# Antioxidant and Antimicrobial Activity of Ophiurasaponin Extracted fromOphiopholis mirabilis

Authors: Rongzhen Wang; Xiaoyu Xue; Jingrong Zhen; Chenghua Guo
Journal: Journal of Chemistry
 (2014)

---

## Abstract

The aim of this study was to analyze antioxidant and antimicrobial activity of ophiurasaponin extracted fromOphiopholis mirabilis  (overall). Ophiurasaponin was extracted with solvent extraction and purified through AB-8 macroporous resin, silica gel column chromatography, Sephadex LH-20 gel column chromatography, and C18 ODS column chromatography. The antioxidant activity of ophiurasaponin was detected by the chemiluminescence assay. The paper filtering method and the modified agar dilution method were used to determine antimicrobial activity. The results showed that the content of crude ophiurasaponin and the refined ophiurasaponin was 46.75% and 96.72%, respectively. The values of the IC50 of hydroxyl-radicals, superoxide anions, and peroxide were 25.54 mg/mL, 9.98 mg/mL, and 1.37 mg/mL, respectively. The refined ophiurasaponin had a good inhibitory effect on Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Aerobacter aerogenes, and Proteusbacillus vulgaris, and the minimum inhibitory concentration (MIC) was 0.0443 mg/mL. In conclusion, ophiurasaponin from Ophiopholis mirabilis had obvious antioxidant activities and antimicrobial activities which could provide the theoretical basis for further research and development of antioxidant and antimicrobial marine drugs.


---

## Body

## 1. Introduction

Ophiopholis mirabilis is normally restricted to the shallow water of the northern and central Yellow Sea of China, such as along the coast of Dalian, Qingdao, and Yantai. Zoologically it belongs to Echinodermata, Ophiuroidea, Gnathophiurina, Ophiactidae, Ophiopholis Muller, and Troschel [1]. In this experiment,Ophiopholis mirabilis was harvested from Changdao archipelago (longitude 120°35′28′′–120°56′36′′, latitude 37°53′30′′–38°23′58′′), the interface between the Yellow Sea and the Bohai Sea.Many kinds of active substances from echinoderms, such as saponins, polysaccharides, proteins, alkaloids, and fatty acids, have been isolated and reported by scholars both at home and abroad. However, they are mostly extracted from Asteroidea, Holothuroidea, and Echinoidea [2, 3]. These researches on Ophiuroidea from Echinodermata focus more on the morphology, taxonomy, regeneration incidence on species level, wrist-regeneration, and ecological fields [4, 5]. Recently, the studies on the preparation and characteristics of the Ophiurasaponin fromOphiura kinbergi have been reported [6]. The four compounds are isolated fromAxtroclarus conferus and identified by spectroscopic analysis [7]. However, there has been no report about antioxidant and antimicrobial activities of ophiurasaponin fromOphiopholis mirabilis.


## 2. Experiments

### 2.1. Extraction and Purification of Ophiurasaponin fromOphiopholis mirabilis

Commonly-used solvent extraction method was applied in the experiment. The marine sample was naturally air-dried and then finely crushed. Afterwards the crushed sample was soaked in 85% ethanol three times. The collected ethanol-extract was concentrated in rotary evaporator. Equal volume of petroleum ether was added to the concentration extracted six times for degreasing and equal volume ofn-butanol was added to the water extracted six times as well. Then the supernatant was collected and concentrated in the rotary evaporator. After concentration, ophiurasaponin was precipitated by adding 10 times of volume of acetone and then centrifuged. After the precipitate was dried, the crude ophiurasaponin was obtained. Subsequently, some crude ophiurasaponin was purified through AB-8 macroporous resin, eluted with water, 20% ethanol, 40% ethanol, 60% ethanol, and 80% ethanol in turn. The eluted part of 40% ethanol was collected, silica gel column chromatography with elution (trichloromethane : methanol:  H2O = 82 : 16 : 2), Sephadex LH-20 gel column chromatography with methanol elution, and C18 ODS column chromatography with elution (methanol : H2O = 1 : 1) were applied to purify ophiurasaponin, and then the refined ophiurasaponin was obtained after being concentrated. The colorimetric method was adopted in this experiment to determinate ophiurasaponin content, compared with the standard saponins.


### 2.2. Identification of Ophiurasaponin

Liebermann-Burchard reaction, melting test, UV-VIS determination of characteristic absorption peak, and infrared spectrum scanning were used to the identification of ophiurasaponin.


