Antibacterial activities, antioxidant contents and antioxidant properties of three traditional Chinese medicinal extracts
Abstract
The present study was carried out to identify antibacterial and antioxidant characteristics of traditional aqueous extracts derived from three traditional Chinese medicinal plants (Scutellaria baicalensis, Coptis chinensis and Sonchus oleraceus). It was indicated that the S. oleraceus showed the highest antibacterial efficacy, especially against Staphylococcus aureus. The minimum inhibitory concentration (MIC) of the S. oleraceus was 5.0 mg/mL what was in correlation with the high total phenolic and flavonoid contents and CUPRAC value, and MIC of both S. baicalensis and C. chinensis was 7.5 mg/mL. The rational pH of the working S. oleraceus was acidic, while the other two preferred to neutral or alkaline environment. The reasonable preservation temperature of S. baicalensis should not beyond 60°C, while the other two below 90°C. Meanwhile, S. baicalensis had significant antioxidant activity with the highest CUPRAC and ·OH scavenging activity. These results had provided useful information on further drug discovery.
Introduction
Currently, naturally derived products play an important role as a source of medicine (Rubio-Moraga et al., 2013). Plants are the sound source of the natural product in a most efficient way, their extracts have attracted a lot of interest due to large contributions to some important applications. Chinese traditional medicinal plants are important parts of Traditional Chinese Medicine (TCM), which are antibacterial and antioxidant and may play a significant role in the prevention and treatment of many diseases, and are considerably used for thousands of years not only in China, but all over the world (Liu et al., 2012; Sun et al., 2014; Zhang and Su, 2014). Despite the widespread use, there are many confusing aspects related to TCM, such as incorrect doses had led to the evolution of drug-resistance, which had limited their anthelmintic efficacy, e.g. Buchmann et al., (1992) revealed that extended exposure to subtherapeutic doses of mebendazole resulted in drug resistant parasite populations.
Based on traditional claims for the treatment of various disorders, the present study selected 3 traditional Chinese medicinal plants Scutellaria baicalensis Georgi, Coptis chinensis Franch and Sonchus oleraceus, which are widely distributed in the in northern China to evaluate the in vitro antibacterial activities and their optimal service conditions in a systematic and consistent manner. Furthermore, the study also aimed to determine their antioxidant contents, and measure their antioxidant activities. The results can provide scientific evidence supporting clinical application of these plants and serve as a starting point for new drug discovery from TCM.
Materials and Methods
Collection of Chinese medicinal plants
Three dried Chinese medicinal plant materials namely S. baicalensis, C. chinensis and S. oleraceus were purchased from Beijing Tong Ren Tang Chinese herbal medicine shop, Beijing, China. These three medicinal plants were harvested and processed and naturally dried according to traditional procedures.
Preparation of aqueous extracts
The collected samples were cleaned, freeze-dried in a freeze dry system (Christ 1-4), and ground into a fine powder by a Kenwood Multi-Mill (Kenwood, Havant, UK). Dried plant samples were further air-dried in a ventilated oven at 40°C for 24 hours, then ground into a fine powder and passed through a sieve (24-mesh). Powdered sample (5 g) was extracted with 100 mL ultra-filtered water at 100°C for 30 min in a water bath shaker (Shaking Bath 5B-16) (Techne, Ltd., UK). The extract was filtered by a Millipore filter with a 0.45 um nylon membrane under vacuum at 23°C, the filter residue was added the 50 mL water to operate heating circumfluence by Soxhlet's extraction equipment at 90°C for 2 times and 30 min per time, then the extract was filtered as the above method. All the filtrates were mixed and concentrated under reduced pressure in a vacuum rotary evaporator (R-201, Shanghai Shenshen, China) until all the solvents had almost evaporated. These extracts were eluted several times by PBS solution, and then adjusted the final volumes to 10 mL (the concentrations was 0.5 g/mL) in 10 mL volumetric flasks. The samples were stored at 4°C until use. Eight different concentrations (2.5, 5, 7.5, 10, 20, 40, 120 and 200 mg/mL) of each plant extract was prepared for screening the minimal inhibitory concentration (MIC) and antibacterial activities.
