Quantitative analysis and In vitro anti-inflammatory effects of gallic acid, ellagic acid, and quercetin from radix sanguisorbae
Chang-Seob Seo1, Soo-Jin Jeong2, Sae-Rom Yoo1, Na-Ri Lee1, Hyeun-Kyoo Shin1
1 K-Herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
2 KM Convergence Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
|Date of Submission||13-Aug-2015|
|Date of Decision||29-Sep-2015|
|Date of Web Publication||2-Mar-2016|
K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054
Republic of Korea
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Radix Sanguisorbae has long been used to treat diarrhea, enteritis, duodenal ulcers, and internal hemorrhage. Objective: We investigated the in vitro anti-inflammatory effects of Radix Sanguisorbae and performed quantitative analyses of three marker components, namely gallic acid, ellagic acid, and quercetin, using high-performance liquid chromatography coupled with a photodiode array detector. Materials and Methods: The three marker components were separated using a reversed-phase Gemini C18 analytical column maintained at 40°C by the gradient elution with two solvent systems. We examined the biological effects of the three marker compounds, gallic acid, ellagic acid, and quercetin, by determining their anti-inflammatory activities in the murine macrophage cell line RAW 264.7. Results: All of the marker compounds exhibited inhibitory effects on prostaglandin E2 production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, with no cytotoxicity. Particularly, ellagic acid significantly inhibited production of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-6 in LPS-treated RAW 264.7 cells. Conclusion: Our results suggest that ellagic acid is the most potent bioactive phytochemical component of radix Sanguisorbae in the treatment of inflammatory diseases.
Keywords: Anti-inflammation, ellagic acid, quantitative analysis, radix Sanguisorbae
|How to cite this article:|
Seo CS, Jeong SJ, Yoo SR, Lee NR, Shin HK. Quantitative analysis and In vitro anti-inflammatory effects of gallic acid, ellagic acid, and quercetin from radix sanguisorbae. Phcog Mag 2016;12:104-8
|How to cite this URL:|
Seo CS, Jeong SJ, Yoo SR, Lee NR, Shin HK. Quantitative analysis and In vitro anti-inflammatory effects of gallic acid, ellagic acid, and quercetin from radix sanguisorbae. Phcog Mag [serial online] 2016 [cited 2021 Jul 26];12:104-8. Available from: http://www.phcog.com/text.asp?2016/12/46/104/177908
- Established high.performance liquid chromatography method was applied in the quantitative analysis of gallic acid, ellagic acid, and quercetin present in an extract from radix Sanguisorbae
- Among the three compounds, the ellagic acid.(7.65.mg/g) is main component in radix Sanguisorbae
- Ellagic acid significantly inhibited production of the proinflammatory cytokines tumor necrosis factor alpha and interleukin.6 in lipopolysaccharide.treated RAW 264.7.cells.
| Introduction|| |
Radix Sanguisorbae is derived from the root of Sanguisorbaofficinalis L. (Rosaceae) which is distributed widely in Korea, Japan, and China. In these countries, it has long been used to treat diarrhea, enteritis, duodenal ulcers, and internal hemorrhage.,, Phytochemical studies of Sanguisorba species have identified phenolic acids (e.g., gallic acid and ellagic acid), flavonoids (e.g., kaempferol and quercetin), triterpene glycosides (e.g., 3β, 19α-dihydroxyurs-12-en-28-oic acid), and dimeric triterpene glycosides (e.g., sanguidio sides A, B, C, and D).,,,,
Inflammation is a self-protective response to various harmful stimuli such as damaged cells, pathogens, and irritants. Radix Sanguisorbae is used to treat inflammatory diseases, and scientific evidence has been obtained to support its effects. In addition, many studies have shown that gallic acid,, ellagic acid,, and quercetin , extracted from various herbal plants have anti-inflammatory effects. Based on previous research, we aimed to determine the bioactive chemical in radix Sanguisorbae that is responsible for its anti-inflammatory effect by testing the marker compounds gallic acid, ellagic acid, and quercetin in the murine macrophage cell line RAW 264.7. In addition, we performed quantitative analyses of two phenolic acids, gallic acid and ellagic acid, and one flavonoid, quercetin, to assess the quality of radix Sanguisorbae using high-performance liquid chromatography (HPLC) coupled with a photodiode array (PDA) detector.
