# Evaluation of Anti-MRSA and Xanthine Oxidase Inhibition Activities of Phenolic Constituents fromPlumula nelumbinis

Authors: Xiao Ding; Ming-An Ouyang; Yuan-Shuai Shen
Journal: Journal of Chemistry
 (2015)

---

## Abstract

Isolation of metabolites fromPlumula nelumbinis led to the discovery of eleven compounds, including six flavonoids and five phenolderivatives. Their structures have been determined on the basis of chemical and spectroscopic data. Most of them, such as compounds1,4,6,8, and10, have shown inhibitory activity against hospital-acquired methicillin-resistantStaphylococcus aureus (HA-MRSA). MICs of compound8 against SA-200195 and SA-300150 were 2 μg/mL and 8 μg/mL, respectively. And the antioxidant activity of isolated compounds was determined by checking the scavenging activity against three different radicals: 2,2-diphenyl-1-picrydrazyl (DPPH) radical, hydroxyl radical (O
H
∙), and superoxide anion (O
2
∙
-), as well as xanthine oxidase inhibition. All flavonoids showed strong antioxidant activity. And compound6 displayed the highest inhibitory effect against xanthine oxidase with IC50 value of 8.2 μg/mL.


---

## Body

## 1. Introduction

Plumula nelumbinis is the dried young cotyledon and radicle of ripe seed ofNelumbo nucifera Gaertn (Nymphaeaceae). It is a well-known traditional herbal heat-clearing and detoxifying medicine which is frequently used to clear the heart fire, stop bleeding, and arrest seminal emission in many countries [1–3].Plumula nelumbinis contains various alkaloids and flavonoids [4]. Previously, rutin, hyperin, and galuteolin were reported as the antioxidative flavonoids. The extraction ofPlumula nelumbinis showed antioxidant effect on superoxide radical and hydroxyl radical [5].Methicillin-resistantStaphylococcus aureus (MRSA) is the most common cause of nosocomial infections. MRSA is defined as difficult-to-treat strain ofStaphylococcus aureus which resists to almost all antibiotics [6]. However infections caused by MTSA have been a major threat to public health in hospitals and the community during the past decade. And there were few studies about anti-MRSA effect ofPlumula nelumbinis. So the discovery of new anti-MRSA agents will greatly help improve healthcare safety.


## 2. Experimental

### 2.1. General Experimental Procedures and Chemicals

All melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were measured with a WZZ-2S digital polarimeter spectrophotometer.1H, 13C NMR, DEPT, 1H-1H COSY, NOESY, HSQC, HMBC, and HSQC-TOCSY spectra were taken with Bruker AM-400 and DRX-500 spectrometer in CD3OD or DMSO-d
6. Negative ion FABMS and HRFABMS were recorded using a matrix of triethanolamine on a JEOL SX-102A spectrometer.Xanthine, xanthine oxidase, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), deoxyribose, nitroblue tetrazolium (NBT), and phenazine methosulphate (PMS), were obtained from Sigma Chem. Co. (St. Louis, USA). And other chemicals used were obtained from China National Medicines Co. Ltd. (Beijing, China).


### 2.2. Plant Material

Plumula nelumbinis, the buds ofNelumbo nucifera Gaertn, were purchased from Fujian Medicine Co. Ltd, Fujian Province, People’s Republic of China, in September 2007 and identified by Professor Ke-Cuo He, College of Plant Protection, Fujian Agriculture and Forestry University. A voucher specimen (No. 070923) was deposited in the College of Plant Protection, Fujian Agriculture and Forestry University.


