Antioxidant activity and mechanism of Rhizoma Cimicifugae
© Li et al.; licensee Chemistry Central Ltd. 2012
Received: 30 September 2012
Accepted: 12 November 2012
Published: 23 November 2012
As a typical Chinese herbal medicine, rhizoma Cimicifugae (RC, 升麻 in Chinese) possesses various pharmacological effects involved in antioxidant activity. However, its antioxidant activity has not been reported so far. The aim of the present study was to systematically evaluate the antioxidant ability of RC in vitro, then discuss the mechanism.
Firstly, five RC extracts (i.e. petroleum ether extract PERC, ethyl acetate extract EARC, absolute ethanol extract AERC, 95% ethanol extract 95ERC, and water extract WRC) were prepared and determined by various antioxidant methods, including anti-lipidperoxidation, protection against DNA damage, ·OH scavenging, ·O2- scavenging, DPPH· (1,1-diphenyl-2-picryl-hydrazl radical) scavenging, ABTS+· (2,2’-azino-bis (3-ethylbenzo- thiazoline-6-sulfonic acid radical ion) scavenging, Cu2+-chelating, and Fe3+ reducing assays. Subsequently, we measured the chemical contents of five RC extracts, including total phenolics, total saponins, total sugars, caffeic acid, ferulic acid and isoferulic acid. Finally, we quantitatively analyzed the correlations between antioxidant levels (1/IC50 values) and chemical contents.
In the study, the antioxidant levels and chemical contents (including total phenolics, total saponins, total sugars, caffeic acid, ferulic acid and isoferulic acid) of five RC extracts were determined by various methods. In all antioxidant assays, five RC extracts increased the antioxidant levels in a dose-dependent manner. However, their antioxidant levels (IC50 values) and chemical contents significantly differed from each other. Quantitative analysis of the correlation showed that total phenolic was of significant positive correlations (average R value was 0.56) with antioxidant levels; In contrast, total sugars and total saponins had no positive correlation with antioxidant (the average R values were −0.20 and −0.26, for total sugars and total saponins, respectively); Among total phenolics, three phenolic acids (caffeic acid, ferulic acid and isoferulic acid) also displayed positive correlations (the average R values were 0.51, 0.50, and 0.51, for caffeic acid, ferulic acid and isoferulic acid, respectively).
As an effective antioxidant, Rhizoma Cimicifugae can protect DNA and lipids against oxidative damage. Its antioxidant ability can be responsible for its various pharmacological effects and may be mainly attributed to the existence of total phenolics, among which caffeic acid, ferulic acid and isoferulic acid are regarded as main bioactive components. Rhizoma Cimicifugae exerts its antioxidant effect through metal-chelating, and radical-scavenging which is via donating hydrogen atom (H·) and donating electron (e).
KeywordsRhizoma Cimicifugae 升麻 Antioxidant activity Metal chelating Radical-scavenging Phenolic acid
As we know, reactive oxygen species (ROS) are various forms of activated oxygen including free radicals and non-free-radical species. ROS can oxidatively damage vital cellular structures such as lipids and DNA [1, 2], then lead to severe biological consequences including mutation, cell death, carcinogenesis, and aging .
Therefore, it is important to search for potential therapeutic agents for oxidative damage. In recent years, medicinal plants especially Chinese medicinal herbals have attracted much attention.
As a typical Chinese herbal medicine, rhizoma Cimicifugae (RC, 升麻 in Chinese, the photo is shown in Additional file 1) has been used for over 2000 years . From the viewpoint of tradition Chinese medicine (TCM),RC can elevate yang, lift qi, clear heat, remove toxic, induce sweats to dispel exopathogens, and promote eruption .
Modern medicine indicated that RC possessed various pharmacological effects. Ye reported that RC possessed antidepressant-like properties in rodents ; Kim pointed out that RC can treat pain and inflammation ; An isopropanolic extract of RC, however, was proved to be able to diminish the urinary content of PYR and DPY and the morphometric correlates of bone loss associated with ovariectomy in rats ; The supply of RC can therefore prevent OVX-induced bone loss in mice . In addition, the extract of RC was found to have protective effect against gastric injury . According to free radical biology & medicine , these pharmacological effects are related to antioxidant ability. However, its antioxidant ability has not been explored so far.