| Materials and Methods|| |
Radix Sanguisorbae was purchased from HMAX (Jecheon, Korea) in October 2008. The botanical origin of this sample was taxonomically confirmed by Prof. Je-Hyun Lee, Dongguk University, Gyeongju, Republic of Korea. A voucher specimen (2008-ST-24) has been deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine.
Reagents and materials
Gallic acid (purity ≥97.5%), ellagic acid (purity ≥96.0%), and quercetin (purity ≥98.0%) were purchased from Sigma-Aldrich Co., (St Louis, MO, USA). HPLC-grade methanol, acetonitrile, and water were obtained from J.T. Baker (Phillipsburg, NJ, USA). Analytical reagent grade glacial acetic acid was procured from Merck (Darmstadt, Germany).
Preparation of 70% ethanol extract
Dried radix Sanguisorbae (200 g) was extracted 3 times with 70% (v/v) ethanol (2.0 L) by sonication for 60 min. The extracted solution was filtered through filter paper, evaporated to dryness at 40°C using a Büchi R-210 rotary evaporator (Flawil, Switzerland) under vacuum, and then freeze-dried. The yield of the freeze-dried 70% ethanol extract was 12.15% (24.30 g).
Quantitative analysis of the marker components in radix Sanguisorbae
The quantitative analysis was performed with a Prominence LC-20A series HPLC system (Shimadzu, Kyoto, Japan) which comprised a solvent delivery unit (LC-20 AT), online degasser (DGU-20A3), column oven (CTO-20A), autosampler (SIL-20 AC), and PDA detector (SPD-M20A). Data processing was performed using LC Solution (version 1.24; Shimadzu, Kyoto, Japan). The analytical column used for separating the three marker compounds was a Gemini C18 column (250 mm × 4.6 mm; particle size, 5 µm; Phenomenex, Torrance, CA, USA), which was maintained at 40°C. The mobile phases for chromatographic separation employed gradient elution with 1.0% v/v acetic acid in water (eluent A) and 1.0% v/v acetic acid in acetonitrile (solvent B). The gradient flow in the two-solvent system was as follows: 10–10% B (5 min), 10–50% B (30 min), 50% B (35 min), and 50–10% B (40 min). The analysis was performed at a flow rate of 1.0 mL/min using detection wavelengths of 254 nm for ellagic acid, 270 nm for gallic acid, and 370 nm for quercetin. The injection volume was 10 µL.
The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco Inc., Grand Island, NY, USA) which was supplemented with 5.5% heat-inactivated fetal bovine serum (Gibco Inc.,), penicillin (100 U/mL), and streptomycin (100 µg/mL) in a 5% CO2 incubator at 37°C.
Cell viability was assessed with the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. RAW 264.7 cells were incubated in 96-well plates with various concentrations of the test materials for 24 h. CCK-8 reagent was added to each well, before incubating for 4 h. The absorbance was measured at 450 nm with a Benchmark plus microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The percentage of cell viability was calculated by the following formula: Cell viability (%) = (mean absorbance in test wells/mean absorbance in control wells) × 100.
Measurement of tumor necrosis factor alpha, interleukin-6, and prostaglandin E2 production
RAW 264.7 cells were treated with various concentrations of marker compounds from radix Sanguisorbae for 4 h before lipopolysaccharide (LPS) (1 µg/mL) stimulation. After incubation for 20 h, the supernatants were analyzed to determine the levels of tumor necrosis factor alpha (TNF-α) (BD Biosciences, Mountain View, CA, USA), interleukin-6 (IL-6) (BD Biosciences, Mountain View, CA, USA), and prostaglandin E2 (PGE2) (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturers' instructions.
