Skip to main content
Download PDF

Synergistic effects and related bioactive mechanism of Potentilla fruticosa L. leaves combined with Ginkgo biloba extracts studied with microbial test system (MTS)

BMC Complementary and Alternative Medicine volume 16, Article number: 495 (2016) Cite this article

Abstract

Background

Potentilla fruticosa, also called “Jinlaomei” and “Gesanghua”, is widely used as folk herbs in traditional Tibetan medicine in China to treat inflammations, wounds, certain forms of cancer, diarrhoea, diabetes and other ailments. Previous research found P. fruticosa leaf extract (C-3) combined with Ginkgo biloba extracts (EGb) showed obvious synergistic effects in a variety of oxidation systems. The aim of the present study was to further confirm the synergy of P. fruticosa combined with EGb viewed from physiological bioavailability and explore the related bioactive mechanism behind the synergism.

Methods

The microbial test system (MTS) was adopted to evaluate the related bioactive mechanism. The synergistic effects were evaluated by isobolographic analysis. The H2O2 production rate and antioxidant enzyme (Catalase (CAT), Peroxidase (POD), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX)) activities were determined by the colorimetric method. Enzyme gene (CAT, SOD) expression was measured by real time-PCR.

Results

The MTS antioxidant activity results showed the combination of C-3 + EGb exhibited synergistic effects especially at the ratio 5:1. Components of isorhamnetin and caffeic acid in C-3 and EGb displayed strong antioxidant activities on MTS and their combination also showed significant synergy in promoting H2O2 production. The combinations of C-3 + EGb and isorhamnetin + caffeic acid promoted CAT and SOD enzyme activities and the ratio 1:1 exhibited the strongest synergy while no obvious promotion on POD and GSH-PX enzyme activities was found. Both combinations above promoted gene expression of CAT and SOD enzymes and the ratio 1:1 exhibited the strongest synergy.

Conclusions

Antioxidant activity results in MTS further confirmed the significant synergy of C-3 combined with EGb and isorhamnetin combined with caffeic acid. The synergy of C-3 combined with EGb may be attributed to the combination of isorhamnetin + caffeic acid, which promoted CAT and SOD enzyme gene expression and further promoted the enzyme activities in E. coli. This study could further provide rational basis for optimizing the physiological bioavailability of P. fruticosa by using natural and safe antioxidants in low doses to produce new medicines and functional products.

Peer Review reports

Background

Previous research has revealed that many plant antioxidants can protect against oxidation damage and they may even have potential applications in preventing various diseases [1]. Their human use has a good safety record when consumed in reasonable amounts, however, when used in large amounts the safety effects are unknown [2]. Considering the high values of plant antioxidants and their related products, a detailed quantitative analysis of the phytochemical profiles and biological properties, especially of antioxidant combinations with known synergistic effects seem to be a useful exercise.

P. fruticosa belonging to the rosaceae family, widely distributed in cool temperate, high altitude regions of the northern hemisphere [3]. In China, the plant species P. fruticosa also called “Jinlaomei” and “Gesanghua”, has long been utilized as folk herbs especially in traditional Tibetan medicine for its superior therapeutic effects [4, 6]. The leaf extracts of P. fruticosa were used to prevent cardiac-cerebral vascular disease, the stem extracts of P. fruticosa were used to treat inflammation, dyspepsia and edema in traditional Tibetan medicine [7–9]. It was also reported that the curative properties of P. fruticosa were used to treat certain types of cancer; bacterial, fungal and viral infections; diabetes and other ailments [10–17]. Furthermore, the extracts of P. fruticosa possess relatively high concentrations of phenolic acids and flavonoids and powerful radical scavenging capacity, which was higher than that of butylated hydroxytoluene [18–20]. Meanwhile, Gingko biloba extracts have gained acceptance in industry as a promising source of antioxidant compounds and the radical scavenging capacities of P. fruticosa extracts were even higher than that of the synthetic antioxidant butylated hydroxytoluene (BHT) used as antioxidant in pharmaceutical preparations and cosmetic formulations [21–23].