Bacteria and culture
Two bacteria were kindly provided by China general microbiological culture collection center, they are Gram-positive Staphylococcus aureus (S. aureus, ATCC25922) and Gram-negative Escherichia coli (E. coli, ATCC 25322). The strains were cultured at 37°C on plate count agar (PCA) medium (Sun et al., 2014).
Antibacterial assays
This assay was carried out on two bacterial cultures using an agar-well diffusion method (Sun et al., 2014), the reference bacterial strains used in this experiment were S. aureus and E. coli. Both bacterium were suspended in sterile water and diluted to 1x106 CFU/mL. The suspension (100 uL) was spread onto the surface of PCA medium. Wells (4.6 mm in diameter) were cut from the agar with a sterile borer and 60 uL prepared medicinal plants extracted solutions above were delivered to them. Negative controls were prepared using PBS solution. The inoculated plates were incubated at 35°C for 24 hours. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (DIZ) of the tested bacteria. DIZ was expressed in millimeters. All tests were performed in triplicate.
For determining the effect of different pH value to antibacterial activity, the extracted samples (the selected concentration above) was adjusted pH value to 3, 5, 7, 9 and 11 using HCl or NaOH to keep 1 hour and then adjusted back to the primary value to test antibacterial activity, respectively.
For determination the effect of different temperature of to antibacterial activity, the extracted samples (the selected concentration above) were kept 1 hour under 30°C, 60°C, 90°C and 100°C on dry bath incubator and then cooled back to the room temperature to test antibacterial activity, respectively.
Antioxidant analyses
The antioxidant contents were determined using spectrophotometric methods. The total phenolic contents (TPCs) of three extracts were determined by Folin-Ciocalteu reagent method according to Terpinc ÄŒeh et al. (2012) with some modifications. Exactly 0.1 mL of each appropriate dilutions of the filtered extracts (approximately 0.2 mg/mL) or gallic acid (10-100 mg/mL, as standard) was mixed with 0.5 mL of 10% Folin-Ciocalteu reagent, 0.4 mL of 7.5% sodium carbonate was added after 5 min. These mixtures were then incubated at 25°C for 40 min. The absorbance was recorded at 765 nm using a Shimadzu UV-1800 spectrometer. The TPC was obtained from a regression equation (R2= 0.9996), and expressed as mg gallic acid equivalents (GAE) per g dried extract.
The total flavonoid contents (TFCs) of three extracts were determined colorimetrically as described previously (Jia et al., 1999) with some modifications. Exactly 0.5 mL of the extracts was added to a tube containing 1.5 mL of distilled water. Then, 0.09 mL of 5% NaNO2 solution was added to the mixture, and 0.18 mL of 10% AlCl3·6H2O solution was added after 6 min later. After 6 min, 0.7 mL of 4% NaOH solution was added, distilled water was added to bring the total volume to 3 mL. The absorbance was determined at 510 nm after 10 min. The total flavonoid content was determined by a rutin standard curve and expressed as mg rutin equivalents (RE) per g dried extract.
Antioxidant activities determination
The cupric reducing antioxidant capacity (CUPRAC) assay, a method for determination of total antioxidant capacity, was applied with some modifications (Celik et al., 2010). Sample mixture was performed by mixing 1 mL CuCl2 (10 mM), 1 mL neocuproine solution (7.5 mM in 96% ethanol (v:v) and 1 mL NH4Ac buffer (1.0 M, pH = 7.0) in a test tube. Volumes of (x) mL extracted sample (or standard) solution and (1-x) mL H2O were added to the initial mixture so as to make the final volume 4.1 mL. The sample mixture was standing at room temperature for 30 min, and then the absorbance of the final solution was read at 450 nm. A standard curve was prepared with gallic acid stock solution (1 mg/mL). Final results were given as mg gallic acid equivalents (GAE) per g dried extract.
