Abstract: Polygonum perfoliatum is commonly used in folk medicine to treat inflammatory disorders in China. Present studies on the anti-inflammation effect of quercetin-3-O-β-D-glucuronide-methyl ester from Polygonum perfoliatum was evaluated in vivo models of both acute inflammations such as xylene-induced ear edema, acetic acid-induced vascular permeability. Quercetin-3-O-β-D-glucuronide-methyl ester showed a significant dose-related inhibition in xylene-induced ear swelling and acid-induced vascular permeability in mice. These results demonstrate that quercetin-3-O-β-D-glucuronide-methyl ester possesses a potent anti-inflammatory function on acute inflammation. These results also support the claims of traditional Chinese medicine practitioners about the use of Polygonum perfoliatum in the treatment of inflammatory disease.
INTRODUCTION
Polygoni Perfoliati Herba, the aerial part of Polygonum perfoliatum L. also known as traditional Chinese medicine (Guangbangui), is a vine-type weed usually found in Asia, especially in Chinese. It is widely found in Guizhou, Hunan, Anhui, Fujian and other South Provinces in Chinese. In Chinese folk medicine, the whole plant of Polygonum perfoliatum is used in the treatment of reliving cough, fever and detoxification, etc. (Delectics Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, 1998; National Commission of Chinese Pharmacopoeia, 2010; Xin and Xue, 2000). P. perfoliatum has been shown to possess pharmacological properties such as anti-inflammatory (Huang et al., 2008), anti-bacterial (Fu et al., 2008) and cough relieving activity (Long and Li, 2010). Previous phytochemical investigations on the plant have revealed the presence of diferuloyl esters of sucrose (Sun et al., 2000), flavonoids (Zhu et al., 2000; Liu et al., 1999; Zhao et al., 2010; Zhang et al., 2008; Wang and Lu, 2004; Li et al., 2009), neoflavonoids (Sun and Sneden, 1999), triterpene, steroid (Li et al., 2009) etc.
P. perfoliatum is widely used in ethnomedicine for the treatment of inflammation disorders, but its chemical compounds have not yet been pharmacologically evaluated for anti-inflammatory activity. In order to find the chemical compounds of anti-inflammatory potential, this study was undertaken to investigate the bioactivity compounds of Polygonum perfoliatum. A flavonoid (Quercetin-3-O-β-D-glucuronide-methyl ester) was isolated from Polygonum perfoliatum. The results from animal experiment indicated that the compound had powerful anti-inflammatory activity.
MATERIALS AND METHODS
Plant materials: The whole plants of Polygonum perfoliatum were collected at Guiyang city of China in August, 2009 and were identified by macroscopic examination, TLC and HPLC methods compared authentic sample. A voucher specimen (herbarium No. 20090815) of the plant is deposited at the herbarium of the Research Center for Quality Control of Natural Medicine, Guizhou Normal University.
Extraction and isolation: The air-dried powder of whole plant (5.0 kg) was extracted with 85% (v/v) EtOH there times under reflux. The combined EtOH extract was evaporated to dryness in vacuo and the residue was suspended in H2O and then extracted with EtOAc at room temperature.
The EtOH-soluble fraction (320 g) was subjected to dry column chromatography over silica gel, eluted with CHCl3-MeOH (50: 1 to 2: 1, v/v) and yielded five fractions based on the TLC profiles. Fraction 3 was subjected to column chromatography on silica gel H, eluted with a gradient of CHCl3-MeOH (15: 1 to 5: 1, v/v) and then purified by Sephadex LH-20 (CHCl3-MeOH, 1: 1, v/v) to yield compound 1(50 mg).
| Table 1: | 1H and 13C NMR spectral data of quercetin-3-O-β-D-glucuronide-methylester (400 MHz, in CD3OD, δ in ppm, J in Hz) |
Quercetin-3-O-β-D-glucuronide-methyl ester (Fig. 1): amorphous yellow power, m.p. 180-181°C; IR(KBr): 3365, 2932, 1612, 1521, 1452, 1085. ESI-MS: m/z 493 [M+H]+, 515 [M+Na]+; 1H NMR (400MHz, CD3OD) and 13C NMR (100MHz, CD3OD): As shown in Table 1.