In consideration of the rational basis for the use of phytochemicals in P. fruticosa and EGb were still poorly explored by chemical methods including DPPH, ABTS, FRAP, and the oxygen radical absorbance capacity (ORAC) assay, a strategy to enhance the physiological bioavailability of P. fruticosa combined with EGb seems to be imperative [4, 24–26]. According to the results of previous study, the combination of P. fruticosa and EGb showed enhanced activity in achieving a superior synergism outcome directed at the free radical oxidant damage. Among the combinations of the 6 phenolic compounds (catechin, caffeic acid, hyperoside, rutin, ellagic acid and quercetin) in P. fruticosa and the 4 phenolic compounds (catechin, quercetin, kaempferide and isorhamnetin) in EGb detected by chromatographic fractionation, isorhamnetin combined with caffeic acid displayed the greatest synergistic effects [6]. However, the observation in antioxidant synergistic effects of P. fruticosa combined with EGb is still not enough to guide in-depth pharmaceutical applications of P. fruticosa. This experiment carried on the modified and improved assay based on microbial test system (MTS) in our lab [27], focused on the physiological bioavailability to further confirm the synergistic effects of P. fruticosa combined with EGb and try to find specific combinations with least concentrations that exhibit synergistic effects. Those compounds may help to produce natural and safe antioxidants products in lower doses instead of synthetic antioxidants.

In this consideration, the objectives for this study were: (1) to further confirm the antioxidant synergistic effects of P. fruticosa (C-3) combined with EGb studied with MTS assay viewed from physiological bioavailability; (2) to evaluate the antioxidant enzyme activities and gene expression to further explore the related bioactive mechanism of P. fruticosa (C-3) combined with EGb; (3) to provide rational basis to optimize the antioxidant capacity and physiological bioavailability of P. fruticosa using natural and safe antioxidants in low doses instead of synthetic antioxidants, which might be useful to produce new medicines and functional products.

Methods

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.

Results and discussion

MTS antioxidant activity of C-3, EGb and their combinations

Previous research of Wang [6] found that phase C-3 was the most effective fraction isolated from P. fruticosa extracts which could be selected as the main section to explore the synergy mechanism. Therefore, C-3 was combined with EGb to produce natural powerful combined antioxidants of P. fruticosa. The protective effects of C-3, EGb and their combinations on MTS was first assessed and quercetin was used as the positive control.

The results showed that C-3, EGb and their combinations could better increase cell growth rate than quercetin under oxidative stress. All combinations of C-3 + EGb exhibited synergistic effect and the ratio 5:1 showed strongest synergy with μ30 value of 1.603 ± 0.017, followed by 3:1 (1.411 ± 0.016), 1:3 (1.400 ± 0.012), 1:5 (1.376 ± 0.010), and 1:1 (1.366 ± 0.018) (Fig. 1).

Fig. 1
figure1

Synergistic effect of C-3, EGb and C-3 combined with EGb on MTS antioxidant activity

Full size image

In summary, C-3 combined with EGb displayed synergistic effects on MTS and the ratio 5:1 (C-3: EGb) displayed the strongest synergistic, which was in accordance with the results showed in Wang’s research. The results showed that the combination of C-3 + EGb displayed significant antioxidant activity in E.coli viewed from bioavailability as well as in antioxidant assays based on chemical methods. This further suggested that EGb could be selected to promote the antioxidant capacity of P. fruticosa in order to produce natural powerful combined antioxidants.

MTS antioxidant activity of individual compounds in C-3 and EGb

To identify above-mentioned phenomena, further study in MTS antioxidant activity of individual compounds in C-3 (catechin, caffeic acid, hyperoside, rutin, ellagic acid and quercetin) and EGb (catechin, quercetin, kaempferide and isorhamnetin) were carried out to explore the mechanism behind the synergism. It is previously reported that these compounds showed relatively high antioxidant capacities respectively [40].

The protective effect of individual compounds, as determined on MTS, was presented in Fig. 2. Among the main phenolics in C-3 and EGb, caffeic acid and isorhamnetin showed a notable protective effect with μ30 values of 1.407 ± 0.021 and 1.317 ± 0.017, which were higher than those of the other metabolites.

Fig. 2
figure2

Antioxidant activity of catechin, quercetin, kaempferide, isorhamnetin, caffeic acid, rutin, ellagic acid and hyperoside on MTS

Full size image

Since the individual compounds of caffeic acid and isorhamnetin displayed significant synergistic effects on MTS, the protective effects in MTS was likely a result of the existing phytochemicals therein C-3 and EGb. Therefore, isorhamnetin and caffeic acid may played a great role in the synergy observed in the combination of C-3 + EGb, and the results further suggests that the related bioactive mechanism of Potentilla fruticosa L. leaves combined with Ginkgo biloba extracts may linked to the reaction of individual compounds therein the C-3 and EGb.