Hydroxyl (·OH) radical scavenging activity was determined according to the method of Smirnoff and Cumbes (1989) with some modifications. Briefly, 0.1 mL of samples (2-10 mg/mL) was mixed with 0.5 mL FeSO4 (1.5 mM) and 0.15 mL sodium salicylate (20 mM), then added 0.25 mL H2O2 (6 mM) to start the reaction. The mixture was incubated at 37°C for 1 hour. The absorbance of the hydroxylated salicylate complex was recorded at 562 nm. The OH radical scavenging activity was calculated using the following equation:
OH scavenging effect (%) = (1-(A sample - A blank ) / A control )x 100
where Asample, Acontrol and Ablank was the absorbance of the sample test, the solvent control, and the reagent blank without sodium salicylate, respectively.
Statistical analysis
All measurements were replicated three times. Statistical analyses were performed with Data Processing System statistical software package using ANOVA followed by Duncan's multiple range tests to evaluate significant differences between different group means, different letter means at a significance level of p = 0.05.
Results
The range of concentrations tested was 0 to 200 mg/mL for three medical plants. There was a significant variation in the antibacterial activities (DIZ values) to the corresponding bacteria between S. oleraceus and other two medical plants extracts (Table I). For Gram-negative E. coli, the DIZ values of S. oleraceus ranged from 7.3 mm to 8.9 mm under the concentration of 5.0 mg/mL and 10.0 mg/mL, respectively. Under the concentration of 2.5 mg/mL, there was no obvious inhibition. However, under the concentration higher than 10 mg/mL, the E. coli was completely inhibited. So, the MIC of S. oleraceus for E. coli was 5.0 mg/mL, the corresponding minimum DIZ was 7.3 mm. Moreover, the MICs of S. baicalensis and C. chinensis extracts for E. coli were 7.5 mg/mL, and the DIZ values of S. baicalensis and C. chinensis were between 7.4 mm and 9.3 mm, 7.1 mm and 9.6 mm respectively.
Table I: Antibacterial activity of three Chinese medicinal plants
| Concentration (mg/mL) |
Diameter of inhibition zone (DIZ)(mm)a | |||||
|---|---|---|---|---|---|---|
| Escherichia coli | Staphylococcus aureus | |||||
| S. baicalensis | C. chinensis | S. oleraceus | S. baicalensis | C. chinensis | S. oleraceus | |
| 2.5 | - | - | - | - | - | - |
| 5.0 | - | - | 7.3 ± 0.4 | - | - | 7.9 ± 0.4 |
| 7.5 | 7.4 ± 0.5 | 7.1 ± 0.8 | 8.4 ± 0.7 | 8.2 ± 0.6 | 7.6 ± 0.5 | 9.1 ± 0.8 |
| 10 | 7.9 ± 0.6 | 7.4 ± 0.6 | 8.9 ± 0.7 | 9.3 ± 0.6 | 8.0 ± 0.6 | 10.7 ± 0.6 |
| 20 | 9.3 ± 0.4 | 9.6 ± 0.3 | + | 12.1 ± 0.5 | 9.4 ± 0.6 | + |
| 40 | + | + | + | + | + | + |
| 120 | + | + | + | + | + | + |
| 200 | + | + | + | + | + | + |
| aAverage of three replicates ± SD; + indicates complete inhibition and - indicates no inhibition | ||||||
For S. aureus, the antibacterial features of these three extracts were similar with those to E. coli, the MIC of S. baicalensis, C. chinensis and S. oleraceus was 7.5, 7.5 and 5.0 mg/mL, with the minimum DIZ 8.2 mm, 7.6 mm and 7.9 mm respectively. Just under the same concentration, the DIZ values to S. aureus were larger than to E. coli, indicating the antibacterial activities of these three extracts to S. aureus were stronger than to E. coli.
The effect of different medical plant extract pH value on antibacterial activity was determined (Figure 1). The DIZ values of three extracts against bacteria were first increased and then decreased with pH values from 3 to 11 and the DIZ values of all extracts ranging from 6.3 mm to 12.8 mm, except for S. baicalensis against S. aureus which DIZ values decreased with increasing pH value, indicating the pH value of the working S. baicalensis against S. aureus should be around 3, while for E. coli it should be 5. The maximum DIZ value of C. chinensis against E. coli and S. aureus was 10 mm and 12 mm, which was under the pH value 9 and 7 respectively, indicating the pH value of the working C. chinensis against E. coli and S. aureus should be around 9 and 7. Moreover, the optimal pH value of S. oleraceus against both bacteria was approximately 7.
