Chemical material
L-Ascorbic acid, aluminium chloride (AlCl3), 2,2’-azino-bis3-ethylbenzothiazoline-6-sulfonic acid (ABTS), calcium chloride (CaCl2), corilagin, dexamethasone, disodium phosphate (Na2HPO4), ferric chloride (FeCl3), ferrous chloride (FeCl2), ferrous sulfate (FeSO4), formic acid, Folin-ciocalteu reagent, 4,4′,5,5′,6,6′-hexahydroxy-diphenic acid 2,6,2′,6′-dilactone (ellagic acid), hyaluronic acid, hydrochloric acid (HCl), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), bovine testicular hyaluronidase (E.C.3.2.1.3.5), potassium acetate (CH3COOK), potassium persulphate (K2S2O8), sodium acetate (C2H3NaO2), sodium carbonate (Na2CO3), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), 2,4,6-tripyridyl-striazine (TPTZ), and 3,4,5-trihydroxybenzoic acid (gallic acid) were analytical grade bought from Sigma-Aldrich (St. Louis, MO, USA). Amphotericin B, Dulbecco modified eagle medium (DMEM), L-glutamine, penicillin/streptomycin, trypan blue, and secondary antibody conjugated with HRP were bought from Invitrogen™ (Carlsbad, CA, USA). Lipopolysaccharide (LPS) was bought from Cell Signaling Technology® (Danvers, MA, USA). GlutaMAX™-I supplement was bought from Thermo Fisher Scientific, Inc. (ThermoFisher Scientific, Waltham, MA, USA). Newborn bovine calf serum, fetal bovine serum (FBS), bovine serum albumin (BSA), and antibiotic-antimycotic solution (100 ×) was bought from Gibco™ (Thermo Fisher Scientific, Waltham, MA, USA). Acetic acid, ethanol, and dimethyl sulfoxide (DMSO), were purchased in analytical grade from Labscan, Ltd. (Dublin, Ireland). Acetonitrile was purchased in HPLC-grade from Labscan, Ltd. (Dublin, Ireland).
Plant Material
The D. longan fruits were collected from Chiang Mai Province in the Northern Thailand during 2020 by the gardeners according to WHO Guidelines on Good Agricultural and Collection Practices (GACP) for Medicinal Plants.39 The preparation of conventional dried and black D. longan was performed by Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, Thailand.
Conventional dried D. longan preparation
Conventional dried D. longan was obtained after the whole fruit of fresh D. longan was incubated in an oven set the temperature of 50 ºC until dryness. The sample of dried D. longan was kept in sealed plastic bag to prevent contact with air and humidity in the room temperature until further experiments.
Black D. longan preparation by thermal and aging process
Black D. longan was obtained after the whole fruit of dried D. longan was incubated for 20 days in a controlled temperature of 70 ºC and controlled humidity of 75% relative humidity. The sample of black D. longan was kept in sealed plastic bag to prevent contact with air and humidity in the room temperature until further experiments.
Preparation of dried D. longan and black D. longan extracts
Seed, aril, and pericarp were separated from each other. Each part of D. longan fruit was ground into fine powder using 20-inch herbal medicine grinder tub with a powerful motor (Thai Pradith Industry Co., Ltd., Bangkok, Thailand). Dried D. longan powder was subsequently macerated in 95% v/v ethanol for 3 cycles of 24 h. The extracting solvent from 3 cycles were combined and removed using a rotary evaporator (Buchi Labortechnik GmbH, Essen, Germany). All extracts were stored in the refrigerator (4 ºC) until further experiments.
Chemical compositions determination of dried D. longan and black D. longan extracts
Total phenolic content determination
Each D. longan extract was analyzed for total phenolic content using the Folin-Ciocalteu method according to the previously described method.40 The results were presented in the form of gallic acid equivalent values (GAE) representing an amount of gallic acid (µg) per g of the D. longan extracts. GAE was calculated following the equation; X = (Y - 0.0075)/0.3812, where X is GAE or µg of gallic acid per g of the D. longan extracts and Y is an absorbance of each sample tested in Folin–Ciocalteu assay. The experiments were triplicately performed.
Total flavonoid content determination
Total flavonoid content of each D. longan extracts was investigated using aluminium chloride method which has been previously described with some modifications.41 Firstly, 20 μl of the ethanolic solution of D. longan extracts was mixed with 80 μl of 10% w/v AlCl3 aqueous solution and 100 μl of 1 M CH3COOK aqueous solution. After the resulting mixtures were incubated for 30 min in the dark, they were measured for an absorbance at 415 nm using multimode detector (SPECTROstar Nano, BMG Labtech, Offenburg, Germany). Quercetin was applied as a standard compound to construct a calibration curve. Finally, the results were presented as quercetin equivalent (QE) values which represented a µg of quercetin per g of the D. longan extracts. QE was calculated following the equation; X = (Y + 0.033)/0.107, where X is QE or µg of quercetin per g of the D. longan extracts and Y is an absorbance of each sample tested in aluminium chloride assay. The experiments were triplicately performed.