### 2.3. Extraction and Isolation

The air-driedPlumula nelumbinis (5.0 kg) were powdered and extracted exhaustively by maceration with MeOH at room temperature. The extract solution was concentrated under diminished pressure to afford 110 g of dried extract. The extract was subjected to column chromatography over macroporous adsorbent resin D101 (Shanghai Anlande Co. Ltd.) and eluted with H2O and MeOH, and MeOH eluent was concentrated to afford MeOH fraction (52 g). MeOH fraction submitted to column chromatography over silica gel and eluted with a gradient CHCl3-MeOH (100 : 1–2 : 1) to afford ten fractions. Fr.3 (2.1 g) submitted to Sephadex LH-20 column chromatography eluted with MeOH to afford three fractions. Afterwards, SFr.3 (265 mg) was analyzed by normal-phase semipreparation HPLC with CHCl3-acetone (9 : 1) to yield8 (26.7 mg) and9 (97.4 mg). Fr.6 (6.2 g) as mentioned above was subjected to step-gradient silica gel column chromatography with a solvent consisting of CHCl3-MeOH to afford six fractions. And then, SFr.2 (356 mg) was subjected to column chromatography over RP-18 and eluted with a gradient 40%–90% MeOH. Further purified by RP-18, semipreparation HPLC eluted with 75% MeOH to yield1 (52.7 mg) and4 (43.7 mg). Fr.7 (308 mg) was purified over RP-18 with 80% MeOH to yield6 (12.8 mg) and11 (39.0 mg). Fr.8 (620 mg) was purified over RP-18 with 65% MeOH to yield2 (82.3 mg) and5 (72.1 mg). Fr.9 (3.9 g) was subjected to step-gradient silica gel column chromatography with a solvent consisting of CHCl3-MeOH to afford10 (49.3 mg),3 (38.0 mg), and three fractions. SFr.3 (216 mg) was purified by Sephadex LH-20 and then purified by RP-18 semipreparation HPLC eluted with 75% MeOH to yield7 (22.6 mg).


### 2.4. Bacterial Strains and Media

Five HA-MRSA strains were provided by Fujian Medical University Union Hospital. All strains were cultured on nutrient agar before determination of MIC values. Mueller-Hinton broth (MHB) was used for susceptibility tests.


### 2.5. Susceptibility Testing against HA-MRSA

Minimum inhibitory concentration(MIC) values were determined by standard microdilution procedures in duplicate, as recommended by the National Committee for Clinical Laboratory Standards Guidelines [7]. Concentration of test compounds ranged from 4 to 128 μg/mL. An inoculum density of 5 × 105–1 × 106 cfu of each test strain was prepared by blood counting chamber. MHB (100 μL) was dispensed into 1–6 wells of a 96-well microtiter plate (Nunc, 300 μL volume per well). All compounds were dissolved in DMSO before diluting into MHB. And the highest concentration of DMSO remaining after diluting (3% v/v) caused no evident inhibition of HA-MRSA growth. Then, compound1 was dispensed into well 1 (128 μg/mL) and serially diluted across the plate (64–4 μg/mL). Then the inoculum (100 μL) was added into wells 1–6. And susceptibility testing of other compounds was determined as described above, leaving 2 wells empty for growth control of each strain and 2 wells being free of inoculum served as the sterile control in a 96-well microtiter plate. Then the plates were incubated at 37°C for 18 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; sigma) was dissolved in phosphate buffered saline (PBS, 5 mg/mL), and it was used to detect bacterial growth by color change from yellow to dark blue. The MICs were defined as the lowest concentration which yielded no visible bacterial growth. All samples were tested in triplicate.


### 2.6. Xanthine Oxidase Inhibition Assay

The xanthine oxidase inhibition activity was evaluated by the slightly modified method of the previously reported method [8]. The conversion of xanthine to uric acid was calculated according to the increasing absorbance at 290 nm. Test compounds were dissolved in a small amount of DMSO (final concentration < 1%) and diluted with phosphate buffer (20 mM, pH 7.5). Test solutions were prepared by adding xanthine (final concentration 50 μM), hydroxylamine (final concentration 200 μM), EDTA (final concentration 100 μM), and test compounds in various concentrations. The reaction was started by adding 2.5 mU/mL xanthine oxidase in a phosphate buffer solution (200 mM, pH 7.5). The mixture was incubated for 30 min at 37°C. The reaction was terminated by adding 100 μL of HCl (0.58 M) and the variation of absorbance was measured against a blank which was prepared in the same way except that the xanthine oxidase was replaced by buffer solution. The absorbance of the mixture was measured at 290 nm. And a test mixture containing no test compounds was prepared to measure the total uric acid production. Allopurinol was used as a reference inhibitor. All tests were carried out in triplicate. The half-maximal inhibitory concentration (IC50) was calculated by linear regression analysis.