Figure 1: Antibacterial activity of three Chinese medicinal plants after different pH values treatment against (a) Escherichia coli and (b, below) Staphylococcus aureus, respectively
The effect of different medical plant extract temperature on antibacterial activity was determined (Figure 2). The DIZ values of these three medical plants extracts against E. coli increased along with the increasing treatment temperature, the turning point was around 90°C, and below 80°C the DIZ values increased gradually with the value about 8 mm, but decreased dramatically beyond 90°C. And the C. chinensis and S. oleraceus against S. aureus as well, with the optimal treatment temperature 90°C. However, the changing trend of DIZ values of S. baicalensis against S. aureus was increased and then decreased with the increasing treatment temperature, and the maximum DIZ value was 11.4 mm under the treatment temperature 60°C.
Figure 2: Antibacterial activity of three Chinese medicinal plants after different temperature treatment against (a) Escherichia coli and (b, below) Staphylococcus aureus, respectively
The comparative evaluation of the antioxidant compounds of the three Chinese medicinal plants extracts was based on the contents of total phenolic content (TPC) and total flavonoid (TFC) (Table II).
Table II: Antioxidant contents and antioxidant properties of three Chinese medicinal plants
| Plant species | TPC (mg GAE/g) |
TFC (mg RE/g) |
CUPRAC (mg GAE/g) |
·OH scavenging activity IC50 value (mg/mL) |
|---|---|---|---|---|
| Scutellaria baicalensis | 176.5 ± 7.4ab | 33.1 ± 2.0c | 82.4 ± 3.1a | 0.2 ± 0.0a |
| Coptis chinensis | 85.6 ± 3.3b | 75.0 ± 4.0b | 42.7 ± 1.3b | 0.1 ± 0.0b |
| Sonchus oleraceus | 185.3 ± 10.4a | 145.2 ± 5.3a | 76.5 ± 4.3ab | 0.1 ± 0.0b |
| Data represent means ± SD of three independent replications (n=3); abc, different letters indicate significant differences (p<0.05) among the 3 different plants and refer to each subset of data. TPC, TFC, GAE, RE, CUPRAC and IC50 correspond to total phenolic content, total flavonoid content, gallic acid equivalent, rutin equivalent, cupric reducing antioxidant capacity and half maximal (50%) inhibitory concentration, respectively | ||||
A significant difference in TPC and TFC content was observed among three plant extracts. The highest phenolic content was 185.3 mg GAE/g dried extract which was found in the plants of S. oleraceus, followed by S. baicalensis. The TPC content of C. chinensis was significantly lower than that of the other two plants. The TFCs of the evaluated extract were presented in Table II, the results indicated a significant difference among three species. For instance, the highest TFC was found in the extract of S. oleraceus also with 145.2 mg RE/g dried extract, followed by C. chinensis, while the S. baicalensis was the lowest with the value of 33.1 mg RE/g dried extract. While all the ratio of TPC in relation to TFC was greater than one, indicating the TPC was higher than TFC content, in the order of S. baicalensis > S. oleraceus > C. chinensis extract, and the S. baicalensis was obviously higher than the other two.
The comparative evaluation of the hydrophilic antioxidant properties of the three Chinese medicinal plants extracts was based on the CUPRAC value and hydroxyl (·OH) radical scavenging activity (Table II).
In terms of the CUPRAC assay, the total antioxidant capacity of three different Chinese medical plants was shown in Table I. The greatest quantity of CUPRAC value was found in S. baicalensis (82.4 mg GAE/g dried extract), followed by S. oleraceus (76.5 mg GAE/g dried extract). The lowest amount of CUPRAC was determined in C. chinensis (42.7 mg GAE/g dried extract).
mined in C. chinensis (42.7 mg GAE/g dried extract).
The result of hydroxyl free radical-scavenging ability of the three medical plants was shown in Table II. For each plant, the IC50 showing the concentration that has the ability to scavenge 50% of hydroxyl radical, which was evaluated. A lower IC50 value corresponds to a larger scavenging activity. The IC50 value of S. oleraceus and C. chinensis for eliminating hydroxyl radicals was 0.1 and 0.1 mg/mL, respectively, while the IC50 value of S. baicalensis was 0.2 mg/mL, which indicated that the scavenging activity of S. baicalensis against hydroxyl radical was significantly lower than that of S. oleraceus and C. chinensis.