Synergistic effects of C-3 with 4 compounds in EGb on MTS antioxidant activity

The results above-mentioned suggested that the synergistic protective effect was likely due to the high activity as revealed in isorhamnetin and caffeic acid. What’s more, currant literature observed that synergistic actions displayed when crude extracts combined with natural antioxidants [36, 38]. Thus the C-3 was added to the other 4 compounds in EGb on MTS and the protective effects of the combinations was analyzed.

Isobolographic plot of the combinations of C-3 with 4 compounds in EGb was showed in Fig. 3. The observed antioxidant capacity of each mixture was compared with the expected value (Table 1) which was based on the isobologram. In general, no antagonistic effect was found in testing of the combinations and the combination of C-3 + isorhamnetin displayed obvious synergism followed by 1:1 (γ = 1.622) > 5:1 (γ = 1.561) > 3:1 (γ = 1.372) > 1:3 (γ = 1.265) > 1:5 (γ = 1.128).

Fig. 3
figure3

Isobolographic plot of C-3 + catechin, C-3 + quercetin, C-3 + kaempferide and C-3 + isorhamnetin on MTS antioxidant activity

Full size image
Table 1 Synergistic effects of C-3 + catechin, C-3 + quercetin, C-3 + kaempferide and C-3 + isorhamnetin on MTS antioxidant activity
Full size table

Accordingly, these results indicated that there was no obvious synergistic effect among C-3 and other 3 compounds, thus isorhamnetin may played an important role in the synergy of C-3 and EGb combination. Therefore, isorhamnetin were chosen as the specific compounds for further explore the synergistic effects of 6 compounds in C-3 with EGb to find specific combinations with least concentrations that exhibit synergistic effects of C-3 combined with EGb.

Synergistic effects of 6 compounds in C-3 with isorhamnetin on MTS antioxidant activity

Similarly, to assess how interactions of C-3 and isorhamnetin contribute to the synergistic protective effects, isorhamnetin was added to the other 6 compounds in C-3 on MTS and the protective effect of the combinations was detected.

The results in Table 2 and Fig. 4 illustrated that majority compounds in C-3 combined with isorhamnetin just showed simple additive effect, there was no significant difference between observed values and expected values (p < 0.05). However, the combination of isorhamnetin + caffeic acid showed obvious synergism and the ratio 1:1 exhibited the strongest antioxidant activity on MTS (γ = 1.488) followed by 3:1 (γ = 1.295) > 5:1 (γ = 1.192) > 1:3 (γ = 1.009) > 1:5 (γ = 1.004).

Table 2 Synergistic effects of isorhamnetin + catechin, isorhamnetin + caffeic acid, isorhamnetin + rutin, isorhamnetin + ellagic acid, isorhamnetin + hyperoside and isorhamnetin + quercetin on MTS antioxidant activity
Full size table
Fig. 4
figure4

Isobolographic plot of isorhamnetin + catechin, isorhamnetin + caffeic acid, isorhamnetin + rutin, isorhamnetin + ellagic acid, isorhamnetin + hyperoside, isorhamnetin + quercetin on MTS antioxidant activity

Full size image

In conclusion, the observation suggested that the synergistic protective effect response in C-3 + EGb may be due to the interacting of isorhamnetin and caffeic acid. Furthermore, the combinations of C-3 + EGb and isorhamnetin + caffeic acid were selected to reveal the related bioactive mechanism behind this synergism. Moerover, the ratio of 1:1 (isorhamnetin: caffeic acid) may be chosen as the best ratio that exhibited the synergistic effects in the combination of C-3 and EGb.

Influence of C-3 + EGb and isorhamnetin + caffeic acid on H2O2 antioxidant production rate

Previous research showed that the antioxidant capacity of the combinations and their autoxidation of H2O2 producing ability are proportional to the relationship [28]. The results in Table 3 showed that the combination of C-3 + EGb generally displayed an additive effect while 5:1 displayed synergism (γ = 1.192). Furthermore, isorhamnetin + caffeic acid showed a trend of synergism in which 1:1 displayed the strongest synergism (γ = 1.536). This suggested that the presence of isorhamnetin and caffeic acid played a great role in the synergy observed.

