Fitoterapia 82 (2011) 805810

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Anti-inammatory, antiviral and quantitative study of quercetin-3-O--D-glucuronide in Polygonum perfoliatum L.

Dongsheng Fan a, Xin Zhou a, b,, Chao Zhao a, b, Huaguo Chen a, b, Yang Zhao a, b, Xiaojian Gong a, b
a b

The Research Center for Quality Control of Natural Medicine, Guizhou Normal University, Guiyang 550001, China Key Laboratory for Information System of Mountainous Areas and Protection of Ecological Environment, Guizhou Normal University, Guiyang 550001, China

a r t i c l e

i n f o

a b s t r a c t
Quercetin-3-O--D-glucuronide, isolated from Polygonum perfoliatum L., was evaluated by antiviral efcacy against inuenza A virus and anti-inammatory activity in vivo in mouse, and it was used for quality evaluation of P. perfoliatum L.. In vivo study, oral administration of quercetin-3-O--D-glucuronide signicantly suppressed ear edema induced by dimethyl benzene and peritoneal permeability induced by acetic acid in mice, and quercetin-3-O--Dglucuronide also showed to possess inhibitory activity against inuenza A virus (FLUAV). In the present study, additionally, a rapid, simple and sensitive method for quantitative analysis of quercetin-3-O--D-glucuronide in P. perfoliatum L. was developed using high performance liquid chromatography (HPLC) coupled with photodiode array detection. The separation was carried out on a Lichrosher-C18 column (250 mm 4.6 mm, 5 m) together with a C18 guard column at isocratic elution systems of methanol (A) and 0.05% aqueous phosphoric acid (B) (43:57, v/v) with detection wavelength at 258 nm and column temperature at 30 C. The method was validated for linearity, repeatability, limit of quantication (LOQ), precision and robustness. The contents of quercetin-3-O--D-glucuronide in 28 samples from different regions of China were between 0.06% and 2.09%. The developed analytical method was applied to investigate P. perfoliatum L. and for quality control of the herb. 2011 Elsevier B.V. All rights reserved.

Article history: Received 9 December 2010 Accepted in revised form 9 April 2011 Available online 5 May 2011

Keywords: Polygonum perfoliatum L. Quercetin-3-O--D-glucuronide Anti-inammation Antivirus HPLC

1. Introduction Polygonum perfoliatum L. is a well-known Traditional Chinese Medicine (TMC), named Gangbangui in Chinese [1]. It is widely used in various regions, especially in Guizhou province, which is distributed throughout the south of China, including Guizhou, Sichuan, Hunan and Guangxi. As reported, it could be used for the treatments on reliving cough, fever, antidote, etc. [1,2]. Recently, contemporary studies have shown that P. perfoliatum L. has a variety of pharmacological functions including anti-inammatory [3], anti-bacterium [3,4], and anti-tumor effects [5,6]. To the best of our knowledge, P. perfoliatum L. comprises a mixture of different bioactive compounds, including volatile oils

Corresponding author. Tel./fax: + 86 851 6690018. E-mail addresses: gznutcm@126.com, alice9800@sina.com (X. Zhou). 0367-326X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.tote.2011.04.007

[7], avonoids [812], phenylpropanoids [1315], anthraquinones [10,15], and terpenoids [15]. It is worth noting that the main compounds in P. perfoliatum L. are avonoids, which can partly represent the chemical characteristics and bioactivities of P. perfoliatum L. [8]. Pharmacological studies indicated that the avonoids in P. perfoliatum L. have anti-inammatory [16], antiviral [17], anti-oxidative [18,19] and anti-tumor [20] activities. So far, the previous investigations have mainly focused on identication of chemical components [715]. Several literatures were reported on quantication of quercetin using HPLC with UV detection [2123]. No analytical method was reported on the analysis of other bioactive ingredients in Gangbangui, as well as anti-inammatory and antiviral study of quercetin-3-O--Dglucuronide. Therefore, it was decided to study potential biological activities of quercetin-3-O--D-glucuronide with experimental animal models, and a simple, rapid and sensitive HPLC method on quantication of quercetin-3-O- -D-

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glucuronide in P. perfoliatum L. was established for control and evaluate the quality of the herb better.