Discussion
In this study, we evaluated the antioxidant contents, antioxidant properties and antibacterial activities of three traditional Chinese medicinal extracts (S. baicalensis, C. chinensis and S. oleraceus) from China using water as solvent solution, and also observed the effect of different pH value and temperature treatment on the antibacterial activity. The results suggested a practical potential for these three Chinese medicinal extracts application as an intervention strategy in mitigating microorganisms infection to human beings, which was crucial for its rational usage in treating disease safely and excavating related new drugs.
Based on previous published studies to date, the most common methods of initial screening of various plant species for potential antimicrobial or antioxidant activities have been started with using crude alcohol extraction methods, such as ethanol or methanol extraction (Zhang et al., 2013; Irshaid et al., 2014; Khaled-Khodja et al., 2014). However, here in this study water extraction was used by reason of in the folk people usually dococts the medicinal herbs using water, also as the water extract showed the strongest antibacterial activity and with high content of total phenols (Wong et al., 2006; LiÄina et al., 2013). Medicinal plants are antibacterial materials to bacteria, which have been used as sources of medicine in virtually all cultures. Although there are many reports of antioxidant capacity and antibacterial activity in TCM preparations, few systematic surveys exist. Both of the three extracts tested in this study exhibited high antibacterial activity against both of the tested bacteria, and there was a highly positive relationship between the concentration of extract and antibacterial activity. To some extent, these results were similar to our previous study (Sun et al., 2014). Moreover, the antibacterial activities of three extracts under the same concentration against S. aureus were stronger than against E. coli, expressed as the DIZ values to S. aureus were larger than to E. coli. Our results suggested that Gram-positive bacteria were generally more sensitive to the herb extracts than Gram-negative bacteria, this was consistent with the previous studies on other spices and herbs (Ceylan and Fung, 2004; Lopez et al., 2005; Sun et al. 2014). A possible explanation for these observations may lie in the significant differences in the outer layers of Gram-negative and Gram-positive bacteria, Gram-negative bacteria possess an outer membrane and a unique periplasmic space not found in Gram-positive bacteria (Duffy and Power, 2001).
Further, this study has shown a link between the concentration of extracts and their antibacterial activity, and the present result indicated that the MIC values of S. baicalensis, C. chinensis and S. oleraceus to E. coli was 7.5, 7.5 and 5.0 mg/mL respectively, and those to S. aureus was similar with E. coli. Among three medicinal plants extracts, in general under the same concentration the C. chinensis displayed the lowest antibacterial activity to both bacteria, with the smaller DIZ values than the other two extracts. Importantly, a positive relationship between total phenol contents and antioxidant capacity and antibacterial activity was observed in these investigated plant extracts, similar to previous reports (Yildirim et al., 2001; Katalinic et al., 2006; Taguri et al., 2006). Moreover, the pH value of the working S. baicalensis extract against E. coli and S. aureus should be around 5 and 3, respectively, indicating this extract was more active under acid environment, while the other two extracts preferred to the neutral or alkaline environment. Antibacterial activity of three Chinese medicinal plants after different temperature treatment against two tested bacteria was shown in Figure 2, if the treated temperature beyond 90°C, all the antibacterial activity of extracts to both bacteria will be rapidly decreased except for S. baicalensis extract against S. aureus which inflection point was 60°C. These results may provide a certain basis on the preservation and use of these three Chinese medicinal plants for us to some extent.