Table 3 H2O2 production rate of C-3 + EGb and isorhamnetin + caffeic acid
Full size table

Among the results detected, the combination of C-3 + EGb and isorhamnetin + caffeic acid showed synergistic on H2O2 antioxidant production rate, which was in accordance with the results above on MTS. It was suggested that the synergism response in C-3 + EGb may be due to the interacting of isorhamnetin and caffeic acid, thus the combination of C-3 + EGb and isorhamnetin + caffeic acid were picked out to in-depth explore the related bioactive mechanism behind synergy through the determination of enzymatic antioxidant activites in E. coli.

Influence of C-3 + EGb and isorhamnetin + caffeic acid on CAT, POD, SOD and GSH enzyme activities

To ascertain the root cause why the combinations above promoted the protective effect on MTS, the activities of four main antioxidant enzymes (CAT, POD, SOD and GSH-PX) in E. coli were determined. Results showed that both the combinations of C-3 + EGb and isorhamnetin + caffeic acid promoted CAT and SOD enzyme activities and the proportion of 1:1 exhibited the strongest effect with value of 13.175 ± 0.071 U/mg prot, 105.125 ± 0.073 U/mg prot, 10.699 ± 0.101 U/mg prot and 125.970 ± 1.681 U/mg prot (Table 4). However, these two combinations did not affect much on POD and GSH enzyme activities. All these experimental results above highlighted that synergy response in the combination of C-3 + EGb had a correlation with isorhamnetin + caffeic acid.

Table 4 Influence of C-3 + EGb and isorhamnetin + caffeic acid on CAT, POD, SOD and GSH enzyme activities
Full size table

In addition, the combinations of C-3 + EGb and isorhamnetin + caffeic acid were evaluated for their antioxidant capacities as isobolographic plots showed in Figs. 5 and 6. Accordingly, the combination of isorhamnetin + caffeic acid showed obvious synergy in the CAT and SOD enzyme activity at the ratio 1:1, suggesting that isorhamnetin and caffeic acid may be the main compounds that affected the enzyme activities in E. coli.

Fig. 5
figure5

Isobolographic plot of C-3 + EGb on CAT, POD, SOD and GSH-PX enzyme activities

Full size image
Fig. 6
figure6

Isobolographic plot of isorhamnetin + caffeic acid on CAT, POD, SOD and GSH-PX enzyme activities

Full size image

Similar to the results in previous chapter, other ratios of the two combinations showed simple additive effects, which could be speculated that the related bioactive mechanism of combined compounds closely linked to the effects of antioxidant enzyme activities.

Influence of C-3 + EGb and isorhamnetin + caffeic acid on CAT, SODa, SODb and SODc gene expression by real-time PCR

To reveal the reason why the combinations of C-3 + EGb and isorhamnetin + caffeic acid promoted CAT and SOD enzyme activities, influence on CAT, SODa, SODb and SODc gene expression were measured by real-time PCR. Among the results, C-3 + EGb and isorhamnetin + caffeic acid promoted CAT and SODa gene expression and the ratio 1:1 of two combinations exhibited the strongest promotion with value of 1.279 ± 0.140, 1.569 ± 0.045, 1.395 ± 0.004 and 1.685 ± 0.019 (γ = 1.122, 1.275, 1.075 and 1.211) respectively (Table 5). However, the combinations of C-3 + EGb and isorhamnetin + caffeic acid did not promote SODb and SODc gene expression so significant.

Table 5 Influence of C-3 + EGb and isorhamnetin + caffeic acid on CAT, SODa, SODb and SODc gene expression
Full size table

Similarly, isobolographic plot of C-3 + EGb and isorhamnetin + caffeic acid on CAT, SODa, SODb and SODc gene expression both showed significant synergistic promoting effect on CAT and SODa gene expression and the synergy was most significant at the ratio 1:1 (Figs. 7 and 8). The results also showed that the combination of C-3 + EGb just displayed an additive effect in SODb and SODc gene expression as well as the combination of isorhamnetin + caffeic acid.

Fig. 7
figure7

Influence of C-3 + EGb on CAT, SODa, SODb and SODc gene expression

Full size image
Fig. 8
figure8

Influence of isorhamnetin + caffeic acid on CAT, SODa, SODb and SODc gene expression

Full size image

In conclusion, the changes in gene expression may be closely linked to those in enzyme activity. The results showed the combination of isorhamnetin + caffeic acid promoted CAT and SODa gene expression and had no obvious promoting effect on SODb and SODc gene expression. This further explain that the synergistic effects of C-3 combined with EGb may have relations with its components of isorhamnetin and caffeic acid. Interactions between isorhamnetin and caffeic acid may played an important role in the related bioactive mechanism behind synergism. However, the problem still needs further research.