2.3. Biological activities of quercetin-3-O--D-glucuronide 2.3.1. Animal Mice (Kunming gene, 1822 g) used in the present study were purchased from the Animal Laboratories of Medical College of Guiyang (Guiyang, China). The experimental animals were randomly divided into several groups and each group had 10 mice. Mice were fed on food and water ad libitum and maintained under standard laboratory conditions. The animal experiments comply with ethical guidelines for the care of laboratory animals. 2.3.2. Drug administration Mouse were orally administered with quercetin-3-O--Dglucuronide (4 mg/kg and 8 mg/kg; 3 mg/kg and 6 mg/kg), Ribavirin (1800 mg/kg, reference drug, Baili Pharmacy of Sichuan, China), and aspirin (100 mg/kg, reference drug, Pingguang Pharmacy of Jiangsu, China) once a day, respectively. The control group was given the same volume of distilled water. These doses selected were carried out according to the method described earlier [25] and were suitable for the study. 2.3.3. Effect of quercetin-3-O--D-glucuronide on inuenza A virus in vivo in mice The effect of quercetin-3-O--D-glucuronide on inuenza A virus in vivo in mice was evaluated according to a report [26,27] with some modications. Under slightly ether anesthesia, mice were inoculated intranasally with approximately 0.005 mL of inuenza A virus (Sinovac Biotech Co., Ltd., Beijing, China). After 2 h virus inoculation, the drug was administered orally in 0.2 mL/10 g bodyweight for 4 days. On the fourth day, mice were prohibited food and water for more than 4 h, then weighted, and killed by dislocation of the neck. The lungs of the sacriced animals were removed, rinsed in normal saline, dried with absorbent paper and weighed. Ribavirin was used as the reference drug. To observe the control group, normal group versus control group was used in this experiment. Lung index and inhibition (%) were calculated according to the following equation. Lung index = Lung weightg = body weightg 100% Inhibition% = control mean of lung index experiment mean of lung index control mean of lung index 100% 2.3.4. Dimethyl benzene induced mice ear edema The anti-inammatory effect of quercetin-3-O--Dglucuronide was assessed in acute inammation method described [28] with some modications. On the 7th day, mice were orally administered with the drugs after 45 min, the right ear of each rat was treated with dimethyl benzene (40 L) on both ear surfaces, and the left ear was used as the control. The animals were killed by cervical dislocation after 15 min. A 6 mm section from each ear was removed with a metal punch and weighed. The edema weight and inhibition percentage are assessed according to the following equation.
Edema weight = weight of the right earweight of the left ear Inhibition% = edema weight of control group edema weight of treated group edema weight of control 100%

2. Experimental 2.1. Chemicals and reagents Analytical grade ethanol (Fuyu, Tianjin, China) was used for sample preparation. HPLC grade methanol (TEDIA, Company Inc. USA), analytical grade phosphoric acid (Kelong, Chendu, China) and HPLC grade water obtained from water purication system (18.2M, Sartorius Germany) were used as mobile phases. Acetic acid (Damao Chemical Reagent Factory, Tianjin, China) and dimethyl benzene (Chuandong Chemical Co., Ltd., Chongqing, China) were of analytical grade. The standard reference of quercetin-3-O--D-glucuronide was extracted, isolated and puried from Gangbangui in our laboratory. The puried compound was identied by 1H NMR, 13C NMR, ESI-MS, UV and IR spectrometric techniques and was compared with literatures [24], of which purity was no less than 98%.

2.2. Plant materials 28 batches of samples were collected from various locations in China, which were authenticated as P. perfoliatum L. (Table 1). The specimen was deposited at the Research Center for Quality Control of Nature Medicine, Guizhou Normal University.

Table 1 Collected information and contents of quercetin-3-O--D-glucuronide in Polygonum perfoliatum L. (n = 3). No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Source Drug plant garden, Guizhou Duyun, Guizhou Huaxi, Guizhou Jiuchang, Guizhou Tongren, Guizhou Bijie, Guizhou Xifeng (1), Guizhou Liuzhi, Guizhou Longli, Guizhou Xifeng (2), Guizhou Menguan, Guizhou Yanhe, Guizhou Zhenfeng, Guizhou Xintianzhai, Guizhou Shuicheng, Guizhou Qiannan (1), Guizhou Qianxi, Guizhou Qiannan (2), Guizhou Xifeng (3), Guizhou Niuliangguandaxingtian, Guizhou Jinping, Guizhou Majiang, Guizhou Danzhai, Guizhou Xinfeng (4), Guizhou Drug material market of Guiyang, Gouzhou Guangshan, Henan Dabieshan, Anhui Chenxi, Hunan Collecting time 2009.01 2008.08. 2003.06 2008.08. 2008.08 2009.07 2008.08. 2008.08 2008.08. 2008.08 2008.08. 2008.08. 2008.08. 2008.08. 2008.08. 2008.08. 2008.08. 2008.08. 2008.08 2008.08. 2008.09 2008.08 2008.09 2008.08 2008.08 2008.08. 2008.08 2008.08 Content, % 1.15 0.77 1.15 2.09 0.22 0.65 1.27 0.06 1.08 0.65 0.80 0.85 0.69 1.29 1.78 0.52 0.28 0.71 1.47 1.29 0.17 0.53 0.69 0.81 1.01 0.52 0.41 0.49