On the other side, phenolic compounds are widely distributed in various plant species (Li et al., 2006), they have received considerable attention. Phenolic antioxidants provided tremendous potential benefits because of their ability to scavenge active oxygen species and free radicals such as hydroxyl radicals as well as their ability to disrupt the free-radical chain reaction of lipid peroxidation (Bakirel et al., 2008). Moreover, antibacterial activity of polyphenols against both Gram negative and Gram positive bacteria have been reported in various plant species (Taguri et al., 2006; Padam et al., 2012). TPC was determined by Folin Ciocalteu method and the result was expressed in terms of mg GAE/g dried extracts. All the three extracts showed containing high contents of phenolic. This result demonstrated that these three medicinal plants rich in phenolic compounds. Many researches have stated clearly that phenolic has strong antioxidant activity (Fu et al., 2014). So, these three medicinal plants may be served as a new source of natural antioxidants. However, there was a significant difference in TPC among three species, S. oleraceus provided the highest value of TPC and C. chinensis showed the lowest among samples studied. Flavonoids are the most common and widely distributed group of plant phenolic compounds, which is ubiquitous in fruits and vegetables (Abu Baker et al., 2009). Total flavonoid was determined in the sample extracts by reaction with sodium nitrite, followed by the development of colored flavonoid-aluminium complex formation using aluminium chloride which can be monitored spectrophotometrically at 510 nm, and which was expressed in terms of mg RE/g dried extracts. S. oleraceus provided the highest value of TFC also, while S. baicalensis contained the lowest amount of TFC. By the way, C. chinensis showed the highest ratio of TFC in relation to TPC as compared to the other two samples.
However, it is difficult to separate and quantify individual antioxidants (i.e., parent compounds, glycosides, polymers and many isomers) due to their chemical diversity. Moreover, the total antioxidant power is often more meaningful to evaluate health beneficial effects because of the cooperative action of antioxidants. Therefore, it is desirable to establish a method that can measure the total antioxidant capacity level directly from plant extracts. CUPRAC method is a versatile, simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E (Apak et al., 2008; Celik et al., 2010). In present work, S. baicalensis and S. oleraceus provided the higher CUPRAC value than C. chinensis, this tread was very similar with the result of TPC, while the TFC result of S. baicalensis was inconformity. It was coincident with the previous report, CUPRAC and total polyphenols measurement results in the extracts of kiwifruit correlated very well (r2=0.81), better than with other total antioxidant capacity assays (such as ABTS/TEAC). And the CUPRAC assay was probably the most consistent method of total antioxidant measurement in relation to Folin reagent-responsive TPC. The low correlation observed between CUPRAC results and flavonoids content was due to the nature of measurement technique. The AlCl3 test for flavonoids does not measure those flavonoids that do not bear the characteristic chelating functional groups for Al binding (Park et al., 2006; Apak et al., 2008).
Antioxidants may play their protective role by different processes, therefore, it is pertinent to use several assays instead of only one to determine the antioxidant activity (Petlevski et al., 2013). In the present study, the in vitro antioxidant activities of three extracts were also determined using ·OH radicals scavenging assays. Among all the reactive oxygen species, hydroxyl radical is the most harmful free radicals, and mainly responsible for oxidative injury to biomolecules. The potential antioxidant activities and hydroxyl radical scavenging abilities have potentially beneficial implications in human health (Li et al., 2006; Fu et al., 2014). ·OH radical scavenging activities of three extracts were shown the activity of extract was compared using IC50 value, a smaller value corresponding to a higher antioxidant activity. S. baicalensis showed a significantly highest IC50 value than C. chinensis and S. oleraceus, they exhibited IC50 as 0.19, 0.11 and 0.12 mg/mL respectively, and no significantly difference was detected between C. chinensis and S. oleraceus. Results of above three radical scavenging assays demonstrated that extracts of these three plants exhibited relatively strong radical scavenging activities, and can serve as an effective radical scavenger.
Conclusion
The aqueous extracts of three traditional Chinese medicinal plants possessed excellent antioxidant and antibacterial potential. Overall, the S. oleraceus extract showed the strongest antibacterial activity with the MIC value 5.0 mg/mL what is in correlation with high TPC, TFC and CUPRAC value, and the most rational pH value of the working antibiotics against bacteria should be around 7, the reasonable preservation temperature should below 90°C. On the other hand, S. baicalensis extract had significant antioxidant activity with the highest CUPRAC and ·OH scavenging activity.
Acknowledgement
Thanks are also due to the Undergraduate Innovation Training of Taiyuan University of Science and Technology (UIT2013004, xj2014092), and UIT of Shanxi Province (2015286). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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