### 2.3. Detection of Antioxidation Capabilities

#### 2.3.1. Determination of the Capacity of Scavenging Hydroxyl Free Radicals

According to the chemiluminescence system of copper sulfate-luminol-vitamin C-hydrogen peroxide [8], Ultra-Weak Luminescence Analyzer was used to detect the capability of the ophiurasaponin scavenging hydroxyl free radicals. The following reagents were added into the sample cell: ophiurasaponin 50 μL (1 mmol/L) (blank sample used as control), ascorbic acid 20 μL (1 mmol/L), luminol 50 μL (1 mmol/L), borate 780 μL (pH 9.24), and hydrogen peroxide 50 μL (1 mmol/L). The analyzers were immediately started and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −800 V. The luminescence kinetic curves of hydroxyl inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydroxyl illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


#### 2.3.2. Determination of the Capacity of Scavenging Superoxide Anion Free Radicals

According to the chemiluminescence system of pyrogallol-luminol [9], various concentrations of ophiurasaponin 50 μL (blank sample used as control) and pyrogallol 50 μL (6.25 mmol/L) were added into the sample cell. The analyzers were started immediately and the illumination-intensity was tested within 20 s under the test conditions of T = 30°C and Hi-V: −900 V. The luminescence kinetic curves of superoxide anion inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited superoxide anion illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


#### 2.3.3. Determination of the Capacity of Scavenging Hydrogen Peroxide Free Radicals

According to the chemiluminescence system of hydrogen peroxide-luminol [10], the following reagents were added into the sample cell: various concentrations of ophiurasaponin 50 μL (blank sample used as control), luminol 50 μL (1 mmol/L), carbonate buffer 800 μL (pH 9.5), and 3% hydrogen peroxide 100 μL. The analyzers were started immediately and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −850 V. The luminescence kinetic curves of hydrogen peroxide inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydrogen peroxide illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value in IC50 was determined as well.


### 2.4. Detection of Antimicrobial Activity

The antimicrobial activity of ophiurasaponin was detected by using circular filter paper method [11]. Cell suspensions were finally diluted to 105 CFU/mL in order to be used in the activity assays. Potato Sucrose Agar (PSA) medium (fungi) and Beef Extract Peptone medium (bacteria) were incubated at 25°C for 48 h (fungi) and 37°C for 24 h (bacteria). Chloramphenicol (0.04 g/mL) and miconazole nitrate (0.04 g/mL) were used as the positive control and normal saline (NS) was used as the negative control. The minimum inhibitory concentration (MIC) of ophiurasaponin was determined by modified agar dilution method [12]. The concentration of ophiurasaponin was prepared to 0.06 g/mL, and then it was diluted into 10−1, 10−2, 10−3, 10−4, 10−5, and 10−6, respectively. All the tests were performed in triplicate (n=3) and the results were expressed as mg/mL.


### 2.5. Statistical Data Processing

SPSS 11.0 software was used in the single factor analysis of variance by statistically analyzing experimental data, and the results were indicated by the mean standard deviation of plus or minus (+/−s).


## 2.1. Extraction and Purification of Ophiurasaponin fromOphiopholis mirabilis

Commonly-used solvent extraction method was applied in the experiment. The marine sample was naturally air-dried and then finely crushed. Afterwards the crushed sample was soaked in 85% ethanol three times. The collected ethanol-extract was concentrated in rotary evaporator. Equal volume of petroleum ether was added to the concentration extracted six times for degreasing and equal volume ofn-butanol was added to the water extracted six times as well. Then the supernatant was collected and concentrated in the rotary evaporator. After concentration, ophiurasaponin was precipitated by adding 10 times of volume of acetone and then centrifuged. After the precipitate was dried, the crude ophiurasaponin was obtained. Subsequently, some crude ophiurasaponin was purified through AB-8 macroporous resin, eluted with water, 20% ethanol, 40% ethanol, 60% ethanol, and 80% ethanol in turn. The eluted part of 40% ethanol was collected, silica gel column chromatography with elution (trichloromethane : methanol:  H2O = 82 : 16 : 2), Sephadex LH-20 gel column chromatography with methanol elution, and C18 ODS column chromatography with elution (methanol : H2O = 1 : 1) were applied to purify ophiurasaponin, and then the refined ophiurasaponin was obtained after being concentrated. The colorimetric method was adopted in this experiment to determinate ophiurasaponin content, compared with the standard saponins.