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2.3.5. Effect of quercetin-3-O--D-glucuronide on acetic acid-induced increase in a capillary permeability in mice Another inammation was induced by the method described [29] with some modications. On the 7th day, 30 min after oral administration of quercetin-3-O--D-glucuronide, each mouse was injected with 0.1 mL of 2% Evans blue (A Johnson Company, Heyshom, Lancs) in saline solution at the tail. Then, 10 min after the i.v. injection of the dye solution, 0.2 mL of 0.6% (v/v) ACOH was injected intraperitoneally. 20 min later, animals were sacriced and the peritoneal cavity washed with 5 mL normal saline in cuvette and centrifuged, and the absorption of the supernatant was measured at 590 nm using a spectrophotometer. 2.3.6. Statistical analysis The results were expressed as means SDs. Comparison between treated groups and the control groups were carried out by ANOVA and StudentsNewmanKeuls post hoc tests. p b 0.05 was considered to be signicant. 2.4. Sample preparation

OH OH

HO

O OH O COOH O OH OH OH
Fig. 1. The chemical structure of quercetin-3-O--D-glucuronide.

The dried material was grounded into powder. 0.1 g powder was accurately weighed and put into a 100 mL volumetric ask. 30 mL of 80% ethanol was added to the ask, which was subsequently distilled for 1 h at 80 C. The extraction step was repeated twice, and the extracted solution was mixed and ltered through analytical paper. Then, the ltered solution was dried and dissolved in 25 mL methanol. The sample was ltrated through a 0.45 m membrane before HPLC analysis. 2.5. Apparatus and chromatographic conditions HPLC analysis was carried out on an Agilent series 1100 liquid chromatography, equipped with vacuum degasser, a quaternary pump, an autosampler and a diode array detector (DAD) system, connected to a reserved-phase column (Lichrosher-C18 column, 250 mm 4.6 mm, 5 m, Jiangsu Hanbang Science & Technology Co. Ltd., China). Data collection was performed by using ChemStation soft (Agilent). The mobile phases consisted of methanol (A) and 0.05% aqueous phosphoric acid (B) (43:57, v/v) was applied. The ow rate was 1 mL/min and column temperature was 30 C. The detection wavelength was set at 258 nm. The injection volume was 20 L. 3. Results and discussion 3.1. Selection of target compound To further investigate its active components, phytochemical and pharmacological studies were performed in our laboratory. It was found that the peak of quercetin-3-O--D-glucuronide is the highest in the HPLC chromatogram obtained from antiinammatory and analgesic extracts of Ganbangui. Then, the compound regarded as the marker was isolated from the effective part by column chromatography and preparative HPLC, and its structure was identied by 1H NMR, 13C NMR, ESIMS, UV, and IR spectrometric techniques and compared with literatures (Fig. 1). In the light of the pharmacological study in

effective part, quercetin-3-O--D-glucuronide tested for antiinammatory and antiviral activities revealed satisfactory results. Moreover, it was reported that quercetin-3-O--Dglucuronide could inhibit lipid peroxidation [30,31], angiotensin II-stimulated JNK activation [32] and the formation of reactive oxygen species (ROS) [33]. In the present study, it was found that the content of quercetin-3-O--D-glucuronide in Gangbangui was over 0.90%, which was more than that of quercetin in Gangbangui [1], indicating that the quercetin-3O--D-glucuronide was another major active compound in Gangbangui. So it was necessary to establish an HPLC method for quantication of quercetin-3-O--D-glucuronide as a compensatory approach for quality control of the herb.

3.2. Effect of quercetin-3-O--D-glucuronide on mice infected with virus Quercetin-3-O--D-glucuronide administrated to FLUAVinduced mice suppressed edema of lung. As indicated by the representative data present in Table 2, statistical difference between the control group and the treated groups were signicant, especially with quercetin-3-O--D-glucuronide at dose of 6 mg/kg.

Table 2 Effect of quercetin-3-O--D-glucuronide on inuenza A virus in vivo in mice. Group Normal Control Ribavirin (1800 mg/kg) Quercetin-3-O--D-glucuronide (6 mg/kg) Quercetin-3-O--D-glucuronide (3 mg/kg) Flung index 1.063 0.21 1.235 0.08 0.939 0.13* 0.890 0.07** 0.982 0.13* Inhibitory (%) 23.97 27.94 20.49

Compared with the control group *p b 0.05, **p b 0.01, ***p b 0.001.