Therefore, the aim of the study was to investigate the antioxidant ability of RC in vitro, then further discuss the antioxidant mechanism.
Results and discussion
The IC 50 values of five RC extracts and the positive controls (μg/mL)
13.99 ± 2.11e
4.55 ± 0.44 b
14.92 ± 4.31 d
10.62 ± 0.97 c
19.18 ± 2.04 f
0.028 ± 0.00 a *
DNA protective effect
1905.51 ± 177.54 c
14397.18 ± 1075.01 d
1090.11 ± 21.25 b
114.42 ± 2.66 a **
561.19 ± 21.16 f
111.55 ± 1.32 c
132.32 ± 0.15 d
84.30 ± 1.02 b
233.45 ± 15.70 e
37.67 ± 0.67 a **
322.81 ± 14.90 b
306.68 ± 0.87 b
284.36 ± 11.43 b
301.08 ± 8.34 b
650.65 ± 59.55 c
81.65 ± 5.21 a
191.86 ± 1.84 b
319.20 ± 6.36 c
872.71 ± 30.63 e
553.34 ± 12.59 d
2289.11 ± 100.00 f
107.86 ± 0.31 a ***
813.00 ± 225.17 d
227.09 ± 10.14 b
260.34 ± 11.37 c
254.73 ± 4.33 c
229.17 ± 5.81 b
5.13 ± 1.19 a **
395.65 ± 77.34 e
72.10 ± 1.32 b
96.39 ± 1.12 d
94.38 ± 2.45 d
83.58 ± 0.81 c
4.76 ± 0.28 a
533.12 ± 54.25 e
159.35 ± 1.37 b
317.06 ± 3.47 c
156.19 ± 2.12 b
488.75 ± 18.6 0 d
51.89 ± 1.29 a
Therefore, the A532nm value is proportional to the produced amount of ·OH radicals. Higher A532nm values indicate higher levels of ·OH radicals. If an antioxidant sample is added, the A532nm value will decrease, suggesting that some ·OH radicals are scavenged and the hydroxyl-induced DNA damage are protected by the antioxidant.
Previous studies have shown that there are two approaches for natural phenolic antioxidant to protect DNA oxidative damage: one is to scavenge the ·OH radicals then to reduce its attack; one is to fast repair the deoxynucleotide radical cations resulting from ·OH radicals attack . In order to further confirm whether the protective effect against DNA oxidative damage was relevant to radical-scavenging ability, the ·OH and ·O2- radical-scavenging abilities of five RC extracts was determined in vitro.
Taken together, the protective effect of RC against hydroxyl-induced DNA oxidative damage can be assumed to be relevant to the ·OH or ·O2- radical-scavenging ability.
The fact that five RC extracts can effectively bind Fe2+ ion, suggests that metal-chelating may be one of mechanisms for scavenging ·OH or ·O2-.
In order to verify whether five RC extracts can directly scavenge radicals. We determined their DPPH· and ABTS·+ radical-scavenging abilities.
Based on the discussion above, it can be deduced that direct radical-scavenging was regarded as another mechanism for five RC extract to scavenge ·OH or ·O2-, and they exerted radical-scavenging action by donating hydrogen atom (H·) and electron (e).