## 2.2. Identification of Ophiurasaponin

Liebermann-Burchard reaction, melting test, UV-VIS determination of characteristic absorption peak, and infrared spectrum scanning were used to the identification of ophiurasaponin.


## 2.3. Detection of Antioxidation Capabilities

### 2.3.1. Determination of the Capacity of Scavenging Hydroxyl Free Radicals

According to the chemiluminescence system of copper sulfate-luminol-vitamin C-hydrogen peroxide [8], Ultra-Weak Luminescence Analyzer was used to detect the capability of the ophiurasaponin scavenging hydroxyl free radicals. The following reagents were added into the sample cell: ophiurasaponin 50 μL (1 mmol/L) (blank sample used as control), ascorbic acid 20 μL (1 mmol/L), luminol 50 μL (1 mmol/L), borate 780 μL (pH 9.24), and hydrogen peroxide 50 μL (1 mmol/L). The analyzers were immediately started and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −800 V. The luminescence kinetic curves of hydroxyl inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydroxyl illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


### 2.3.2. Determination of the Capacity of Scavenging Superoxide Anion Free Radicals

According to the chemiluminescence system of pyrogallol-luminol [9], various concentrations of ophiurasaponin 50 μL (blank sample used as control) and pyrogallol 50 μL (6.25 mmol/L) were added into the sample cell. The analyzers were started immediately and the illumination-intensity was tested within 20 s under the test conditions of T = 30°C and Hi-V: −900 V. The luminescence kinetic curves of superoxide anion inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited superoxide anion illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


### 2.3.3. Determination of the Capacity of Scavenging Hydrogen Peroxide Free Radicals

According to the chemiluminescence system of hydrogen peroxide-luminol [10], the following reagents were added into the sample cell: various concentrations of ophiurasaponin 50 μL (blank sample used as control), luminol 50 μL (1 mmol/L), carbonate buffer 800 μL (pH 9.5), and 3% hydrogen peroxide 100 μL. The analyzers were started immediately and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −850 V. The luminescence kinetic curves of hydrogen peroxide inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydrogen peroxide illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value in IC50 was determined as well.


## 2.3.1. Determination of the Capacity of Scavenging Hydroxyl Free Radicals

According to the chemiluminescence system of copper sulfate-luminol-vitamin C-hydrogen peroxide [8], Ultra-Weak Luminescence Analyzer was used to detect the capability of the ophiurasaponin scavenging hydroxyl free radicals. The following reagents were added into the sample cell: ophiurasaponin 50 μL (1 mmol/L) (blank sample used as control), ascorbic acid 20 μL (1 mmol/L), luminol 50 μL (1 mmol/L), borate 780 μL (pH 9.24), and hydrogen peroxide 50 μL (1 mmol/L). The analyzers were immediately started and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −800 V. The luminescence kinetic curves of hydroxyl inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydroxyl illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


## 2.3.2. Determination of the Capacity of Scavenging Superoxide Anion Free Radicals

According to the chemiluminescence system of pyrogallol-luminol [9], various concentrations of ophiurasaponin 50 μL (blank sample used as control) and pyrogallol 50 μL (6.25 mmol/L) were added into the sample cell. The analyzers were started immediately and the illumination-intensity was tested within 20 s under the test conditions of T = 30°C and Hi-V: −900 V. The luminescence kinetic curves of superoxide anion inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited superoxide anion illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value of IC50 was determined as well.


## 2.3.3. Determination of the Capacity of Scavenging Hydrogen Peroxide Free Radicals

According to the chemiluminescence system of hydrogen peroxide-luminol [10], the following reagents were added into the sample cell: various concentrations of ophiurasaponin 50 μL (blank sample used as control), luminol 50 μL (1 mmol/L), carbonate buffer 800 μL (pH 9.5), and 3% hydrogen peroxide 100 μL. The analyzers were started immediately and the illumination-intensity was tested within 100 s under the test conditions of T = 30°C and Hi-V: −850 V. The luminescence kinetic curves of hydrogen peroxide inhibited by ophiurasaponin were indicated with illumination time as X-axis and illumination intensity integral as Y-axis. Origin 7.5 was applied to draw the kinetic curve of inhibited hydrogen peroxide illumination by ophiurasaponin. According to the luminescence kinetic curves, the ratio of the inhibition capacities to ophiurasaponin concentration was calculated, and the value in IC50 was determined as well.