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D. Fan et al. / Fitoterapia 82 (2011) 805810 Table 4 Effect of quercetin-3-O--D-glucuronide on vascular permeability induced by acetic acid in mice. Group Control Aspirin (100 mg/kg) Quercetin-3-O--D-glucuronide (8 mg/kg) Quercetin-3-O--D-glucuronide (4 mg/kg) Increase in vascular permeability (OD590) 1.92 1.36 1.26 0.24* 1.15 0.18* 1.26 0.36* Inhibitory (%) 34.38 40.10 34.38

3.3. Effect of quercetin-3-O--D-glucuronide on dimethyl benzene induced mice ear edema The results were showed in Table 3, and it was found that the compound signicantly suppressed the dimethyl benzene induced ear edema at high and low dose. Aspirin signicantly inhibited the edema, also. Therefore, the effect of quercetin3-O--D-glucuronide was obviously better than that of aspirin. 3.4. Effect of quercetin-3-O--D-glucuronide on vascular permeability induced by acetic acid in mice As shown in Table 4, quercetin-3-O--D-glucuronide and aspirin inhibited the acetic acid-induced increase in vascular permeability in mice which was another acute inammatory model. The high dose of quercetin-3-O--D-glucuronide produced a better inhibitory effect than the low dose of quercetin-3-O--D-glucuronide and aspirin. These results suggest that quercetin-3-O--D-glucuronide has an antiacute inammatory action. 3.5. Selection of extraction procedure The efciency of exaction was inuenced by several variables including solvent, extraction method, extraction time, temperature of exaction, and solvent volume, etc. Firstly, the solvents including water, ethanol and different aqueous ethanol were examined. The results indicated that 80% ethanol was better than other solvents. Secondly, extraction method was measured with reuxing and ultrasonic method, and it was found that the former was better than the latter. Then, the temperature of extraction was set at 50, 60, 70, 80, and 90 C, and it was shown that content of the marker was highest at 80 C. The other variables were considered in the following optimization step. To further optimize the condition of the extraction, an orthogonal array experimental design (OAD) was applied to screen conditions for the extraction of the quercetin-3-O-D-glucuronide. Factors including solvent volume (10, 20, 30 mL); extraction times (1, 2, 3); and reaction time (1, 2, 3 h) were displayed in L9 (3 4) matrix and the variables investigated were set at three levels. So attention is placed on the main effects of the most important factors and the interactions among different variables were ignored in the matrix. According to results, the order of inuencing factor was A N BNC N D, which indicated that factor A was the most important factor contributing to the extraction efciency. The result of analysis of variance (ANOVA) was employed to estimate the effects of factors, and it could be inferred that
Table 3 Effect of quercetin-3-O--D-glucuronide on oedema induced by dimethylbenzene in mice. Group Control Aspirin (100 mg/kg) Quercetin-3-O--D-glucuronide (8 mg/kg) Quercetin-3-O--D-glucuronide (4 mg/kg) Oedema 7.31 0.77 5.51 1.00* 3.95 1.06*** 4.95 0.96** Inhibitory (%) 24.62 45.96 32.28

Compared with the control group *p b 0.05, **p b 0.01, ***p b 0.001.

solvent volume (A) as a signicant variable has effects on the content of quercetin-3-O- -D-glucuronide which was extracted from Gangbangui. Moreover, other factors have insignicant effects. Thus, all factors at each level were xed. The optimal condition was presented in detail in the sample preparation (Section 2.4). 3.6. Selection of chromatographic conditions To develop an excellent chromatographic condition for Gangbangui, an optimized strategy for HPLC conditions was performed. The various mixtures of watermethanol and wateracetonitrile were used for obtaining the best resolution and the shortest analytical time. In order to improve peak shape, different concentrations of acids including formic acid, acetic acid and phosphoric acid were added into mobile phases. The results indicated that the mixtures of water methanol and wateracetonitrile containing 0.05% phosphoric acid obtained satisfactory baseline resolution and symmetry (Fig. 2). Considering applicability economy of the method, and friendliness to environment, we selected methanolwater obtaining 0.05% phosphoric acid (43/57, v/v) as mobile phase. The wavelength for detection is optimized by using photodiode array detection (DAD) and UV spectrum. On the basis of baseline and abundance of chromatographic peaks, 258 nm was chosen as detection wavelength. 3.7. Method validation 3.7.1. Linearity and limit of quantication (LOQ) The linearity of the HPLC system detector response for determination of quercetin-3-O--D-glucuronide was estimated by different standard solutions. Nine different concentrations were performed, and each concentration was repeated three times and mean value was employed, which ranged from 0.038 g to 2.376 g. The equation was y = 43694x 19.744, where y = peak area and x = concentration with a correlation coefcient (R 2) = 0.9999. The signal-to-noise (10:1) was employed to establish LOQ that was 16.28 ng. 3.7.2. Precision, repeatability and stability The intra- and inter-precision were measured by analyzing standard solution injected six times in one day and twice as many times on successive three days. The relative standard deviations (RSDs) of intra- and inter-precision were 1.75 and 1.67%, respectively. Repeatability was evaluated by six sample solutions of the same sample; Stability was examined