### 2.7. DPPH Free Radical Scavenging Assay

1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), a stable free radical, shows strong absorption at 515 nm (in ethanol), as a deep violet color. The concentration of DPPH was 300μM. The test compounds were dissolved in DMSO and DPPH solution was prepared in ethanol. After incubation, decrease in absorption was measured at 515 nm by using a multiplate reader [9]. Percent radical scavenging activity of compounds was determined in comparison with a DMSO treated control group. The compounds were done in triplicate. The following formula was used as calculating % radical scavenging activity:(1)
%
R
S
A
=
O
D
c
o
n
t
r
o
l
-
O
D
t
e
s
t
c
o
m
p
o
u
n
d
O
D
c
o
n
t
r
o
l
×
100
.


### 2.8. Hydroxyl Radicals Scavenging Assay

Hydroxyl radicals scavenging activity was evaluated using the hydroxyl radical system generated by the Fenton reaction [10]. The reaction mixture containing 2 mM deoxyribose, 0.1 mM ferric chloride, 0.1 mM ascorbic acid, 0.1 mM EDTA, and 2 mM H2O2 in 20 mM phosphate buffer (pH 7.4) was added to every concentration of the test compounds. The reaction was started by the addition of H2O2. After incubation at 37°C for 3 h, the reaction was stopped by adding 0.75 mL of 3.0% trichloroacetic acid and 0.75 mL of 1.0% of 2-thiobarbituric acid in 50 mM NaOH. Then the solution was boiled for 10 min and cooled in water. The absorbance of the solution was measured at 520 nm. Scavenging capacity was expressed as inhibitory concentration of test compound required to produce 50% inhibition of hydroxyl radicals (IC50 values).


### 2.9. Superoxide Anion Radical Scavenging Assay

Superoxide anion radical scavenging activities of test compound were determined by monitoring the competition of those with NBT for the superoxide anion generated by the PMS-NADH system [11]. Superoxide anion radical was generated in 20 mM Tris-HCl buffer (pH 7.8) containing 50 μM nitroblue tetrazolium (NBT), 10 μM phenazine methosulphate (PMS), and test compounds in various concentrations. After at 25°C for 5 min, the reaction was started by adding 78 μM NADH. Blue chromogen, formed due to NBT reduction, was read at 560 nm. Results were expressed as percentage of inhibition.


## 2.1. General Experimental Procedures and Chemicals

All melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were measured with a WZZ-2S digital polarimeter spectrophotometer.1H, 13C NMR, DEPT, 1H-1H COSY, NOESY, HSQC, HMBC, and HSQC-TOCSY spectra were taken with Bruker AM-400 and DRX-500 spectrometer in CD3OD or DMSO-d
6. Negative ion FABMS and HRFABMS were recorded using a matrix of triethanolamine on a JEOL SX-102A spectrometer.Xanthine, xanthine oxidase, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), deoxyribose, nitroblue tetrazolium (NBT), and phenazine methosulphate (PMS), were obtained from Sigma Chem. Co. (St. Louis, USA). And other chemicals used were obtained from China National Medicines Co. Ltd. (Beijing, China).


## 2.2. Plant Material

Plumula nelumbinis, the buds ofNelumbo nucifera Gaertn, were purchased from Fujian Medicine Co. Ltd, Fujian Province, People’s Republic of China, in September 2007 and identified by Professor Ke-Cuo He, College of Plant Protection, Fujian Agriculture and Forestry University. A voucher specimen (No. 070923) was deposited in the College of Plant Protection, Fujian Agriculture and Forestry University.