## 2.4. Detection of Antimicrobial Activity

The antimicrobial activity of ophiurasaponin was detected by using circular filter paper method [11]. Cell suspensions were finally diluted to 105 CFU/mL in order to be used in the activity assays. Potato Sucrose Agar (PSA) medium (fungi) and Beef Extract Peptone medium (bacteria) were incubated at 25°C for 48 h (fungi) and 37°C for 24 h (bacteria). Chloramphenicol (0.04 g/mL) and miconazole nitrate (0.04 g/mL) were used as the positive control and normal saline (NS) was used as the negative control. The minimum inhibitory concentration (MIC) of ophiurasaponin was determined by modified agar dilution method [12]. The concentration of ophiurasaponin was prepared to 0.06 g/mL, and then it was diluted into 10−1, 10−2, 10−3, 10−4, 10−5, and 10−6, respectively. All the tests were performed in triplicate (n=3) and the results were expressed as mg/mL.


## 2.5. Statistical Data Processing

SPSS 11.0 software was used in the single factor analysis of variance by statistically analyzing experimental data, and the results were indicated by the mean standard deviation of plus or minus (+/−s).


## 3. Results and Discussion

### 3.1. The Chemical Features of Ophiurasaponin

Measured with ultraviolet absorption method, the content of crude ophiurasaponin was 46.75% and content of the refined ophiurasaponin was 96.72%. Identified by color reaction, ophiurasaponin was determined as steroidal saponins, whose melting points range from 245.4°C to 260.6°C. With ultraviolet visible light scanning within the scope of the 200 nm to 800 nm wavelength, compared with the standard saponins, a maximum absorption peak was identified at 276 nm. In 400 cm−1~4000 cm−1 range scan, characteristic absorption peak was found similar to the standard saponins in 1030 cm−1, 1336 cm−1, and 1610 cm−1. The result suggested that ophiurasaponin and standard saponins were analogous, but ophiurasaponin showed some differences in the fact thatOphiopholis mirabilis came from marine environment and had special structure.


### 3.2. Antioxidant Capacity

Metabolism of normal physiological process in human body could produce a small quantity of Oxo-free radicalism such as hydroxyl, superoxide anion, and hydrogen peroxide. The amount of free radicals in human body is usually in a homeostasis. A small amount of ROS (reactive oxygen species) free radicals can help transmit energy to maintain the vitality, strengthen the immunity, rid of inflammation, and inhibit tumor, and so forth, while excessive free radicals could interfere with the normal metabolism of human body and lead to sickness [13]. Therefore, the study of free radical scavengers on disease prevention and control is of great significance.The luminescence kinetic curves of ophiurasaponin inhibiting hydroxyl, superoxide anion, and hydrogen peroxide were shown in Figures1, 2, and 3, respectively.Figure 1
Inhibition of different concentrations of ophiurasaponin to hydroxyl: 20 mg/mL, 23 mg/mL, 25 mg/mL, 27 mg/mL, and 30 mg/mL.Figure 2
Inhibition of different concentrations of ophiurasaponin to superoxide anion: 1.1 mg/mL, 1.5 mg/mL, 2.2 mg/mL, 4.4 mg/mL, and 22 mg/mL.Figure 3
Inhibition of different concentrations of ophiurasaponin to hydrogen peroxide: 0.05 mg/mL, 0.5 mg/mL, 1 mg/mL, 3 mg/mL, and 5 mg/mL.The results showed that the antioxidant capabilities increased linearly with the increase of the concentration of ophiurasaponin fromOphiopholis mirabilis in a certain range. The inhibition was constant when it increased to a specific concentration. Usually the concentration of ophiurasaponin at 50% of illumination inhibition (IC50) is taken to measure antioxidant capacity of the samples. Lower IC50 indicates that it has stronger antioxidant capacity.As shown in Table1, the result showed that ophiurasaponin fromOphiopholis mirabilis had the strongest inhibition to hydroxyl, the weakest to hydroxyl radicals, and medium to superoxide.Table 1
Capacities of antioxidation of ophiurasaponinn=6.
Superoxide anion
Hydroxyl
Hydrogen peroxide
Concentration (mg/mL)
Inhibition (%)
Concentration (mg/mL)
Inhibition (%)
Concentration (mg/mL)
Inhibition (%)
22
98.32 ± 0.09
30
77.28 ± 0.10
5
94.01 ± 0.07
4.4
32.05 ± 0.15
27
59.63 ± 0.11
3
72.14 ± 0.08
2.2
19.83 ± 0.06
25
47.45 ± 0.10
1
49.19 ± 0.11
1.5
16.32 ± 0.11
23
34.36 ± 0.05
0.5
38.58 ± 0.06
1.1
10.17 ± 0.08
20
10.14 ± 0.13
0.05
31.86 ± 0.12
The linear regression
Y
=
0.04
x
+
0.1012
Y
=
0.0666
x
-
1.2017
Y
=
0.1239
x
+
0.335
R
2
=
0.9928
R
2
=
0.9941
R
2
=
0.9918
IC50 (mg/mL)
9.98 ± 0.12
25.54 ± 0.07
1.37 ± 0.03