Determination of gallic acid, corilagin, and ellagic acid content by high performance liquid chromatography (HPLC)
The quantitative analysis of gallic acid, corilagin, and ellagic acid were performed using an HP 1100 chromatographic system (Hewlett-Packard, Waldbronn, Germany). A gradient mobile phase system composed of two phases, including phase A (0.05% formic acid in acetonitrile) and phase B (0.05% formic acid aqueous solution). The program was set for gradient elution of 10% A (0–8 min), 20% A (8–28 min), 30% A (28-30 min), and 10% 10 (30-35 min), eluting the sample at a flow rate of 1.0 ml/min. The UV detector was set at 280 nm with a Eurospher II 100-5 C18 (250 ´ 4.6 mm, i.d. 5 µm, Knauer, Berlin, Germany). All samples, standard solution, and mobile phase had been filtrated through a 0.45 mm millipore filter, type GV (Millipore, Bedford, MA) before injection to the HPLC system. The injected volume was set at 20 mL. The sample of D. longan extracts was prepared at the concentration of 1 mg/ml. Various concentrations of standard gallic acid (10-150 µg/ml), ellagic acid (5-100 µg/ml), and corilagin (2-80 µg/ml) solution were used for the construction of standard curves for quantitative determination. Subsequently, the content of gallic acid, corilagin, and ellagic acid was then calculated.
Gallic acid content was calculated following the equation; X1 = (100A + 1,296)/26.8C (R2 = 0.9964), where X1 is the gallic acid concentration, A is the area under the curve (AUC) of the gallic acid peak detected around 4 min, and C is the concentration of the respective sample solution.
Corilagin content was calculated following the equation; X2 = (100A + 2,325)/17.8C (R2 = 0.9996), where X2 is the concentration of corilagin, A is the AUC of the corilagin peak detected around 10 min, and C is the concentration of the respective sample solution.
Ellagic acid was calculated following the equation; X3 = (100A + 13,372)/33.7C (R2 = 0.9957), where X3 is the concentration of ellagic acid, A is the AUC of the ellagic acid peak detected around 20 min, and C is the concentration of the respective sample solution.
Antioxidant activities determination of dried D. longan and black D. longan extracts
2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay
The radical scavenging effects against ABTS•+ of D. longan extracts, gallic acid, corilagin, and ellagic acid were evaluated by ABTS assay and reported in the term of Trolox equivalent antioxidant capacity (TEAC) which represented the quantity of Trolox that equivalent to 1 mg of the D. longan extracts.42 TEAC values were calculated following the equation; X = (Y - 1.2028)/7.9964, where X is TEAC value and Y is an absorbance of each sample tested in ABTS assay. L-Ascorbic acid was used as a positive control. The experiments were triplicately performed.
Ferric reducing/antioxidant power (FRAP) assay
The reducing capacity of D. longan extracts, gallic acid, corilagin, and ellagic acid were investigated by means of ferric ion reducing activities.40 The ferric reducing/antioxidant power of each D. longan extracts were expressed in the form of equivalent concentrations (EC1) representing the amount of ferric-TPTZ reducing capacity, which is equivalent to 1 mg of the D. longan extract. EC1 values were calculated following the equation; X = (Y - 0.0287)/0.01405, where X is EC1 value and Y is an absorbance of each sample tested in FRAP assay. L-Ascorbic acid was used as a positive control. The experiments were triplicately performed.
Anti-inflammatory activities determination of dried D. longan and black D. longan extracts
Murine monocyte-macrophage (RAW 264.7) cells (American Type Culture Collection, ATCCTIB-71) treated with LPS were used to investigate the effect of D. longan extracts and their chemical compositions on the pro-inflammatory cytokine secretion (IL-6 and TNF-). The cell culture was performed according to a method previously described with some modifications.43,44 The treated cells along with its supernatant were divided into two parts. The first withdrawal part was undergone centrifugation for 10 min at 13,500´ g and the supernatant was investigated for the cytokine secretion by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s protocol (R&D Systems). On the other hand, the second part, which was leftover in the wells, was investigated for the cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To reduce variation due to cell density differences, secretion of IL-6 and TNF-α from RAW 264.7 cells were normalized to MTT levels.44 RAW 264.7 cells without LPS treatment served as a negative control, while 100% of cytokine secretion was from positive control of which RAW 264.7 cells treated with LPS. The inhibitory activities of each samples were was calculated following the equation; %inhibition = 100 – A, where A is the amount of cytokines secretion. Dexamethasone was served as a positive control for both IL-6 and TNF-α secretory inhibition. The experiments were triplicately performed.
Anti-hyaluronidase activity determination of dried D. longan and black D. longan extracts
The hyaluronidase inhibitory activity of D. longan extracts, gallic acid, corilagin, and ellagic acid was evaluated by measuring a product from the cleavage of sodium hyaluronate by hyaluronidase.45 Prior to the experiment, the enzyme activity of hyaluronidase was determined. Only more than 90% enzyme activity was used in the anti-hyaluronidase activity determination. The hyaluronidase inhibitory activity were calculated according to the following equation; % Inhibition = [1 – X/Y)] ´ 100, where X is the absorbance of the mixtures with sample; Y is the absorbance of the mixtures without sample. Oleanolic acid was used as a positive control. The experiments were triplicately performed.
Statistical analysis
All values were given as means ± standard and analyzed. The statistical analysis was used t-test and ANOVA by using SPSS software (SPSS Statistics 21.0, IBM Corporations, New York, NY, USA). A value of p < 0.05 were accepted as significant.