## 2.3. Extraction and Isolation

The air-driedPlumula nelumbinis (5.0 kg) were powdered and extracted exhaustively by maceration with MeOH at room temperature. The extract solution was concentrated under diminished pressure to afford 110 g of dried extract. The extract was subjected to column chromatography over macroporous adsorbent resin D101 (Shanghai Anlande Co. Ltd.) and eluted with H2O and MeOH, and MeOH eluent was concentrated to afford MeOH fraction (52 g). MeOH fraction submitted to column chromatography over silica gel and eluted with a gradient CHCl3-MeOH (100 : 1–2 : 1) to afford ten fractions. Fr.3 (2.1 g) submitted to Sephadex LH-20 column chromatography eluted with MeOH to afford three fractions. Afterwards, SFr.3 (265 mg) was analyzed by normal-phase semipreparation HPLC with CHCl3-acetone (9 : 1) to yield8 (26.7 mg) and9 (97.4 mg). Fr.6 (6.2 g) as mentioned above was subjected to step-gradient silica gel column chromatography with a solvent consisting of CHCl3-MeOH to afford six fractions. And then, SFr.2 (356 mg) was subjected to column chromatography over RP-18 and eluted with a gradient 40%–90% MeOH. Further purified by RP-18, semipreparation HPLC eluted with 75% MeOH to yield1 (52.7 mg) and4 (43.7 mg). Fr.7 (308 mg) was purified over RP-18 with 80% MeOH to yield6 (12.8 mg) and11 (39.0 mg). Fr.8 (620 mg) was purified over RP-18 with 65% MeOH to yield2 (82.3 mg) and5 (72.1 mg). Fr.9 (3.9 g) was subjected to step-gradient silica gel column chromatography with a solvent consisting of CHCl3-MeOH to afford10 (49.3 mg),3 (38.0 mg), and three fractions. SFr.3 (216 mg) was purified by Sephadex LH-20 and then purified by RP-18 semipreparation HPLC eluted with 75% MeOH to yield7 (22.6 mg).


## 2.4. Bacterial Strains and Media

Five HA-MRSA strains were provided by Fujian Medical University Union Hospital. All strains were cultured on nutrient agar before determination of MIC values. Mueller-Hinton broth (MHB) was used for susceptibility tests.


## 2.5. Susceptibility Testing against HA-MRSA

Minimum inhibitory concentration(MIC) values were determined by standard microdilution procedures in duplicate, as recommended by the National Committee for Clinical Laboratory Standards Guidelines [7]. Concentration of test compounds ranged from 4 to 128 μg/mL. An inoculum density of 5 × 105–1 × 106 cfu of each test strain was prepared by blood counting chamber. MHB (100 μL) was dispensed into 1–6 wells of a 96-well microtiter plate (Nunc, 300 μL volume per well). All compounds were dissolved in DMSO before diluting into MHB. And the highest concentration of DMSO remaining after diluting (3% v/v) caused no evident inhibition of HA-MRSA growth. Then, compound1 was dispensed into well 1 (128 μg/mL) and serially diluted across the plate (64–4 μg/mL). Then the inoculum (100 μL) was added into wells 1–6. And susceptibility testing of other compounds was determined as described above, leaving 2 wells empty for growth control of each strain and 2 wells being free of inoculum served as the sterile control in a 96-well microtiter plate. Then the plates were incubated at 37°C for 18 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; sigma) was dissolved in phosphate buffered saline (PBS, 5 mg/mL), and it was used to detect bacterial growth by color change from yellow to dark blue. The MICs were defined as the lowest concentration which yielded no visible bacterial growth. All samples were tested in triplicate.


## 2.6. Xanthine Oxidase Inhibition Assay

The xanthine oxidase inhibition activity was evaluated by the slightly modified method of the previously reported method [8]. The conversion of xanthine to uric acid was calculated according to the increasing absorbance at 290 nm. Test compounds were dissolved in a small amount of DMSO (final concentration < 1%) and diluted with phosphate buffer (20 mM, pH 7.5). Test solutions were prepared by adding xanthine (final concentration 50 μM), hydroxylamine (final concentration 200 μM), EDTA (final concentration 100 μM), and test compounds in various concentrations. The reaction was started by adding 2.5 mU/mL xanthine oxidase in a phosphate buffer solution (200 mM, pH 7.5). The mixture was incubated for 30 min at 37°C. The reaction was terminated by adding 100 μL of HCl (0.58 M) and the variation of absorbance was measured against a blank which was prepared in the same way except that the xanthine oxidase was replaced by buffer solution. The absorbance of the mixture was measured at 290 nm. And a test mixture containing no test compounds was prepared to measure the total uric acid production. Allopurinol was used as a reference inhibitor. All tests were carried out in triplicate. The half-maximal inhibitory concentration (IC50) was calculated by linear regression analysis.


