Plant materials and preparation on the C-3 extracts of P. fruticosa
P. fruticosa leaves were collected from Huzhu Northern Mountain, Qinghai during 2014 at an altitude of 2940 m (E 102°21.149′, N 36°55.807′) [5]. The voucher specimen was identified by the professor Dengwu Li and was deposited at Herbarium of the Northwest A&F University, Yangling, China (WUK0780381). The crude extracts of P. fruticosa were the acetone extraction phase (C-3) extracted by Wang in our lab [6]. The air-dried, ground leaves of P. fruticosa were extracted with 80% acetone for three days at room temperature to obtain the crude acetone extracts. Part of them were further partitioned with ethyl acetate extracts and then subjected to column chromatography (15 cm diameter, 120 cm length) with silica gel as stationary phase. The elution of components present in the extract was then dissolved in different concentration gradient of ether/acetone (4:1, 1:1, 1:5 and 0:1 v/v). The third fraction labeled as C-3 was found to be most active in the assay, so the C-3 fraction was selected for further research. Both of the C-3 and EGb extracts were dissolved in the initial concentration of 5 mg/ml and the individual compounds were dissolved in the concentration of 1 mM.
Bacteria and chemicals
E.coli (ATCC No.25922) (Microbial Culture Collection Center of Guangdong Institute of Microbiology, China); Ginkgo biloba extracts, EGb 761, standard at European Pharmacopeia (Shanghai Youxin Biological Science and Technology Co., Ltd. PR China); quercetin, catechin, caffeic acid, rutin, hyperoside, kaempferide, ellagic acid and isorhamnetin (Shanghai Yuanye Industrial Co., Ltd. PR China); sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, potassium dihydrogen phosphate, hydrogen peroxide 30% (Guangdong Guanghua Chemical Factory Co., Ltd. PR China), dimethyl sulfoxide (DMSO) (Tianjin Bodi Chemical Factroy Co., Ltd. PR China), horseradish peroxidase; Yeast extract, Tryptone (OXOID Ltd., Basingstoke, Hampshire, England). All reagents and solvents used were of analytical grade. Deionized water (0.055 μS/cm) was used to prepare aqueous solutions.
Determination of antioxidant synergy effects on MTS
To circumvent the limitations of the assays based on chemical mechanism to determine antioxidant synergistic effects, the MTS assay was adopted to measure the bioavailability behind synergism with some modification [27]. Bacteria were cultivated aerobically overnight in LB medium in shaking bed (37 °C, 120 rpm). Cell growth was monitored by measurement of the optical density at 600 nm. One milliliter of cell suspension added to 9.0 ml of LB medium to final OD600 = 0.250 ± 0.004. Subsequently, 100 μL of sample (5 mg/ml), 1 ml of the diluted cell suspension and 8.9 ml LB medium were mixed and incubated at 37 °C (180 rpm, 1.5 h).121 After the addition of 6.0 mM H2O2 we calculated the μ value at 30 min to assess the antioxidant activity of extracts. The specific growth rate for each sample was calculated according to the following equation:
$$ \upmu =\mathrm{In}\left(\frac{N}{N_0}\right)/t $$
Where μ was the specific growth rate, N0 and N were the optical density at time zero and t. The protective activity of each sample was calculated as follows: the specific growth rate of the E. coli-containing samples and 6.0 mM H2O2 was divided by the specific growth rate of the samples containing only H2O2. All measurements were done in triplicate.
Measurement of H2O2 production rate
Rate of hydrogen peroxide production in phosphate medium was assayed by Amplex Red-horseradish peroxidase detecting system (AR/HRP) [28]. The solutions of studied compounds was prepared freshly. One milligram of AR was dissolved in 0.78 ml of DMSO, and 0.75 ml of this solution was then diluted into 18 ml of 50 mM potassium phosphate (KPi, pH 7.8) to generate a 200 μM stock solution, which was shielded from light. HRP was dissolved in 50 mM KPi (pH 7.8) to 0.02 mg/ml. In order to measure H2O2, 1 ml sample was mixed with 20 μL HRP and 200 μL AR. H2O2 concentration in samples was measured using a Shimadzu RF Mini-150 fluorometer (λex563 nm and λem587 nm) at 0 and 15 min after incubation had started. Note that a small amount of H2O2 is generated by the dye/HRP detection system itself, this amount was accounted for by the standard curve.
