Plant Material, Reagents and Cells
The C. chinense var. simplex(Bin Hao), M. tenacissima(Dai Bai Jie) and A. graminifolia(Zhu Ye Lan) were purchased from the Institute of Ethnic Medicine (Xishuangbanna, Yunnan Province) and identified by Mrs. Lin Yanfang, Chief Expert of Dai Medicine. They were washed and dried for two weeks in the shade. Before extraction, the plants were cut into small pieces and crushed using a floor-standing continuous feed grinder (DF-35, Wenling Linda Machinery, Zhejiang, China).
DPPH, ABTS, hydrogen peroxide (H2O2, 30% wt), rutin, gallic acid and potassium persulfate were purchased from Aladdin (Shanghai, China); Luria–Bertani (LB) broth, from Hopebio (Qingdao, China); and E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 25923), from Huankai Guangzhou Microbial. RAW 264.7 macrophage cells were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China).
Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco-Thermo-Fisher Scientific (Grand Island, NY, USA). LPS was purchased from Sigma Chemical (St. Louis, USA). NO Griess reagent was acquired from Beyotime Institute of Biotechnology (Shanghai, China). IL-1β, IL-6 and TNF-α PicoKine ELISA kits were purchased from Boster Biological Technology (Wuhan, China). Deionized water (18.2 MΩ·cm) was used to prepare all aqueous solutions. All other reagents were purchased as analytical reagent grade and used without further purification.
Extraction of Dai antidotes
Crushed Bin Hao, Zhu Ye Lan and Dai Bai Jie (100 g) were extracted three times with 95% ethanol at a mass-to-volume ratio of 1:10 for 2 h at 98 ℃. The extracts were filtered, combined, and evaporated under reduced pressure to obtain crude ethanol extract.
The polar extracts were obtained from the corresponding crude extract at room temperature by successive extraction with the same volume of solvents of increasing polarity: petroleum ether, ethyl acetate, n-butanol and distilled water. The solvent was evaporated to dryness under reduced pressure in a rotary evaporator.
In this way, we obtained from Bin Hao a petroleum ether extract (PE, 1.1 g), ethyl acetate extract (EE, 1.3 g), n-butanol extract (BE, 4.2 g), and water extract (WE, 10.3 g). We obtained from Zhu Ye Lan a PE (1.7 g), EE (6.4 g), BE (8.6 g), and WE (9.3 g). We obtained from Dai Bai Jie an EE (13.5 g), BE (4.2 g), and WE (7.6 g). All polar extracts were stored at 4 ℃ until use.
Aqueous decoctions of the three Dai antidotes were prepared by mixing 100 g dried Bin Hao, Zhu Ye Lan or Dai Bai Jie with 1000 mL of distilled water and boiling for 0.5 h. This process was performed three times. The decoctions were filtered with gauze, combined and concentrated to 50 mL, giving crude Dai antidotes 2 g/mL. The extracts were stored at -20 ℃ until use.
Determination of total polyphenols content
The total polyphenols content of Dai antidotes was determined by using Folin–Ciocalteu reagent as described by Mohammad et al[9] with modifications. Briefly, 0.2 mL of samples, 6 mL of ethanol and 0.5 mL of Folin reagent was mixed to the 10 mL volumetric flask. After 5 minutes, 1.5mL of 20% (w/v)Na₂CO₃ was added,dilute with water to volume and incubated at room temperature for 60 minutes, then absorbance was measured at 765 nm. In the same way, the standard solution was prepared with gallic acid in a series of concentration gradients, and the standard curve was drawn to calculate the polyphenols content (mg GAE /g DW).
The linear equation was obtained by taking gallic acid concentration (x) as the horizontal coordinate and absorbance (y) as the ordinate. The linear equation was: y = 0.0374x-0.02602 and the correlation coefficient R2 = 0.9995. The experimental results showed that the absorbance of gallic acid is good linear relation in the range of 0.001 ~ 0.006 mg / mL. According to linear equation, the content of total polyphenols in three Dai antidotes was obtained.
In Vitro Anti-oxidant Activity
The in vitro antioxidant activities of different polar extracts and decoctions of the three Dai antidotes were evaluated based on ability to scavenge DPPH free radicals, ·OH radicals and ABTS radicals. Absorbance was determined on an ultraviolet spectrophotometer (UV-2600, Techcomp, Shanghai, China).
The scavenging effects on DPPH free radical was determined by the method as described by Shimada et al [10]with modifications 3.0 mL of DPPH(0.1mM)was intermingled with 1 mL of each sample (with final concentrations ranging from 0.2 to 1.2mg/mL) and allowed to stand at 37℃ for 30 min. The absorbance was then measured at 517 nm.