## 2.7. DPPH Free Radical Scavenging Assay

1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), a stable free radical, shows strong absorption at 515 nm (in ethanol), as a deep violet color. The concentration of DPPH was 300μM. The test compounds were dissolved in DMSO and DPPH solution was prepared in ethanol. After incubation, decrease in absorption was measured at 515 nm by using a multiplate reader [9]. Percent radical scavenging activity of compounds was determined in comparison with a DMSO treated control group. The compounds were done in triplicate. The following formula was used as calculating % radical scavenging activity:(1)
%
R
S
A
=
O
D
c
o
n
t
r
o
l
-
O
D
t
e
s
t
c
o
m
p
o
u
n
d
O
D
c
o
n
t
r
o
l
×
100
.


## 2.8. Hydroxyl Radicals Scavenging Assay

Hydroxyl radicals scavenging activity was evaluated using the hydroxyl radical system generated by the Fenton reaction [10]. The reaction mixture containing 2 mM deoxyribose, 0.1 mM ferric chloride, 0.1 mM ascorbic acid, 0.1 mM EDTA, and 2 mM H2O2 in 20 mM phosphate buffer (pH 7.4) was added to every concentration of the test compounds. The reaction was started by the addition of H2O2. After incubation at 37°C for 3 h, the reaction was stopped by adding 0.75 mL of 3.0% trichloroacetic acid and 0.75 mL of 1.0% of 2-thiobarbituric acid in 50 mM NaOH. Then the solution was boiled for 10 min and cooled in water. The absorbance of the solution was measured at 520 nm. Scavenging capacity was expressed as inhibitory concentration of test compound required to produce 50% inhibition of hydroxyl radicals (IC50 values).


## 2.9. Superoxide Anion Radical Scavenging Assay

Superoxide anion radical scavenging activities of test compound were determined by monitoring the competition of those with NBT for the superoxide anion generated by the PMS-NADH system [11]. Superoxide anion radical was generated in 20 mM Tris-HCl buffer (pH 7.8) containing 50 μM nitroblue tetrazolium (NBT), 10 μM phenazine methosulphate (PMS), and test compounds in various concentrations. After at 25°C for 5 min, the reaction was started by adding 78 μM NADH. Blue chromogen, formed due to NBT reduction, was read at 560 nm. Results were expressed as percentage of inhibition.


## 3. Results and Discussion

The MeOH extract and isolated compounds1–11 were screened for anti-MRSA and xanthine oxidase inhibition activities. The MeOH extract was proved to be effective with MIC at 64, 64, 32, 64, and 32 μg/mL, respectively, against these five HA-MRSA strains and with IC50 = 76.5 μg/mL against xanthine oxidase. In order to substantiate the result and find out which compound inhibited HA-MRSA, the extract was subjected to column chromatography over silica gel, RP-18, Sephadex LH-20, and semipreparation HPLC to yield the active compounds1–11.


### 3.1. Structural Elucidation of Compounds1–11

Structural elucidation of compounds1–11obtained fromPlumula nelumbinis (Figure 1) makes them identified as follows: six flavonoids, namely, quercetin-3-O-β-D-glucopyranoside (1), isorhamnetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (2) [12], acacetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (3) [13], isovitexin (4) [14], lonicerin (5) [15], quercetin (6) [16]. Five phenolderivatives, namely, phenol-1-O-β-D-glucopyranoside (7) [17], 4-hydroxybenzyl alcohol (8), 4-hydroxyphenethyl alcohol (9) [18], 4-hydroxycinnamic acid (10), 4-O-β-D-glucopyranosyl-trans-cinnamic acid (11) [19] were obtained fromPlumula nelumbinis. The structures determination of compounds1–11 was established using NMR spectral method and their spectral data were compared to previous literature values.Figure 1
Structure of compounds1–11.