Preparation of crude enzyme liquid
Bacteria suspension was put into ultrasonic cell crusher and set the parameter as: 5 s chill time, 2 s cell broke time, 11 min total work time, 15 °C liquid temperature. The instrument power must be guaranteed at 350 Hz. Centrifuge the cell disruption liquid for 8000 rpm in 8 min and filter to get the solution.
Determination of enzyme activities
Catalase (CAT) enzyme
CAT enzyme activity was assayed according to the method of Beers with some modification [29]. Reaction system consists of the following substances: 3 ml PBS buffer, 0.1 ml 300 mM/L H2O2, 0.5 ml crude enzyme liquid. Add the PBS buffer into the control groups. Immediately detect absorbance values under 240 nm in every 30 s for 2 minutes. Take the 0.1 decrease of OD240 in one minute for one unit of enzyme activity. CAT enzyme activity was calculated according to the following equation:
$$ \mathrm{Y}\left(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot { \min}^{-1}\right)=\frac{\varDelta {\mathrm{A}}_{240}\times {\mathrm{V}}_{\mathrm{t}}}{0.1\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}\times {\mathrm{F}}_{\mathrm{m}}} $$
Above the formula,
$$ {\Delta \mathrm{A}}_{240}={A}_0-\frac{{\mathrm{A}}_{\mathrm{S}1}+{\mathrm{A}}_{\mathrm{S}2}}{2} $$
A0 was the initial absorbance, AS1, AS2 were the sample absorbance, Vt was the enzyme liquid volume, Fm was sample mass, t was the total time of detection (2 min).
Peroxidase (POD) enzyme
The method to determinate the activity of POD enzyme was performed according to Huang with some modification [30]. All measurements were done in triplicate. Substances followed: 2 ml 0.3% H2O2, 1 ml 0.2% guaiacol, 0.5 ml crude enzyme liquid and 1 ml PBS buffer were added into the reaction system. The PBS buffer was added into the control groups instead. Put the reaction system into water bath for 10 min then add the metaphosphate termination reaction. The reaction value was monitored under the 470 nm absorbance before and after water bath. POD enzyme activity was calculated according to the following equation:
$$ \mathrm{Y}\left(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot { \min}^{-1}\right)=\frac{{\Delta \mathrm{A}}_{470}\times {\mathrm{V}}_{\mathrm{t}}}{0.01\times \mathrm{m}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}} $$
Above the formula, ∆A470 was the change of the absorbance during reaction, m was the sample mass, Vt was the total volume of enzyme liquid, Vs was the consuming enzyme liquid volume of reaction, t was the response time.
Superoxide dismutase (SOD) enzyme
The method to determinate the activity of SOD enzyme was performed according to Weng with some modification [31]. Included in the reaction system, there were 0.5 ml Met (130 mmol/L), 0.5 ml NBT (750 μmol/L), 0.5 ml EDTA•Na2 (100 μmol/L), 0.5 ml riboflavin (20 μmol/L), 1 ml PBS buffer and 0.5 ml crude enzyme liquid. The tubes were exposed evenly under 4000 xl fluorescent for 20 min, and then monitored under 560 nm absorbance. The 50% nitroblue tetrazolium reduction inhibition under light was taken as one unit of enzyme activity. SOD enzyme activity was calculated according to the following equation:
$$ \mathrm{Y}\left(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot { \min}^{-1}\right)=\frac{\left({\mathrm{A}}_t-{\mathrm{A}}_{\mathrm{s}}\right)\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{V}}{0.5\times {\mathrm{A}}_{\mathrm{ck}}\times {\mathrm{V}}_{\mathrm{t}}\times \mathrm{t}\times {\mathrm{F}}_{\mathrm{m}}} $$
Above the formula, At was the absorbance of the control group, As was the sample absorbance, Vt was the volume of samples, Vs was the total volume of enzyme liquid, t was the response time.
Glutathione peroxidase (GSH-PX)
GSH-PX enzyme activity of the tested compounds was assayed by the method described by Xing with some modification [32]. Reaction system consists of the following substances: 0.5 ml GSH-PX standard substances, 1.5 ml double distilled water, 2 ml PBS buffer and 0.1 ml TDNB. All measurements were done in three parallel and the PBS buffer was added into control groups. The samples were reacted for 5 min at room temperature, then the optical absorbance was monitored under 412 nm. GSH-PX enzyme activity was calculated according to the following equation:
$$ \mathrm{Y}\left(\upmu \cdot {\mathrm{g}}^{-1}\mathrm{F}\mathrm{W}\right)=\frac{{\mathrm{C}\times \mathrm{V}}_{\mathrm{t}}}{{\mathrm{V}}_{\mathrm{s}}\times {\mathrm{F}}_{\mathrm{m}}} $$
Above the formula, C was the GSH-PX enzyme concentration in samples calculated from the standard curves, Vt was the total volume of enzyme liquid, Vs was the volume of samples, Fm was the sample mass.