### 3.3. Results of Antimicrobial Activity

The antimicrobial activity was expressed as the size of the inhibition zone. The larger the size of inhibition zone is, the stronger the antimicrobial activities. As is shown in Table2, the inhibitory effect on the tested bacteria of refined ophiurasaponin was enhanced significantly compared with the crude. The result showed that the refined ophiurasaponin had stronger antibacterial activity. what is more, the antibacterial ability ofAerobacter aerogenes (28.2 mm) was close to the positive control (31 mm), which declared that ophiurasaponin had strong inhibition effect onAerobacter aerogenes.Table 2
Inhibition of ophiurasaponin fromOphiopholis mirabilis to bacteria n=3.
Species
Positive control(chloramphenicol)
Crude(mm)
OphiurasaponinMIC (mg/mL)
Refined(mm)
OphiurasaponinMIC (mg/mL)
Bacillus subtilis
44.5 ± 0.02
12.2 ± 0.10
1.1 ± 0.05
26.9 ± 0.01
0.0443 ± 0.03
Escherichia coli
51.0 ± 0.04
11.5 ± 0.08
1.1 ± 0.03
32.5 ± 0.03
0.0443 ± 0.03
Staphylococcus aureus
46.0 ± 0.06
11.2 ± 0.07
0.11 ± 0.01
30.9 ± 0.09
0.0443 ± 0.07
Proteusbacillus vulgaris
48.0 ± 0.05
11.2 ± 0.01
—
29.6 ± 0.05
—
Aerobacter aerogenes
31.0 ± 0.06
16.0 ± 0.05
1.1 ± 0.01
28.2 ± 0.04
0.0443 ± 0.01
Note: —: no inhibitory effect.As shown in Table3, ophiurasaponin fromOphiopholis mirabilis could inhibit Mucor circinelloides proliferation; however, it had no inhibition onPenicillium digitatum,Aspergillus flavus, andSaccharomyces cerevisiae. The result showed thatcrude ophiurasaponin inhibited the growth ofSaccharomyces cerevisiaeHansen, but the refined ophiurasaponin did not inhibit its growth, because saponins of other substances in crude ophiurasaponin may be reactive. In addition, theinhibition of ophiurasaponin fromOphiopholis mirabilis on fungi was much less effective than that on bacteria.Table 3
Inhibition of ophiurasaponin fromOphiopholis mirabilis on fungi n=3.
Species
Positive control(miconazole nitrate)
Crude(mm)
OphiurasaponinMIC (mg/mL)
Refined(mm)
OphiurasaponinMIC (mg/mL)
Penicillium  digitatum
21.4 ± 0.03
—
—
—
—
Mucor circinelloides
30.7 ± 0.02
11.5 ± 0.06
1.1 ± 0.03
13.0 ± 0.02
4.43 ± 0.02
Aspergillus flavus
28.7 ± 0.01
—
—
—
—
Saccharomyces cerevisiae
20.7 ± 0.04
—
—
—
—
Saccharomyces cerevisiaeHansen
19.8 ± 0.03
11.2 ± 0.04
1.1 ± 0.04
—
—
Note: —: no inhibitory effect.The minimum inhibitory concentration (MIC) was defined as the lowest concentration of ophiurasaponin that inhibited growth. The MIC value of ophiurasaponin was evidently demonstrated in Table3. The MIC of refined ophiurasaponin was 0.0443 mg/mL, much lower than that of the crude on bacterial.Staphylococcus aureus was the most sensitive bacteria to ophiurasaponin, the MIC value of the crude ophiurasaponin was 0.11 mg/mL, and the MIC value of the refined ophiurasaponin was 0.0443 mg/mL.Mucor circinelloides was less sensitive to ophiurasaponin (4.43 mg/mL MIC). Ophiurasaponin was not sensitive toProteusbacillus vulgaris, Penicillium digitatum, Aspergillus flavus, and Saccharomyces cerevisiae. The result illuminated that the antimicrobial ability of ophiurasaponin had a certain selectivity and the antimicrobial ability of ophiurasaponin had a stronger effect on bacteria.


