Evaluation of Anti-MRSA and Xanthine Oxidase Inhibition Activities of Phenolic Constituents from Plumula nelumbinis

1Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China 2State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China 3Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, China


Introduction
Plumula nelumbinis is the dried young cotyledon and radicle of ripe seed of Nelumbo 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][2][3].Plumula nelumbinis contains various alkaloids and flavonoids [4].Previously, rutin, hyperin, and galuteolin were reported as the antioxidative flavonoids.The extraction of Plumula nelumbinis showed antioxidant effect on superoxide radical and hydroxyl radical [5].
Methicillin-resistant Staphylococcus aureus (MRSA) is the most common cause of nosocomial infections.MRSA is defined as difficult-to-treat strain of Staphylococcus 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 of Plumula nelumbinis.So the discovery of new anti-MRSA agents will greatly help improve healthcare safety.

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. 1 H, 13 C NMR, DEPT, 1 H-1 H COSY, NOESY, HSQC, HMBC, and HSQC-TOCSY spectra were taken with Bruker AM-400 and DRX-500 spectrometer in CD 3 OD or DMSO- 6 .Negative ion FABMS and HRFABMS were recorded using a matrix of triethanolamine on a JEOL SX-102A spectrometer.

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.

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 g/mL.An inoculum density of 5 × 10 5 -1 × 10 6 cfu of each test strain was prepared by blood counting chamber.MHB (100 L) was dispensed into 1-6 wells of a 96-well microtiter plate (Nunc, 300 L 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, compound 1 was dispensed into well 1 (128 g/mL) and serially diluted across the plate (64-4 g/mL).Then the inoculum (100 L) 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.

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 M), hydroxylamine (final concentration 200 M), EDTA (final concentration 100 M), 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 L 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 (IC 50 ) was calculated by linear regression analysis.

DPPH Free Radical Scavenging
Assay.1,1-Diphenyl-2picrylhydrazyl radical (DPPH), a stable free radical, shows strong absorption at 515 nm (in ethanol), as a deep violet color.The concentration of DPPH was 300 M.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: 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.  H 2 O 2 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 H 2 O 2 .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 (IC 50 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 M nitroblue tetrazolium (NBT), 10 M phenazine methosulphate (PMS), and test compounds in various concentrations.After at 25 ∘ C for 5 min, the reaction was started by adding 78 M NADH.Blue chromogen, formed due to NBT reduction, was read at 560 nm.Results were expressed as percentage of inhibition.

Results and Discussion
The MeOH extract and isolated compounds 1-11 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 g/mL, respectively, against these five HA-MRSA strains and with IC 50 = 76.5 g/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 compounds 1-11.

MICs of Compounds 1-11
against HA-MRSA.The five HA-MRSA strains were isolated from patients in the hospital.Among the isolated compounds, compound 8 exerted anti-MRSA activities against all the strains tested with MIC values ranging from 2 to 64 g/mL which has not been reported yet.What is more, the inhibitory effect of compound 8 was obviously observed by treating it against strain SA-200195 (2 g/mL) and SA-300150 (8 g/mL), respectively.Compounds 1, 4, 6, and 10 showed weak anti-MRSA effect against five HA-MRSA strains.Other compounds did not show any statistical significance in anti-MRSA activity (Table 1).

Xanthine Oxidase Inhibition Activity.
All isolates were tested by the xanthine oxidase inhibitory activity assay (Table 1).Compounds 1-6 that were isolated from the methanol extract of Plumula nelumbinis showed xanthine oxidase inhibitory activity.In addition to the known xanthine oxidase inhibitory flavone [20], compounds 6 showed the most effective inhibitory activity with IC 50 value of 8.2 g/mL.IC 50 of the others flavonoids ranged from 23.9 to 68.9 g/mL as shown in Table 1.Interestingly, compound 4 showed lower xanthine oxidase inhibitory activity than the others flavonoids, because of the presence of 6-C-glycosyl  [21].As described in Table 1, compounds 1-3, 5, and 6 showed higher hydroxyl radical scavenging activity than 4 in a concentration-dependent manner.The result provided evidence that flavonoids from Plumula nelumbinis had 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.Compounds 1-3, 5, and 6 showed higher superoxide anion scavenging activity, and this activity was decreased in the order 6 > 5 > 1 > 2 > 3. Specifically, compound 9 showed the highest scavenging activity (IC 50 = 123.4g/mL).Moreover, other compounds showed the scavenging activities by IC 50 value which ranged from 143.9 to 248.3 g/mL.The IC 50 value of ascorbic acid was shown to be 111.9g/mL.

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 ( • OH).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 from Lupinus argenteus [25], genistein, orobol, and biochanin A reduced the MIC of berberine (16-fold) and norfloxacin (4fold).As results described above, compound 8 showed anti-MRSA effect against each strain, and compound 9 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.
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.
a cpd: compound.b