### 3.2. MICs of Compounds1–11 against HA-MRSA

The five HA-MRSA strains were isolated from patients in the hospital. Among the isolated compounds, compound8 exerted anti-MRSA activities against all the strains tested with MIC values ranging from 2 to 64 μg/mL which has not been reported yet. What is more, the inhibitory effect of compound8 was obviously observed by treating it against strain SA-200195 (2 μg/mL) and SA-300150 (8 μg/mL), respectively. Compounds1,4,6, and10 showed weak anti-MRSA effect against five HA-MRSA strains. Other compounds did not show any statistical significance in anti-MRSA activity (Table 1).Table 1
Xanthine oxidase inhibitory activity, scavenging effect, and susceptibility of HA-MRSA to compounds1–11.
Cpda
Xanthine oxidaseIC50 (μg/mL)
MIC
Scavenging effect
(μg/mL)
IC50 (μg/mL)
SA-100180b
SA-100220
SA-200195
SA-211510
SA-300150
DPPH
∙OH
O
2
∙
-
1
24.6
>128
64
32
64
64
2.3
273.9
150.7
2
23.9
>128
>128
64
>128
128
5.8
264.1
158.9
3
28.2
>128
>128
64
>128
>128
3.4
266.7
167.4
4
68.9
128
64
32
64
64
23.3
348.5
186.3
5
37.4
>128
128
64
>128
64
9.2
236.8
143.9
6
8.2
64
64
32
32
16
1.1
206.7
123.4
7
>100
>128
>128
>128
>128
>128
8
>100
64
64
2
64
8
9
>100
>128
>128
128
>128
>128
10
>100
32
32
32
64
32
11
>100
>128
>128
128
>128
>128
Allopurinol
3.2
Rifampicin
2
1
1
2
1
Ascorbic acid
1.3
166.5
111.9
acpd: compound.
bSA-100180, SA-100220, SA-200195, SA-211510, and SA-300150 are all methicillin-resistant Staphylococcus aureus (HA-MRSA).


### 3.3. Xanthine Oxidase Inhibition Activity

All isolates were tested by the xanthine oxidase inhibitory activity assay (Table1). Compounds1–6 that were isolated from the methanol extract ofPlumula nelumbinisshowed xanthine oxidase inhibitory activity. In addition to the known xanthine oxidase inhibitory flavone [20], compounds6 showed the most effective inhibitory activity with IC50 value of 8.2 μg/mL. IC50 of the others flavonoids ranged from 23.9 to 68.9 μg/mL as shown in Table 1. Interestingly, compound4 showed lower xanthine oxidase inhibitory activity than the others flavonoids, because of the presence of 6-C-glycosyl groups. The IC50 value of allopurinol, which coassayed in the study as positive control, was shown to be 3.2 μg/mL.


### 3.4. DPPH Scavenging Activity

The DPPH scavenging activity of tested compounds is shown in Table1. The antioxidative activity of the flavonoids (1–6) was analyzed using the scavenging radicals activity test. All compounds showed potent DPPH scavenging activity. Compound6 was more potent than ascorbic acid, with IC50 values of 1.1 μg/mL. Other compounds with IC50 values ranged from 2.3 to 23.3 μg/mL. And ascorbic acid was used as positive control. The results indicated that a carbonyl group at C-4 and a double bond between C-2 and C-3 were essential for DPPH scavenging activity.


### 3.5. Hydroxyl Radicals Scavenging Activity

Increased production of reactive oxygen species (ROS) beyond the body’s antioxidant capacity gives rise to oxidative stress. Much of the oxidative damage to biomolecules is induced by hydroxyl radical (O
H
∙), the most reactive one among ROS species [21]. As described in Table 1, compounds1–3,5, and6showed higher hydroxyl radical scavenging activity than4 in a concentration-dependent manner. The result provided evidence that flavonoids fromPlumula nelumbinishad significant effect on scavenging hydroxyl radical.