## 3.1. The Chemical Features of Ophiurasaponin

Measured with ultraviolet absorption method, the content of crude ophiurasaponin was 46.75% and content of the refined ophiurasaponin was 96.72%. Identified by color reaction, ophiurasaponin was determined as steroidal saponins, whose melting points range from 245.4°C to 260.6°C. With ultraviolet visible light scanning within the scope of the 200 nm to 800 nm wavelength, compared with the standard saponins, a maximum absorption peak was identified at 276 nm. In 400 cm−1~4000 cm−1 range scan, characteristic absorption peak was found similar to the standard saponins in 1030 cm−1, 1336 cm−1, and 1610 cm−1. The result suggested that ophiurasaponin and standard saponins were analogous, but ophiurasaponin showed some differences in the fact thatOphiopholis mirabilis came from marine environment and had special structure.


## 3.2. Antioxidant Capacity

Metabolism of normal physiological process in human body could produce a small quantity of Oxo-free radicalism such as hydroxyl, superoxide anion, and hydrogen peroxide. The amount of free radicals in human body is usually in a homeostasis. A small amount of ROS (reactive oxygen species) free radicals can help transmit energy to maintain the vitality, strengthen the immunity, rid of inflammation, and inhibit tumor, and so forth, while excessive free radicals could interfere with the normal metabolism of human body and lead to sickness [13]. Therefore, the study of free radical scavengers on disease prevention and control is of great significance.The luminescence kinetic curves of ophiurasaponin inhibiting hydroxyl, superoxide anion, and hydrogen peroxide were shown in Figures1, 2, and 3, respectively.Figure 1
Inhibition of different concentrations of ophiurasaponin to hydroxyl: 20 mg/mL, 23 mg/mL, 25 mg/mL, 27 mg/mL, and 30 mg/mL.Figure 2
Inhibition of different concentrations of ophiurasaponin to superoxide anion: 1.1 mg/mL, 1.5 mg/mL, 2.2 mg/mL, 4.4 mg/mL, and 22 mg/mL.Figure 3
Inhibition of different concentrations of ophiurasaponin to hydrogen peroxide: 0.05 mg/mL, 0.5 mg/mL, 1 mg/mL, 3 mg/mL, and 5 mg/mL.The results showed that the antioxidant capabilities increased linearly with the increase of the concentration of ophiurasaponin fromOphiopholis mirabilis in a certain range. The inhibition was constant when it increased to a specific concentration. Usually the concentration of ophiurasaponin at 50% of illumination inhibition (IC50) is taken to measure antioxidant capacity of the samples. Lower IC50 indicates that it has stronger antioxidant capacity.As shown in Table1, the result showed that ophiurasaponin fromOphiopholis mirabilis had the strongest inhibition to hydroxyl, the weakest to hydroxyl radicals, and medium to superoxide.Table 1
Capacities of antioxidation of ophiurasaponinn=6.
Superoxide anion
Hydroxyl
Hydrogen peroxide
Concentration (mg/mL)
Inhibition (%)
Concentration (mg/mL)
Inhibition (%)
Concentration (mg/mL)
Inhibition (%)
22
98.32 ± 0.09
30
77.28 ± 0.10
5
94.01 ± 0.07
4.4
32.05 ± 0.15
27
59.63 ± 0.11
3
72.14 ± 0.08
2.2
19.83 ± 0.06
25
47.45 ± 0.10
1
49.19 ± 0.11
1.5
16.32 ± 0.11
23
34.36 ± 0.05
0.5
38.58 ± 0.06
1.1
10.17 ± 0.08
20
10.14 ± 0.13
0.05
31.86 ± 0.12
The linear regression
Y
=
0.04
x
+
0.1012
Y
=
0.0666
x
-
1.2017
Y
=
0.1239
x
+
0.335
R
2
=
0.9928
R
2
=
0.9941
R
2
=
0.9918
IC50 (mg/mL)
9.98 ± 0.12
25.54 ± 0.07
1.37 ± 0.03