Conclusions

In conclusion, the combination of C-3 + EGb in various ratios exhibited significant synergistic effect on MTS and the ratio 5:1(C-3: EGb) showed the strongest synergy. Similarly, the combination of isorhamnetin + caffeic acid also displayed synergistic effect on MTS and the ratio 1:1 exhibited the strongest synergy. These results were in accordance with that determined in H2O2 antioxidant production rate. Furthermore, both combinations of C-3 + EGb and isorhamnetin + caffeic acid promoted CAT and SOD enzyme activities and the ratio 1:1 exhibited the strongest synergy. What’s more, both combinations above promoted gene expression of CAT and SOD enzyme and the ratio 1:1 exhibited the strongest synergy. Therefore, the related bioactive mechanism behind the synergism of C-3 combined with EGb may be attributed to the combination of isorhamnetin and caffeic acid which promoted gene expression of CAT and SOD enzyme as well as further promoted CAT and SOD enzyme activities thus showed synergistic effect.

P. fruticosa extracts have long been used for its superior therapeutic in traditional Tibetan medicine in China. This study provided relevant theoretical basis for maximizing biological antioxidant capacity and physiological bioavailability of P. fruticosa using EGb in lower doses to avoid the side effects. Moreover, isorhamnetin + caffeic acid could be used as the specific combination that exhibit synergistic effect of P. fruticosa combined with EGb. These results could also be useful to elaborate the related bioactive mechanism behind the synergy of P. fruticosa combined with EGb for theoretical guidance to develop new medicines and natural products. Nevertheless, more researches need to be conducted to explain fundamental reason.

Abbreviations

ANOVA:

Analysis of variance

C-3:

P. fruticosa leaf extracts (the acetone extraction phase)

CAT:

Catalase enzyme

EGb:

Ginkgo biloba extracts

GSH-PX:

Glutathione peroxidase enzyme

MTS:

Microbial test system

POD:

Peroxidase enzyme

SOD:

Superoxide dismutase enzyme

References

  1. 1.

    Capecka E, Mareczek A, Leja M. Antioxidant activity of fresh and dry herbs of some Lamiaceae species. Food Chem. 2005;93:223–6.

    CAS  Article  Google Scholar 

  2. 2.

    Elham RN, Alireza SM, Mohammad G, Mehdi K. Evaluation of antioxidant interactions in combined extracts of green tea (Camellia sinensis), rosemary (Rosmarinus officinalis) and oak fruit (Quercus branti). J Food Sci Technol. 2015;52(7):4565–71.

    Article  Google Scholar 

  3. 3.

    Liu W, Wang DM, Liu JJ, Li DW, Yin DX. Quality Evaluation of Potentilla fruticosa L. by High Performance Liquid Chromatography Fingerprinting Associated with Chemometric Methods. PLoS One. http://dx.doi.org/10.1371/journal.pone.0149197.

  4. 4.

    Wang SS, Wang DM, Pu WJ, Li DW. Phytochemical profiles, antioxidant and antimicrobial activities of three Potentilla species. BMC Complement Altern Med. 2013;13:321.

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Flora reipublicae popularis sinicae. Chinese: 1985. Tomus 37. P.244.

  6. 6.

    Wang SS, Wang DM, Liu ZH. Synergistic, additive and antagonistic effects of Potentilla fruticosa combined with EGb761 on antioxidant capacities and the possible mechanism. Ind Crop Prod. 2015;67:227–38.

    CAS  Article  Google Scholar 

  7. 7.

    Bai DY, Ma MC, Zhang ZH. Analysis of leaf ingredient in wild Potentilla fruticosa L. of different elevation. Chin Agric Sci Bulletin. 2007;16:371–3.

    Google Scholar 

  8. 8.

    Li HC, Sun HZ, Hu X. Analysis on total flavonoid in leaves of Potentiila fruticosa in different environment and related mechanism. Western forestry science. 2007;36:71–4.

    CAS  Google Scholar 

  9. 9.

    Qiao Yin. Research on pharmacological activity of Potentilla L. China modern medicine. 2009; 16: 107.

  10. 10.