All of the values were expressed as the mean ± standard error mean of three independent samples of each compound from radix Sanguisorbae. One-way analysis of variance was used to identify significant differences between the treatment groups. Dunnett's test was used for multiple group comparisons. Differences were considered significant at P < 0.05.
| Results and Discussion|| |
We obtained good separation chromatograms using two solvent systems: 1.0% (v/v) acetic acid in distilled water (A) and 1.0% (v/v) acetic acid in acetonitrile (B), where we employed gradient elution. [Figure 1]a and [Figure 1]b show typical HPLC chromatograms of the standards and the extract. Each compound in the HPLC chromatogram was identified by comparing the retention times and UV spectra with those of reference standards [Figure 1]c. The retention times of gallic acid, ellagic acid, and quercetin under the optimized conditions were 4.26, 19.42, and 26.52 min, respectively. The calibration curves of the three marker components exhibited good linearity with coefficients of determination ≥0.9997 in the seven different concentration ranges that we tested. Thus, the established HPLC analysis method was applied in the quantitative analysis of three compounds present in an extract from radix Sanguisorbae. The concentrations of gallic acid, ellagic acid, and quercetin were 2.37, 7.65, and 0.07 mg/g, respectively.
|Figure 1: (a) Typical high-performance liquid chromatography chromatogram of the standard mixture, (b) 70% ethanol extract of radix Sanguisorbae, and (c) ultraviolet spectra of marker components|
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Before determining anti-inflammatory effects of the marker compounds, we examined the cytotoxic effects of radix Sanguisorbae in RAW 264.7 macrophages. Cells were treated with various concentrations of the marker compounds for 24 h and subjected to an assay using CCK-8. Gallic acid produced no cytotoxic effects at up to 10 µM in RAW 264.7 cells [Figure 2]a. Ellagic acid had no cytotoxic effects up to 5 µM, but it decreased the cell viability by 85.44 ± 0.60% and 40.48 ± 2.48% at 10 and 20 µM, respectively [Figure 2]b. Quercetin increased the proliferation rate at up to 10 µM but reduced the cell viability at 20 µM. No cytotoxicity was observed in RAW 264.7 cells treated with quercetin at up to 20 µM [Figure 2]c. Thus, nontoxic concentrations of the three marker compounds were used in the subsequent experiments.
|Figure 2: (a-c) Cytotoxicity of gallic acid, ellagic acid, and quercetin from radix Sanguisorbae in RAW 264.7 cells. Cells were seeded into 96-well plates and treated with various concentrations of gallic acid, ellagic acid, or quercetin for 24 h. Cell viability was assessed using a Cell Counting Kit-8 assay|
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Various cytokines play key roles in mediating inflammatory responses. Thus, we measured the production of the cytokines, and TNF-α and IL-6, which are related to humoral and cellular inflammation, respectively. An inflammatory reaction was induced by treating RAW 264.7 cells with LPS, which significantly increased TNF-α production. Gallic acid and quercetin had no significant effects on TNF-α production in LPS-stimulated RAW 264.7 cells [Figure 3]a and [Figure 3]c, respectively]. By contrast, ellagic acid significantly inhibited LPS-stimulated TNF-α production at 6.25 and 12.5 µM, but not at 25 µM [Figure 3]b. LPS also significantly enhanced IL-6 production in RAW 264.7 cells [Figure 4]. Ellagic acid had a significant inhibitory effect on LPS-induced IL-6 production at 25 µM, but not at lower concentrations of 6.25 and 12.5 µM [Figure 4]b. Quercetin significantly suppressed LPS-stimulated IL-6 production at concentrations of 6.25–25 µM [Figure 4]c. However, gallic acid did not affect IL-6 production in LPS-treated RAW 264.7 cells [Figure 4]a.