CAT and SOD gene expression by real-time PCR
Gene sequences of CAT and SOD enzyme from E. coli were obtained from the GenBank database and gyrB was used as a reference gene. The primers were designed by Primer premier 5 and Oligo 6.0. The primer sequence of CAT enzyme is: forward primer: 5′-TGGAGTGAATACCACGACGAT-3′, reverse primer: 5′-CATGGAAGC CATCACAAACG-3′, product size is 286 bp; The primer sequence of SODa is: forward primer: 5′-CCCTGCCATCCCTGCCGTAT-3′, reverse primer: 5′-GTGACC GCCAGCGTTGTTGC-3′, product size is 227 bp; The primer sequence of SODb is: forward primer: 5′-TCACTACGGCAAGCACCA-3′, reverse primer: 5′- CAGG CAGTTCCAGTAGAAAGTA-3′, product size is 166 bp; The primer sequence of SODc is: forward primer: 5′-CCAGCGGTTTAGGTTGAT-3′, reverse primer: 5′-TGA AGGGCCAGAAGGTG-3′, product size is 179 bp; The primer sequence of gyrB is: forward primer: 5′-CGGAATGTTGTTGGTAAAGC-3′, reverse primer: 5′-CGTGACGGCAAAGAAGAC-3′, product size is 198 bp.
Total RNA samples from E. coli strains were extracted by total RNA isolation kit and treated with DNaseI (Beijing Kang Wei, Biotechnology Industrial Co., Ltd. PR China) to remove genomic DNA contamination. Reverse transcription was performed in a total volume of 20 μL with HiFiScript cDNA first chain synthesis kit (Beijing Kang Wei, Biotechnology Industrial Co., Ltd. PR China). The reaction system contains 4 μL dNTP Mix (2.5 mM each), 2 μL Primer Mix, 2 μL RNA Template, 4 μL 5 × RT Buffer, 2 μL DTT (0.1 M), 1 μL HiFiScript (200 U/μL) and 5 μL RNase-Free Water. The condition was followed by 42 °C for 40 min, 85 °C for 5 min, as recommended by the manufacturer.
Real-time PCR reactions were performed with Ultra SYBR Mixture, using IQ5 fluorescence quantitative analysis software. The thermal cycling conditions comprised an initial step at 94 °C for 4 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and then 61 cycles of temperature programming at 65 °C. The change in fluorescence of Ultra SYBR Mixture in every cycle was monitored by the system software, and the threshold cycle (CT) was measured. Gene expression level of the cells exposed to FLC relative to that of the control cells was calculated using the formula 2-∆∆CT, where ∆CT was the CT value of genes of interest minus that of the internal control, and ∆∆CT was the mean ∆CT value of the cells exposed to FLC and/or BBR minus that of the control cells. Reactions were performed in triplicate with independent RNA isolations.
Isobolographic analysis
The isobolographic analysis was performed according to the method described by Wang in our lab with some modification [6]. To show the interactions (synergistic, additive or antagonistic), all extracts were prepared at 5 mg/ml and individual compounds at 10 mmol/L in various combinations (ratios of 5:1, 3:1, 1:1, 1:3 and 1:5, v/v). If the observed dose of combination was on the additivity isobole or close to it, this indicates no interaction; measurements below and above the additivity isobole indicates antagonism and synergism, respectively [33–37]. In addition, the interaction index, denoted γ, was introduced to further measure the effects of different combinations:
$$ \left(\mathrm{a}/\mathrm{A}\right)+\left(\mathrm{b}/\mathrm{B}\right)=\upgamma $$
Where A and B are the doses of drug A (alone) and B (alone), respectively, that give the specified effect and (a, b) is the combination dose that produces this effect level. If γ = 1, the interaction is additive, if γ < 1, it is antagonistic, and if γ > 1, it is synergistic.
Statistical analysis
Expected values (E) were calculated as the average of individual observed amounts for each one of two combined extracts or compounds, and observed values(O) came through the observed amounts for combined extracts or compounds [38, 39]. All results were expressed as the mean ± standard deviation (SD). The significant difference was calculated by SPSS 20.0 one-way ANOVA followed by Duncan’s test; values < 0.05 were considered to be significant. All diagrams were draw by Sigmaplot 12.0 and Photoshop 8.0.