    Tomczyk M, Leszczyńska K, Jakoniuk P. Antimicrobial activity of Potentilla species. Fitoterapia. 2008;79:592–4.

    Article  PubMed  Google Scholar 

  11. 11.

    Wang QJ, Zhou XM, Zhang YQ. Structure characteristics and biomass of Potentilla fruticosa shrub in Qinghai-Tibet Plateau. Acta Bot Boreali - occiden Sin. 1991;11:333–40.

    CAS  Google Scholar 

  12. 12.

    Shimono A, Ueno S, Gu S, Zhao X, Tsumura Y, Tang Y. Range shifts of Potentilla fruticosa on the Qinghai-Tibetan Plateau during glacial and interglacial periods revealed by chloroplast DNA sequence variation. Heredity. 2009;104:534–42.

    Article  PubMed  Google Scholar 

  13. 13.

    Shushunov S, Balashov L, Kravtsova A, Krasnogorsky I, Latte KP, Vasiliev A. Detemination of acute toxicity of the aqueous extracts of Potential erecta (Tormentil) rhizomes in rats and mice. J Med Food. 2009;12:1173–6.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Tomczyk M, Latte KP. Potentilla-A review of its phytochemical profile. J Ethnopharmacol. 2009;122:184–204.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Tomczyk M, Drozdowska D, Bielawska A, Bielawski K, Gudej J. Human DNA topoisomerases inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7. Pharmazie. 2008;63:389–93.

    CAS  PubMed  Google Scholar 

  16. 16.

    Tomczyk M, Leszczynska K, Tomczykowa M, Jakoniuk P. Screening of antimicrobial activity of aqueous extracts of the selected Potentilla L. species. Planta Med. 2009;73:854–5.

    Google Scholar 

  17. 17.

    Tomczyk M. Secondary metabolites from Potentilla argentea. Biochem Syst Ecol. 2006;34:770–3.

    CAS  Article  Google Scholar 

  18. 18.

    Miliauskas G, Venskutonis P, Van Beek T. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chem. 2004a; 85: 231–237.

  19. 19.

    Miliauskas G, Van Beek TA, Venskutonis PR, Linssen JP, de Waard P, Sudhölter EJ. Antioxidant activity of Potentilla fruticosa. J. Sci. Food Agric. 2004b; 84: 1997–2009.

  20. 20.

    Tomczyk M, Pleszczynska M, Wiater A. Variation in total polyphenolics contents of aerial parts of Potentilla species and their anticariogenic activity. Molecules. 2010;15:4639–51.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Haramaki N, Aggarwal S, Kawabata T, Droy MTT, Packer L. Effects of natural antioxidant Ginkgo biloba extract (EGB 761) on myocardial ischemia-reperfusion injury. Free Radical Biol Med. 1994;16:789–94.

    CAS  Article  Google Scholar 

  22. 22.

    Van Beek TA. Chemical analysis of Ginkgo biloba leaves and extracts. J Chromatogr A. 2002;967:21–55.

    Article  PubMed  Google Scholar 

  23. 23.

    Diamond BJ, Bailey MR. Ginkgo biloba: Indications, Mechanisms, and Safety. Psychiatr. Clin N Am. 2013;36:73–83.

    Google Scholar 

  24. 24.

    Giedrius M, Teris A, Petras RV, Jozef PH, Pieter W, Ernst JR, Sudhölter. Antioxidant activity of Potentilla fruticosa. J Sci Food Agric. 2004;84:1997–2009.

    Article  Google Scholar 

  25. 25.

    Ding XP, Wang XT, Xu T, Qi J, Wang H, Yu BY. Comparison of Two On-Line Analysis Techniques Used for the Screening of Antioxidants in EGb 761. J Chromatogr A. 2010;71:493–7.

    CAS  Article  Google Scholar 

  26. 26.

    Zhang J, Yue L, Khizar H, Xia S, Zhang XM, Ding BM, Tong JM, Chen ZX. Purification of flavonoid from Gingko biloba extract by zinc complexation method and its effect on antioxidant activity. Sep Purif Technol. 2010;71:273–8.

    CAS  Article  Google Scholar 

  27. 27.

    Zhou D, Wang DM, Yang LN, Liu ZH, Zhang YW. A Modified and Improved Assay Based on Microbial Test System (MTS) to Evaluate Antioxidant Activity. Food Anal Methods. 2015;9(4):895–904.