## 3.3. Results of Antimicrobial Activity

The antimicrobial activity was expressed as the size of the inhibition zone. The larger the size of inhibition zone is, the stronger the antimicrobial activities. As is shown in Table2, the inhibitory effect on the tested bacteria of refined ophiurasaponin was enhanced significantly compared with the crude. The result showed that the refined ophiurasaponin had stronger antibacterial activity. what is more, the antibacterial ability ofAerobacter aerogenes (28.2 mm) was close to the positive control (31 mm), which declared that ophiurasaponin had strong inhibition effect onAerobacter aerogenes.Table 2
Inhibition of ophiurasaponin fromOphiopholis mirabilis to bacteria n=3.
Species
Positive control(chloramphenicol)
Crude(mm)
OphiurasaponinMIC (mg/mL)
Refined(mm)
OphiurasaponinMIC (mg/mL)
Bacillus subtilis
44.5 ± 0.02
12.2 ± 0.10
1.1 ± 0.05
26.9 ± 0.01
0.0443 ± 0.03
Escherichia coli
51.0 ± 0.04
11.5 ± 0.08
1.1 ± 0.03
32.5 ± 0.03
0.0443 ± 0.03
Staphylococcus aureus
46.0 ± 0.06
11.2 ± 0.07
0.11 ± 0.01
30.9 ± 0.09
0.0443 ± 0.07
Proteusbacillus vulgaris
48.0 ± 0.05
11.2 ± 0.01
—
29.6 ± 0.05
—
Aerobacter aerogenes
31.0 ± 0.06
16.0 ± 0.05
1.1 ± 0.01
28.2 ± 0.04
0.0443 ± 0.01
Note: —: no inhibitory effect.As shown in Table3, ophiurasaponin fromOphiopholis mirabilis could inhibit Mucor circinelloides proliferation; however, it had no inhibition onPenicillium digitatum,Aspergillus flavus, andSaccharomyces cerevisiae. The result showed thatcrude ophiurasaponin inhibited the growth ofSaccharomyces cerevisiaeHansen, but the refined ophiurasaponin did not inhibit its growth, because saponins of other substances in crude ophiurasaponin may be reactive. In addition, theinhibition of ophiurasaponin fromOphiopholis mirabilis on fungi was much less effective than that on bacteria.Table 3
Inhibition of ophiurasaponin fromOphiopholis mirabilis on fungi n=3.
Species
Positive control(miconazole nitrate)
Crude(mm)
OphiurasaponinMIC (mg/mL)
Refined(mm)
OphiurasaponinMIC (mg/mL)
Penicillium  digitatum
21.4 ± 0.03
—
—
—
—
Mucor circinelloides
30.7 ± 0.02
11.5 ± 0.06
1.1 ± 0.03
13.0 ± 0.02
4.43 ± 0.02
Aspergillus flavus
28.7 ± 0.01
—
—
—
—
Saccharomyces cerevisiae
20.7 ± 0.04
—
—
—
—
Saccharomyces cerevisiaeHansen
19.8 ± 0.03
11.2 ± 0.04
1.1 ± 0.04
—
—
Note: —: no inhibitory effect.The minimum inhibitory concentration (MIC) was defined as the lowest concentration of ophiurasaponin that inhibited growth. The MIC value of ophiurasaponin was evidently demonstrated in Table3. The MIC of refined ophiurasaponin was 0.0443 mg/mL, much lower than that of the crude on bacterial.Staphylococcus aureus was the most sensitive bacteria to ophiurasaponin, the MIC value of the crude ophiurasaponin was 0.11 mg/mL, and the MIC value of the refined ophiurasaponin was 0.0443 mg/mL.Mucor circinelloides was less sensitive to ophiurasaponin (4.43 mg/mL MIC). Ophiurasaponin was not sensitive toProteusbacillus vulgaris, Penicillium digitatum, Aspergillus flavus, and Saccharomyces cerevisiae. The result illuminated that the antimicrobial ability of ophiurasaponin had a certain selectivity and the antimicrobial ability of ophiurasaponin had a stronger effect on bacteria.


## 4. Conclusions

The paper showed that ophiurasaponin extracted fromOphiopholis mirabilis had excellent antioxidant activity and inhibition on microbial activity. Although further studies are needed to better evaluate its activities and mechanism(s), our data suggest that ophiurasaponin might be a candidate for the antioxidant and antimicrobial marine drugs.



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*Source: 646537-2014-06-04.xml*