The scavenging DPPH free radical effect was calculated according to the following equations:
$$\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\left(\text{%}\right)=\left(1-\frac{{A}_{1}-{A}_{2}}{{A}_{0}}\right)\times 100\text{%}$$
Where A0 is the absorbance of the control (water rather than the sample), and A1 is the absorbance of the samples, A2 is the absorbance of the sample only(water rather than DPPH).
The scavenging effects on hydroxyl radical(·OH) was determined based on Fenton’s reaction as described by Aquino-Martins et al [11] with modifications. 1 mL of samples with different concentrations was mixed with 1mL 9 mM FeSO4 solution, 9 mM salicylic acid ethanol solution, and 8.8 mM H2O2 solution, respectively, and incubated at 37℃ for 30min, then absorbance was measured at 510 nm.
The scavenging ·OH effect was calculated according to the following equations:
$$\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\left(\text{%}\right)=\left(1-\frac{{A}_{1}-{A}_{2}}{{A}_{0}}\right)\times 100\text{%}$$
Where A0 is the absorbance of the control, A1 is the absorbance of the samples, A2 is the absorbance of the sample background.
The protocol of scavenging effects on ABTS free radical was adapted from Roberta Re et al.[12], ABTS reagent (7.0 mM) was mixed with 2.45 mM potassium persulfate in a volume ratio of 1: 1, and allowing the mixture to stand in the dark at room temperature overnight to obtain an ABTS stock solution. Then the ABTS stock solution was diluted with deionized water to obtain ABTS working solution with an absorbance value of 0.70 ± 0.05 at 734 nm. 4.0 mL of ABTS working solution was intermingled with 1 mL of each sample, and incubated at 37℃ in dark for 30 min. The absorbance was then measured at 734 nm. The scavenging ABTS free-radical effect according to the following equations:
$$\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\left(\text{%}\right)=\left(1-\frac{{A}_{1}-{A}_{2}}{{A}_{0}}\right)\times 100\text{%}$$
Where A0 is the absorbance of the control (ABTS), A1 is the absorbance of the samples, A2 is the absorbance of the sample background.
In Vitro Anti-bacterial Activity
The antibacterial activity of three Dai antidotes were evaluated by determining the MICs, MBCs and ZOI against E. coli, P. aeruginosa and S. aureus.
The MICs was determined by a microtiter broth dilution method. In brief, 100 µL of bacteria suspension with the dilution of 1: 10 was inoculated in the 96-well plates, the extracts were diluted serially from 50 to 0.196 mg/mL, then 100 µL of the diluted extracts solutions were added subsequently. The inoculated microplates were incubated under microaerobic conditions at 37℃ for 24 h with shaking (100 rpm). The lowest concentration resulting in no visible growth of tested organisms was recognized as MIC.
For determination of MBC, an aliquot (10 µL) of the bacterial suspension and sample (which shown no visible growth) inoculated onto the appropriated agar and incubated at 37℃ for 24 h. The lowest concentration that completely prevented microbial growth in LB Broth agar was recognized as MBC.
To assess ZOI in an agar diffusion model, bacterial lawns (50 µL) were prepared on a nutrient agar plate using the spread plate method. After soaking the sterile double-layer circular filter paper (diameter 6 mm) in each sample solution(with the concentration of 25mg/mL) for 2 h, the filter paper was removed, dried, and gently put it on the corresponding position of the plate. Then, these petri dishes were incubated at 37℃ for 24 h. Then, the ZOI diameter was measured by digital calipers, and was recorded in cm. Each bacterium was tested separately; three repeats of each material-bacteria combination were assessed.
In Vitro Anti-Inflammatory Activity
Cell Culture
The RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS and antibiotics (streptomycin 100 U/mL and penicillin 100 U/mL) in a humidified atmosphere of 5% CO2 at 37°C.
Determination of NO Production
Herein, we evaluated the ability of the three Dai antidotes to inhibit the NO production by the Griess method. The nitrite assay was carried out according to the manufacturer’s instructions. Briefly, Cells were seeded in 96 well plates at a density of 5 × 104 cells/mL and incubated for 24 h. Then, the cells were incubated with respective extracts of three Dai antidotes at different concentrations and exposed to LPS (1 µg/mL) for 24 h. The blank control cells were treated with DMEM only. LPS-induced NO production was determined by using Griess reagent, and the absorbance at 540 nm was measured using a microplate reader (Molecular Devices, Flex Station 3).
Determination of IL-1β,IL-6, TNF-α Production
The generation of IL-1β,IL-6, TNF-α was detected according to the manufacturer’s instructions. Briefly, Cells were seeded in 96 well plates at a density of 5 × 104 cells/mL and incubated for 24 h. Then, the cells were incubated with respective extracts of three Dai antidotes and exposed to LPS (1 µg/mL) for 24 h. The blank control cells were treated with DMEM only. LPS-induced IL-1β༌IL-6, TNF-α production was determined by using ELISA Kit, and the absorbance at 450 nm was measured using a microplate reader.