### 3.6. Superoxide Anion Radical Scavenging Activity

Superoxide anion scavenging abilities of tested compounds were estimated using PMS-NADH system (NBT method). Results were used to indicate the superoxide productivity. Compounds1–3,5, and6 showed higher superoxide anion scavenging activity, and this activity was decreased in the order6 >5 >1 >2 >3. Specifically, compound9 showed the highest scavenging activity (IC50 = 123.4 μg/mL). Moreover, other compounds showed the scavenging activities by IC50 value which ranged from 143.9 to 248.3 μg/mL. The IC50 value of ascorbic acid was shown to be 111.9 μg/mL.


## 3.1. Structural Elucidation of Compounds1–11

Structural elucidation of compounds1–11obtained fromPlumula nelumbinis (Figure 1) makes them identified as follows: six flavonoids, namely, quercetin-3-O-β-D-glucopyranoside (1), isorhamnetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (2) [12], acacetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (3) [13], isovitexin (4) [14], lonicerin (5) [15], quercetin (6) [16]. Five phenolderivatives, namely, phenol-1-O-β-D-glucopyranoside (7) [17], 4-hydroxybenzyl alcohol (8), 4-hydroxyphenethyl alcohol (9) [18], 4-hydroxycinnamic acid (10), 4-O-β-D-glucopyranosyl-trans-cinnamic acid (11) [19] were obtained fromPlumula nelumbinis. The structures determination of compounds1–11 was established using NMR spectral method and their spectral data were compared to previous literature values.Figure 1
Structure of compounds1–11.


## 3.2. MICs of Compounds1–11 against HA-MRSA

The five HA-MRSA strains were isolated from patients in the hospital. Among the isolated compounds, compound8 exerted anti-MRSA activities against all the strains tested with MIC values ranging from 2 to 64 μg/mL which has not been reported yet. What is more, the inhibitory effect of compound8 was obviously observed by treating it against strain SA-200195 (2 μg/mL) and SA-300150 (8 μg/mL), respectively. Compounds1,4,6, and10 showed weak anti-MRSA effect against five HA-MRSA strains. Other compounds did not show any statistical significance in anti-MRSA activity (Table 1).Table 1
Xanthine oxidase inhibitory activity, scavenging effect, and susceptibility of HA-MRSA to compounds1–11.
Cpda
Xanthine oxidaseIC50 (μg/mL)
MIC
Scavenging effect
(μg/mL)
IC50 (μg/mL)
SA-100180b
SA-100220
SA-200195
SA-211510
SA-300150
DPPH
∙OH
O
2
∙
-
1
24.6
>128
64
32
64
64
2.3
273.9
150.7
2
23.9
>128
>128
64
>128
128
5.8
264.1
158.9
3
28.2
>128
>128
64
>128
>128
3.4
266.7
167.4
4
68.9
128
64
32
64
64
23.3
348.5
186.3
5
37.4
>128
128
64
>128
64
9.2
236.8
143.9
6
8.2
64
64
32
32
16
1.1
206.7
123.4
7
>100
>128
>128
>128
>128
>128
8
>100
64
64
2
64
8
9
>100
>128
>128
128
>128
>128
10
>100
32
32
32
64
32
11
>100
>128
>128
128
>128
>128
Allopurinol
3.2
Rifampicin
2
1
1
2
1
Ascorbic acid
1.3
166.5
111.9
acpd: compound.
bSA-100180, SA-100220, SA-200195, SA-211510, and SA-300150 are all methicillin-resistant Staphylococcus aureus (HA-MRSA).


## 3.3. Xanthine Oxidase Inhibition Activity

All isolates were tested by the xanthine oxidase inhibitory activity assay (Table1). Compounds1–6 that were isolated from the methanol extract ofPlumula nelumbinisshowed xanthine oxidase inhibitory activity. In addition to the known xanthine oxidase inhibitory flavone [20], compounds6 showed the most effective inhibitory activity with IC50 value of 8.2 μg/mL. IC50 of the others flavonoids ranged from 23.9 to 68.9 μg/mL as shown in Table 1. Interestingly, compound4 showed lower xanthine oxidase inhibitory activity than the others flavonoids, because of the presence of 6-C-glycosyl groups. The IC50 value of allopurinol, which coassayed in the study as positive control, was shown to be 3.2 μg/mL.