Chemical contents of five RC extracts
Total phenolics (mg Pyr./g)
13.17 ± 0.43a
65.01 ± 0.34 d
15.73 ± 0.34 b
31.10 ± 1.30 c
12.46 ± 1.00 a
Total sugars (mg Glucose/g)
237.16 ± 38.67 a
175.46 ± 21.50 a
624.75 ± 17.62 b
603.48 ± 18.74 b
804.54 ± 50.53 c
Total saponins (mg Ole./g)
917.52 ± 87.83 c
379.46 ± 15.46 b
185.16 ± 4.10 a
180.44 ± 1.95 a
198.34 ± 12.30 a
Caffeic acid (peak area)
2.96 ± 0.85 a
125.22 ± 8.84 c
6.45 ± 2.93 b
14.19 ± 10.14 b
2.79 ± 1.23 a
Ferulic acid (peak area)
0.89 ± 0.44 c
79.31 ± 3.22 d
0.39 ± 0.37 b
0.68 ± 1.18 c
0.0040 ± 0.0010 a
Isoferulic acid (peak area)
4.92 ± 1.68 a
33.24 ± 0.44 d
12.77 ± 2.56 c
8.68 ± 0.28 b
The R values between chemical contents and 1/IC 50
Then, we used the peak areas to plot the correlation graphs vs 1/IC50 values of five RC extracts, to obtain the correlation coefficients (R values). As shown in Additional file 5 and Table 3, three phenolic acids all presented significant positive correlations (average R value = 0.47, 0.44, and 0.51, respectively for caffeic acid, ferulic acid and isoferulic acid). On the other hand, previous works have shown that three phenolic acids possess strong antioxidant ability [22–24]. Hence, they are considered as three of main bioactive compounds relevant to antioxidant in RC.
Chemicals and plant material
Trolox (± − 6-hydroxyl-2,5,7,8-tetramethlyhromane-2-carboxylic acid), Ferrozin [3-(2-pyridyl)-5,6-bis (4-phenylsulfonicacid)-1,2,4-triazine], DPPH · (1,1-Diphenyl-2-picrylhydrazyl radical), pyrogallol, linoleic acid, BHT (2.6-ditertiary butyl-p-cresol) and murexide (5,5′-Nitrilodibarbituric acid monoammonium salt) were purchased from Sigma Co. (Sigmaaldrich Trading Co., Shanghai, China); ABTS diammonium salt [2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt)], D-2-deoxyribose, and GSH (glutathione) were Amresco Inc. (Solon, OH, USA); DNA sodium salt (fish sperm) was purchased from Aladdin Chemistry Co. (Shanghai, China); Ferulic acid, caffeic acid were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Acetonitrile, methanol and water were of HPLC grade; All other chemicals used were in analytical grade.
Rhizoma Cimicifugae was purchased from Guangzhou University of Chinese Medicine Yanghe Interlink Limited Company and identified by Prof. Shuhui Tan. A voucher specimen was deposited in our laboratory.
Preparation of different extracts of rhizoma Cimicifugae
Protective effect against lipid-peroxidation
Where A is the absorbance with samples, while A 0 is the absorbance without samples.
Protective effect against hydroxyl-induced DNA damage
Where A is the absorbance with samples, while A 0 is the absorbance without samples.
Hydroxyl (·OH) radical-scavenging assay
Where A is the absorbance containing samples, while A 0 is the absorbance without samples.
Superoxide anion (·O2-) radical-scavenging assay
Here, ΔA 325nm, control is the increase in A 325nm of the mixture without the sample and ΔA 325nm, sample is that for the mixture with the sample; T = 5 min. The experiment temperature was 37°C.
Chelating activity on Cu2+
Where is the absorbance ratio of the sample, while is the maximum absorbance ratio and is the minimum absorbance ratio in the test.
DPPH· scavenging assay
Where A 0 is the absorbance without samples, while A is the absorbance with samples.
Where A 0 is the absorbance at 734 nm without samples, while A is the absorbance at 734 nm with samples.
Reducing power (Fe3+) assay
Ferric cyanide (Fe3+) reducing power was determined according to the method of Oyaizu [23, 29]. In brief, sample solutions x μL (4 mg/mL, x = 20, 40, 60, 80, and 100) were mixed with (350-x) μL Na2HPO4/KH2PO4 buffer (0.2 M, pH 6.6) and 250 μL K3Fe(CN)6 aqueous solution (1 g/100 mL).
Here, A max is the maximum absorbance and A min is the minimum absorbance in the test. A is the absorbance of sample.
HPLC analysis for caffeic acid, ferulic acid, and isoferulic acid
Caffeic acid, ferulic acid, and isoferulic acid were identified by comparing their retention times using HPLC method. HPLC analysis was performed on a Syltech P510 system (Los Angeles, California, USA) equipped with Dikma Diamonsil C18 (250 mm × 4.6 mm, 5 μm) (Beijing, China). The mobile phase consisted of acetonitrile-0.5% acetic acid in water (17:83, v: v), the flow rate was 1.0 mL/min, injection volume was 30 μL and absorption was measured at 316 nm.