    Article  Google Scholar 

  28. 28.

    Seaver L, Imlay J. Alkylhydro peroxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol. 2001;183:7173–81.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Beers R, Sizer I. Colorimetric method for estimation of catalase. J Biol Chem. 1952;195:133–9.

    CAS  PubMed  Google Scholar 

  30. 30.

    Huang XS, Wang W, Zhang Q, Liu JH. A basic helix-loop-helix transcription factor, PtrbHLH, of poncirus trifoliate confers cold tolerance and modulates peroxidase-mediated scavenging of hydrogen peroxide. Plant Physiol. 2013;162:1178–94.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Weng SF, Zhang XL. The measurement of serum SOD xanthine oxidase conditions. J Foshan Univ. 2011;29(3):65–7.

    Google Scholar 

  32. 32.

    Xing BY, Zhu N, Zhang HP, Yang XL, Dong JE. Effects of methyl viologen on the antioxidant system in cultured Salvia miltiorrhiza cells. Chin J Plant Ecol. 2014;38(5):507–14.

    Article  Google Scholar 

  33. 33.

    Tallarida RJ. Drug synergism: its detection and applications. J Pharmacol Exp Ther. 2001;298:865–72.

    CAS  PubMed  Google Scholar 

  34. 34.

    Tallarida RJ. An overview of drug combination analysis with isobolograms. J Pharmacol Exp Ther. 2006;319:1–7.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Lansky EP, Jiang W, Mo H, Bravo L, Froom P, Yu W, Harris NM, Neeman I, Campbell MJ. Possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions. Invest New Drugs. 2005;23:11–20.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Li SM, Campbell BL, Katz JL. Interactions of cocaine with dopamine uptake inhibitors or dopamine releasers in rats discriminating cocaine. J Pharmacol Exp Ther. 2006;317:1088–96.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Chen CY, Milbury PE, Lapsley K, Blumberg JB. Flavonoids from almond skins are bioavailable and act synergistically with vitamins C and E to enhance hamster and human LDL resistance to oxidation. J Nutr. 2005;135:1366–73.

    CAS  PubMed  Google Scholar 

  38. 38.

    Viera L, Marques A, Barros L, Barriera J, Ferriera I. Insights in the antioxidant synergistic effects of combined edible mushrooms: phenolis and polysaccharridic extracts of Boletus edulis and Marasmius oreades. J Food Nutr Res. 2012;51(2):109–16.

    Google Scholar 

  39. 39.

    Heo HJ, Kim YJ, Chung D, Kim DO. Antioxidant capacities of individual and combined phenolics in a model system. Food Chem. 2007;104:87–92.

    CAS  Article  Google Scholar 

  40. 40.

    Kirakosyan A, Mitchell Seymour E, Noon KR, Urcuyo Llanes DE, Kaufman PB, Warber SL, Bolling SF. Interactions of antioxidants isolated from tart cherry (Prunus cerasus) fruits. Food Chem. 2010;122:78–83.

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the program from the Fundamental Research Funds for the Central Universities (ZD2013010).

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Authors’ contributions

DMW and DWL conceived and designed the experiments. ZHL performed the experiments and analyzed the data. SFF and ZWL helped with the experiment. ZHL, DWL and DMW wrote the paper. DMW critically revised the paper. All authors read and approved the final manuscript.

Competing interests

All the authors declare no present or potential conflicts of interest. All authors are responsible for the content and writing of the paper and approved of its publication.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Author information

Affiliations

  1. College of Forestry, Northwest A & F University, Yangling, Shaanxi, 712100, China

    Ze-Hua Liu, Dong-Mei Wang, Su-Fang Fan, Deng-Wu Li & Zi-Wen Luo

Authors
  1. Ze-Hua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong-Mei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Su-Fang Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Deng-Wu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zi-Wen Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Dong-Mei Wang or Deng-Wu Li.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, Z., Wang, D., Fan, S. et al. Synergistic effects and related bioactive mechanism of Potentilla fruticosa L. leaves combined with Ginkgo biloba extracts studied with microbial test system (MTS). BMC Complement Altern Med 16, 495 (2016). https://doi.org/10.1186/s12906-016-1485-2

Download citation

Keywords

  • Potentilla fruticosa L. leaves
  • Ginkgo biloba extracts
  • Synergistic mechanism
  • Microbial test system (MTS)
  • Real-time PCR

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

Contact us