## 3.4. DPPH Scavenging Activity

The DPPH scavenging activity of tested compounds is shown in Table1. The antioxidative activity of the flavonoids (1–6) was analyzed using the scavenging radicals activity test. All compounds showed potent DPPH scavenging activity. Compound6 was more potent than ascorbic acid, with IC50 values of 1.1 μg/mL. Other compounds with IC50 values ranged from 2.3 to 23.3 μg/mL. And ascorbic acid was used as positive control. The results indicated that a carbonyl group at C-4 and a double bond between C-2 and C-3 were essential for DPPH scavenging activity.


## 3.5. Hydroxyl Radicals Scavenging Activity

Increased production of reactive oxygen species (ROS) beyond the body’s antioxidant capacity gives rise to oxidative stress. Much of the oxidative damage to biomolecules is induced by hydroxyl radical (O
H
∙), the most reactive one among ROS species [21]. As described in Table 1, compounds1–3,5, and6showed higher hydroxyl radical scavenging activity than4 in a concentration-dependent manner. The result provided evidence that flavonoids fromPlumula nelumbinishad significant effect on scavenging hydroxyl radical.


## 3.6. Superoxide Anion Radical Scavenging Activity

Superoxide anion scavenging abilities of tested compounds were estimated using PMS-NADH system (NBT method). Results were used to indicate the superoxide productivity. Compounds1–3,5, and6 showed higher superoxide anion scavenging activity, and this activity was decreased in the order6 >5 >1 >2 >3. Specifically, compound9 showed the highest scavenging activity (IC50 = 123.4 μg/mL). Moreover, other compounds showed the scavenging activities by IC50 value which ranged from 143.9 to 248.3 μg/mL. The IC50 value of ascorbic acid was shown to be 111.9 μg/mL.


## 4. Conclusion

Flavonoids have been shown to act as scavengers of various oxidising species, 2,2-diphenyl-1-picrydrazyl radical (DPPH), superoxide anion (O
2
∙
-), and hydroxyl radical (O
H
∙). Previous studies [22] have revealed that a catechol moiety in B-ring is the molecular structural basis of the antioxidant activity. Meanwhile, a carbonyl group at C-4 and a double bond between C-2 and C-3 are also important characters for high antioxidant activity in flavonoids. Similarly, isoflavones are often more active than flavones because of the stabilization effects of the 4-carbonyl and 5-hydroxyl in the former [23].Some flavonoids also show inhibitory effect on the enzyme activity, such as NADH-oxidase and xanthine oxidase. As the reoxidation of xanthine oxidase, both superoxide radicals and hydrogen peroxide are produced. In a previous structure-activity study, Cos et al. [20] found that flavonols showed lower inhibitory activity than flavones. And hydroxyl groups at both C-3 and C-3′ were essential for high superoxide scavenging activity. The flavonoids could be classified into three groups according to their ability: inhibit xanthine oxidase, scavenge for superoxide radicals, or show no activity. And C-glycosyl groups at C-6 and C-8 strongly decreased the inhibitory effect on xanthine oxidase. This indicated that steric interactions could reduce the inhibitory effect of flavonoids on xanthine oxidase.In a number of structure-activity studies, flavonoids have been tested for their anti-MRSA effect [24]. Flavonoids not only exert a direct antibacterial effect but also show a potentiation effect against MRSA. The active isoflavones fromLupinus argenteus [25], genistein, orobol, and biochanin A reduced the MIC of berberine (16-fold) and norfloxacin (4-fold). As results described above, compound8 showed anti-MRSA effect against each strain, and compound9 did not show similar effect. A difference of one methylene group between the two compounds was detected. However the mechanisms of the different anti-MRSA activity of the two compounds require further elucidation.



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*Source: 825792-2015-12-24.xml*