouse macrophage cell (RAW264.7) was cultured in Dulbecco’s modified Eagle’s medium, DMEM (Invitrogen, USA), containing fetal bovine serum, FBS (10%) and penicillin/streptomycin (1%), in a humidified incubator (5% CO2) at 37°C for 24 h. The cell culture was transferred to a 24-well plate and suspended to 1 x 105 cells with 500 µl of medium/well. Each cell culture well was treated with different concentrations of extract (or controls) for 1 h and with LPS for an additional 24 h. After that, the supernatant of the culture medium from each well was determined for NO production. Culture medium (50 µl) and Griess reagent (100 µl) were mixed in a 96-well plate and stood for 10 min at room temperature. The absorbance of the reaction mixture was monitored by a microtiter plate reader. The positive control was triamcinolone acetonide (0.1 mg/ml).
Data analysis
The bioactive compounds of dried chili pepper and Capsicum extract were represented as the mean and standard deviation of concentration.
In vitro biological activities of chili pepper extract were also analyzed by descriptive statistics and compared with controls. Data were calculated from triplicate experiments for each parameter.