|Figure 3: (a-c) Effect of gallic acid, ellagic acid, and quercetin from radix Sanguisorbae on lipopolysaccharide-stimulated TNF-α production in RAW 264.7 cells. TNF-α production was measured in the culture medium of cells that had been pretreated with various concentrations (6.25, 12.5, or 25 µM) of each compound for 4 h and then stimulated with lipopolysaccharide (1 µg/mL) for 20 h. Each bar represents the mean of three independent experiments. ##P < 0.01 versus untreated control, and *P < 0.05 and **P < 0.01 versus lipopolysaccharide-treated cells|
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|Figure 4: (a-c) Effects of gallic acid, ellagic acid, and quercetin from radix Sanguisorbae on lipopolysaccharide-stimulated IL-6 production in RAW 264.7 cells. IL-6 production was measured in the culture medium of cells that had been pretreated with various concentrations (6.25, 12.5, or 25 µM) of each compound for 4 h and then stimulated with lipopolysaccharide (1 µg/mL) for 20 h. Each bar represents the mean of three independent experiments. ##P < 0.01 versus untreated control; and **P < 0.01 versus lipopolysaccharide-treated cells|
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We also examined whether gallic acid, ellagic acid, and quercetin from radix Sanguisorbae can regulate the proinflammatory mediator PGE2, which is known to control multiple aspects of inflammation. In this experiment, indomethacin was used as a positive control. As expected, LPS markedly increased the level of PGE2 in RAW 264.7 cells. By contrast, indomethacin significantly reduced LPS-induced PGE2 production. Gallic acid, ellagic acid, and quercetin also significantly inhibited PGE2 production in LPS-treated RAW 264.7 cells [Figure 5]. Overall, these results suggest that ellagic acid may be a major bioactive compound that is responsible for the anti-inflammatory effects of radix Sanguisorbae.
|Figure 5: (a-c) Effects of gallic acid, ellagic acid, and quercetin from radix Sanguisorbae on lipopolysaccharide-stimulated prostaglandin E2 production in RAW 264.7 cells. Prostaglandin E2 production was measured in the culture medium of cells that had been pretreated with various concentrations (6.25, 12.5, or 25 µM) of each compound for 4 h and then stimulated with lipopolysaccharide (1 µg/mL) for 20 h. Indomethacin (1.25 ng/mL) was used as the positive control. Each bar represents the mean of three independent experiments. ##P < 0.01 versus untreated control; and *P < 0.05 and **P < 0.01 versus lipopolysaccharide-treated cells|
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Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and naproxen, are generally used to treat inflammation. NSAIDs target cyclooxygenase (COX) which synthesizes prostaglandin. However, recent studies suggest that a multitarget approach should be considered in the development of anti-inflammatory drugs because inflammation is a complex disease. Natural products that contain multiple components are attractive candidates as multitarget drugs. Indeed, many herbal plants and their bioactive compounds have been shown to produce anti-inflammatory effects by targeting various inflammatory molecules, including COX, nuclear factor kappa B, mitogen-activated protein kinases, proinflammatory cytokines, and mediators.,, In addition, the low frequency of side effects and low toxicity of natural products would be helpful in addressing the safety problem associated with NSAIDs. Thus, further studies should also identify the multitargeting efficacy of the active anti-inflammatory compound ellagic acid from radix Sanguisorbae.
| Conclusion|| |
We successfully established a rapid, accurate, and convenient HPLC–PDA method for the quantitative analysis of two phenols, gallic acid and ellagic acid, and one flavonoid, quercetin, in radix Sanguisorbae. Among these components, we found that ellagic acid was the most abundant, i.e., 7.65 mg/g. Our HPLC–PDA method may be helpful for the quantitative analysis of radix Sanguisorbae. Furthermore, our findings suggest the potential role of ellagic acid from radix Sanguisorbae as an anti-inflammatory drug.
Financial support and sponsorship
This research was supported by a grant (No. K15251) from the Korea Institute of Oriental Medicine.
Conflicts of interest
There are no conflicts of interest.
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| Authors|| |
Hyeun-Kyoo Shin, Korean medicine doctor, is a principal researcher at K-herb Research Center, Korea Institute of Oriental Medicine. Her major is Korean medicine. He is interested in the area of traditional herbal medicines.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
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