Compared with the control group *p b 0.05, **p b 0.01, ***p b 0.001.

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Fig. 2. HPLC chromatograms of quercetin-3-O--D-glucuronide (A) and sample (B) from Polygonum perfoliatum L.

at 0, 2, 4, 8, 12 and 24 h on 1 day. The RSDs of repeatability and stability were 2.00 and 2.53%, respectively. 3.7.3. Accuracy The recovery test was used to evaluate accuracy of the extraction method. The standards of three different concentrations operated were added into samples. The samples were pretreated in accordance with the method established above (Section 2.4). Each concentration levels were repeated three times. The recovery was calculated with the rate of detected and spiked amounts. The RSD of recoveries (n = 9) was 2.52%. 3.7.4. Robustness The parameters of chromatographic condition that are likely to be slightly changed are impossible to affect analytical results. To evaluate robustness of the method, different columns, proportion of the mobile phase, different aqueous phosphoric acid, ow rate, column temperature, and detection wavelength at three levels in the same day were tested. The samples prepared were in triplicate, and mean value of recovery was used to illustrate the robustness. The results were calculated by value of relative standard deviations (RSDs) of three levels in the same variation. The RSDs were less than 3.00%. Therefore, the results demonstrated that small variations of chromatographic conditions have no effect on the analytical method. 3.7.5. Sample analysis In this experiment, an HPLCDAD method was employed for the determination of quercetin-3-O--D-glucuronide in 28 batches of Gangbangui, which were collected from

different habits. The results were given in Table 1 and Fig. 3 and showed that the content of quercetin-3-O--D-glucuronide in sample no.4 was higher than the others, and that of sample no.8 was the lowest. The mean contents of quercetin-3O--D-glucuronide from various locations were all over 0.90%. From the results, it was easy to note that the contents of quercetin-3-O--D-glucuronide were between 0.06% and 2.09%. Therefore, different contents were affected by a number of variances, such as climate, geography, harvesting time, producing areas and man-made environments. Moreover, Gangbangui from different habits were likely to have the variation of therapeutic effects. Hence, original compounds existing in the herb were more representative, specic, and helpful to guide rational herb use. 4. Conclusion In this paper, activity tests were performed which indicated that quercetin-3-O--D-glucuronide could suppress ear edema induced by dimethyl benzene, peritoneal permeability induced by acetic acid, and lung edema induced by inuenza A virus in mice. Because the quercetin-3-O--Dglucuronide was administrated to mice, the metabolites of urine and serum were certain to change compared to those of control. The research on the analyses of metabolites in urine and serum from tested animals are being carried out now, and the objective of the study is to reveal mechanism of the action of quercetin-3-O--D-glucuronide in vivo. Then a practical, accurate and specic HPLCDAD method was also developed to quantify bioactive component of quercetin-3-O--D-glucuronide

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2.5 2

content(%)

1.5 1 0.5 0

11

13

15

17

19

21

23

25

27

Fig. 3. The contents of quercetin-3-O--D-glucuronide in Polygonum perfoliatum L. from 28 batches.

in Gangbangui for the rst time. The satisfactory linearity, intraand inter-day precision, accuracy, repeatability, and robustness of the established method were showed in the method validation procedure. The proposed analytical method was successfully used to determine the contents of quercetin-3O--D-glucuronide in Gangbangui from various regions of China which was known for wide use of Guizhou. The results demonstrated that the developed method was sensitive and reproducible and could be readily utilized as a suitable tool for quality assessment on quantication of quercetin-3-O--Dglucuronide in Gangbangui.

Acknowledgements The study was supported by grants from the National Natural Foundation of China (no. 30760293 and no. 81060340) and The Special Program for Scientic Technology Research and Development of Modernization of Traditional Chinese Medicine of Guizhou Province (no. 20095019).

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