Apparatus: Melting points were determined on an XT-4 micro melting point apparatus without correction. FT-IR spectra were obtained on a Bruker TENSOR 27 spectrophotometer. UV spectra were acquired on a Shimadzu instrument, UV-1700 UV-visible spectrophotometer. ESI-MS experiment was performed on a Thermo TSQ Quantum Ultra mass spectrometer. The 1H and 13C NMR spectra were recorded on an Inova-400 instrument (Varian Inc., Palo Alto, CA) with tetramethylsilane (TMS) as internal standard. TLC was performed on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Column chromatography was carried out with silica gel H (Qingdao Haiyang Chemical Co. Ltd.) and Sephadex LH-20 (Pharmacia, Sweden). Acetic acid (Damao Chemical reagent Factary, Tianjin, China) and dimethyl benzene (Chuandong Chemical Co., Ltd, Chongqing, China) were analytical grade.
Animal: Mice of both sex weighing 18-22 g were obtained from the Animal Laboratory of Medical College of Guiyang (Guiyang, China).
| Fig. 1(a-b): | (a) HMBC and (b) H1, H1-COSY correlations
of Quercetin-3-O-β-D-glucuronide-methyl ester |
The mice were housed in plastic cages, with food and tap water available ad libitum. The experimental animals were randomly divided into four groups of 10 animals each. The animal experiments comply with ethical guidelines for the care of laboratory animals.
Anti-inflammatory activity:
Xylene-induced ear swelling in mice: The effect of quercetin-3-O-β-D-glucuronide-methyl
ester on acute topical inflammation was evaluated by the method previously described
(Wang et al., 2010). Quercetin-3-O-β-D-glucuronide-methyl
ester (4 and 8 mg kg-1) was orally administered daily for seven days
before the experiments. Blank control and positive control animals were treated
with the corresponding distilled water and aspirin (100 mg kg-1).
The 45 min preceding the final administration, both surfaces of the mice=s right
ear were given 40 μL of xylene by topical application. The left ear was
used as a control. Mice were sacrificed by cervical dislocation 15 min after
xylene application. Following this, both ears were removed for analysis. Circular
section were taken using a cork borer with a diameter of 8 mm and weighed. The
ear edema was evaluated as the difference in weight between the right ear section
and the left ear section. The anti- inflammatory activity was evaluated in accordance
with the following equation:
Edema weight = weight of the right ear-weight of the left ear
Acetic acid-induced peritoneal capillary permeability in mice: The method of Whittle (1964) was used. Quercetin-3-O-β-D-glucuronide-methyl ester (3 and 6 mg kg-1) was orally administered daily for 8 days before the experiments. Blank control and positive control animals were administered with corresponding distilled water and aspirin (100 mg kg-1). The 30 min after the final administration, each mouse was given an intravenous injection of 2% Evans blue saline solution at 0.1 mL/10 g body weight to the tail, immediately followed by an intraperitoneal injection of 0.6% acetic acid at 0.1 mL/10 g body weight was injected intravenously. Mice were sacrificed by cervical dislocation 20 min after acetic acid injection and the peritoneal cavity of each animal was washed with 5 mL normal saline into tubes. Saline washes from the same animal were centrifuged for 5 min at 1000Hg. Supernatants were collected and their absorbance at 590 nm was measured with a spectrophotometer. The inhibitory activity was calculated in accordance with following formula:
where, Et is average values of absorbance of the treated group and Ec is average values of absorbance of the control group.