Determination of total phenolics, total sugars and total saponins
The total phenolics of five RC extracts were determined by the Folin-Ciocalteu method with a little modifications . Firstly, 0.5 mL extract methanolic solution ( 0.4 mg/mL ) was mixed with 0.5 mL 0.25 M Folin-Ciocalteu reagent. After incubation for 3 min, 1 mL of Na2CO3 solution (15%, w/v) was added. After standing at the room temperature for 30 min, the mixture was centrifuged at 3500 r/min for 3 min. The absorbance of the supernatant was measured at 760 nm (Unico 2100, Shanghai, China). The determinations were performed in triplicate, and the calculations were based on a calibration curve obtained with pyrogallol. The result was expressed as pyrogallol equivalents (Pyr.) in milligrams per gram of extract.
The total sugars were evaluated according to the phenol-sulfuric acid method . A 40-μL aliquot of sample solution (1 mg/mL) was placed in a flask, then 210 μL distilled water, 250 μL phenol solution (5%, w/v) and 250 μL concentrated sulfuric acid were added. After incubation for 20 min at room temperature, the absorbance of reaction mixture was measured at 490 nm (Unico 2100, Shanghai, China). The measurements were performed in triplicate, and the calculations were based on a calibration curve obtained with glucose. The result was expressed as glucose equivalents in milligrams per gram of extract.
The total saponins were measured according to the method . RC extract was dissolved in methanol to prepare the sample solution (4 mg/mL), then a 20-μL aliquot of sample solution was taken into a mini tube. After the sample solution in tube was evaporated to dryness (water bath, 80°C), 0.1 mL vanillin-acetic acid solution (5 mg/mL) and 0.4 mL perchloric acid were added to the sample residue. The reaction mixture was incubated at 70°C for 15 min, then diluted by 1.25 mL acetic acid. The mixture was measured using a spectrophotometer (Unico 2100, Shanghai, China) at 540 nm against a blank control, which contained all reagents except sample. All analyses were run in triplicate. Quantification was based on the standard curve for oleanic acid (10–79 μg/mL) and the results were expressed in milligrams of oleanic acid (Ole.) equivalents per gram of extract.
The correlation graphs and R values
In order to investigate the correlation between antioxidant activities and chemical components of five RC extracts, 48 correlation graphs were plotted between 1/IC50 values (including of LPO, DNA, ·OH, ·O2- ,Cu-chelating, DPPH·, ABTS·+, and reducing power assays) and chemical contents (including total phenolics, caffeic acid, ferulic acid, isoferulic acid, total sugars, and total saponins). In the correlation graphs, the correlation coefficients (R values) were calculated by Origin 6.0 professional software.
Data are given as the mean ± SD of three measurements. The IC50 values were calculated by linear regression analysis. All linear regression in this paper was analyzed by Origin 6.0 professional software. Significant differences were performed using the T-test (p < 0.05). The analysis was performed using SPSS software (v.12, SPSS, USA).
As an effective antioxidant, rhizoma Cimicifugae can protect DNA and lipids against oxidative damage. Its antioxidant ability can be responsible for its various pharmacological effects and may be mainly attributed to the existence of total phenolics, among which caffeic acid, ferulic acid and isoferulic acid are regarded as main bioactive components. Rhizoma Cimicifugae exerts its antioxidant effect through metal-chelating, and radical-scavenging which is via donating hydrogen atom (H·) and donating electron (e).
Petroleum ether extract from rhizoma Cimicifugae
Ethyl acetate extract from rhizoma Cimicifugae
Absolute ethanol extract from rhizoma Cimicifugae
95% ethanol extract from rhizoma Cimicifugae
Water extract from rhizoma Cimicifugae
2,2′-azino-bis (3-ethylbenzo- thiazoline-6-sulfonic acid salt)
Reactive oxygen species
Traditional Chinese medicine
Thiobarbituric acid reactive substances
This work was supported by the National Nature Science Foundation of China (81273896) and National Key Technologies R&D Program of China (No. 2008BAI51B01).
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