RESULTS
Effect of quercetin-3-O-β-D-glucuronide-methyl ester on xylene-induced ear swelling in mice: The effect of quercetin-3-O-β-D-glucuronide-methyl ester on xylene-induced ear swelling in mice was summarized in Table 2. Aspirin (100 mg kg-1, oral) significantly decreased the xylene-induced ear swelling in mice as compared to the control group (p<0.05). Quercetin-3-O-β-D-glucuronide-methyl ester showed a significant dose-dependent decrease effects in this test.
| Table 2: | Effect of quercetin-3-O-β-D-glucuronide-methylester on xylene-induced ear swelling in mice |
| Data presented as Mean±SD for N = 10, *Significantly different from the control group at p<0.05, **Significantly different from the control group at p<0.01, ***Significantly different from the control group at p<0.001 | |
| Table 3: | Effect of quercetin-3-O-β-D-glucuronide-methylester on vascular permeability induced by acetic acid in mice |
| Data presented as Mean±SD for N = 10, *Significantly different from the control group at p<0.05, **Significantly different from the control group at p<0.01, ***Significantly different from the control group at p<0.001 | |
The 8 and 4 mg kg-1 of quercetin-3-O-β-D-glucuronide- methylester showed inhibition at 48.56 and 35.15%, respectively being better than the results obtained by aspirin.
Effect of quercetin-3-O-β-D-glucuronide ethyl ester on vascular permeability induced by acetic acid in mice: It is well known that the increase of vascular permeability induced by acetic acid corresponds to the early exudative stage of inflammation, one of the most important processes in the inflammatory pathological mechanism (Barros et al., 2010). In this study, the effects of aspirin and quercetin-3-O-β-D-glucuronide-methyl ester on acetic acid-induced vascular permeability in mice are shown in Table 3. Aspirin (100 mg kg-1) treated positive control demonstrated inhibitions of dye leakage by 34.38%. Quercetin-3-O-β-D-glucuronide-methyl ester evoked a significant dose-related inhibition of vascular permeability compared to the control group and produced 59.9 and 47.4% inhibition at the respective doses of 6 and 3 mg kg-1 compared to the control group.
DISCUSSION
Polygonum perfoliatum is a commonly-use herb in traditional Chinese medicine for treatment of various inflammatory diseases. The present studies verified that quercetin-3-O-β-D-glucuronide-methyl ester from Polygonum perfoliatum possesses anti-inflammatory effects in acute inflammation.
Inflammation is a complex process that involves several events, such as: Enzyme action, mediator release, sextravasations of fluid, cell migration, tissue breakdown and repair. This fact makes the use of different experimental models essential when conducting pharmacological trials (Vane and Botting, 1995). In the present study, quercetin-3-O-β-D-glucuronide- methylester significant decrease the edema induced by xylene. Xylene can cause instant irritation to the mice’s ear which leads to fluid accumulation and triggers an acute inflammatory response. Suppression of this response is likely an indication of antiphlogistic effect.
Furthermore another well-characterized acute inflammation model, the vascular permeability test, was used to confirm the anti-inflammation activity. Increased vascular permeability occurs as a result of the overexposure of basement membrane due to the concentration and separation of endothelial cells at their boundaries, which allows plasma protein such as immunoglobulins, coagulation factors and fluid to pass the base membrane freely into the injured tissue (Carlson et al., 1985). Quercetin-3-O-β-D-glucuronide-methyl ester evoked a significant dose-related inhibition of vascular permeability induced by acetic acid in mice. So quercetin-3-O-β-D-glucuronide-methyl ester may effectively suppress the exudative phase of acute inflammation.
In conclusion, the results of this study provide the evidence for the in vivo anti-inflammation activity of quercetin-3-O-β-D-glucuronide- methyl ester in acute inflammation. The anti-inflammation activity may be derived from an inhibition of vascular permeability in addition to the antioxidant activity, just as indomethacin works as a cyclooxygenase inhibitor. Further studies are needed to clarify the mechanisms responsible for the anti-inflammatory activity of quercetin-3-O-β-D- glucuronide-methyl ester. The results also support the claims of traditional Chinese medicine practitioners about the use of Polygonum perfoliatum in inflammation disease.
ACKNOWLEDGEMENTS
The study was supported by grants from National Natural Foundation of China (No.81060340), the Guizhou Provincial Special Program of Research and Development of Science and Technology Industry on Modernization of TCMs (No. ZY 2011 3013), Guizhou Provincial Program of Science and Technology Innovation Talent Team on Pharmaceutical Analysis (No. 2011 4008) and the Characteristic Key Laboratory of Guizhou Province Education Department. (No. KY [2012] 005).