Functional foods play an important role in promoting health and preventing diseases.1) In this regard, tree sprouts have been used as nutritious and beneficial foods.2) The increasing demand for healthy food products has promoted the use of tree sprouts to produce supplemented foods. Phenolic acids have recently gained recognition owing to their diverse, practical, biological, and pharmacological activities.3, 4) Phenolic acids in foods are considered a vital dietary component for humans and exhibit potent antioxidant activity as well as other health benefits.5-7) Among the phenolic acids, chlorogenic (1), caffeic (2), and p-coumaric acids (3) are the most abundant and readily available and are naturally found in a variety of foods.7-9) These acids are indispensable and biologically active dietary polyphenols with many reported pharmacological effects, including anti-cancer, anti-inflammatory, and anti-obesity.10)
Here, we aimed to evaluate the cytotoxicity in the human adenocarcinoma gastric AGS cell line, inhibition of nitric oxide (NO) secretion in lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 macrophage cell line, and α- glucosidase inhibitory activity of the tree sprout extracts, all of which are preliminary studies of anti-cancer, anti-inflam- matory, and anti-obesity effects, respectively. Moreover, chlorogenic (1), caffeic (2), and p-coumaric acid (3) contents in the 12 species of tree sprout extracts were assessed and quantified using high-performance liquid chromatography (HPLC) and an ultraviolet (UV) detector. Lastly, HPLC-UV analysis was conducted on the extract that had the highest concentration of each phenolic acid to determine whether its bioactivity was related to the content of these phenolic acids.
Materials and methods
Plant materials − The 12 species of tree sprouts of Actinidia arguta Planch. (AAG), A. polygama Maxim. (APG), A. kolomikta Maxim. (AKM), Rhus verniciflua Stokes (RVF), Staphylea bumalda DC. (SBD), Morus alba L. (MAB), Securinega suffruticosa Rehder (SST), Cedrela sinensis A. Juss. (CSS), Euonymus alatus (Thunb.) Sieb. (EAT), Fraxinus mandschurica Rupr. (FMS), Philadelphus schrenkii Rupr. (PSR), and Lycium chinense Miller (LCS) were collected from Experimental Forest of National Institute of Forest Science, Hwasung, Korea, from April to May 2020 after considering the growth condition of each species. A voucher specimen was deposited at Division of Special Forest Products, National Institute of Forest Science, Suwon, Korea.
Instruments and reagents − Chromatographic analysis was performed using an HPLC system equipped with a PerkinElmer Flexar QUATERNARY Pump (Waltham, MA, USA), auto-sampler, and PerkinElmer PDA LC Detector. Standards of chlorogenic acid (1), caffeic acid (2), and pcoumaric acid (3) were obtained from Natural Product Institute of Science and Technology (www.nist.re.kr), Anseong, Korea (Fig. 1). HPLC-grade solvents (water and acetonitrile) were purchased from J. T. Baker (Phillipsburg, PA, USA). Acetic acid (99.7%) and ethanol were purchased from Samchun Pure Chemicals (Pyeongtaek, Korea).
Fig. 1. Chemical structures of chlorogenic (1), caffeic (2), and p-coumaric acids (3).
Extraction from the samples − Extracts from the dried samples of tree sprouts (each 10 g) were obtained using ethanol (200 mL) for 3 h and three times under reflux. Sub- sequently, the samples were filtered and evaporated to obtain the extract. The weights of the tree sprouts extracts are as follows: AAG (0.9 g), APG (2.3 g), AKM (0.8 g), RVF (2.2 g), SBD (1.2 g), MAB (1.0 g), SST (2.3 g), CSS (1.3 g), EAT (2.0 g), FMS (5.0 g), PSR (3.1 g), and LCS (2.9 g).
Cytotoxicity assay using the human gastric adenocarcinoma AGS cells − AGS cells were plated on 96-well plates at a density of 1 × 104 cells/well for 24 h and then treated with different concentrations of the extracts. After 24 h of treatment, the medium was replenished with fresh medium, and cytotoxicity was determined using the 10% Ez-Cytox Cell Proliferation Assay Kit (Daeil Lab Service Co., Seoul, Korea). After 1 h of incubation, cell proliferation was assessed by measuring the optical density (OD) at 450 nm using a micro plate reader (PowerWave XS, Bito-Tek Instruments, Win- ooski, VT, USA).
NO assay using RAW264.7 cells − RAW cells were inoculated in 96-well plates at a cell density of 5 × 104 cells/well for 24 h and treated with different concentrations of the sam- ples. After 24 h of incubation, to evaluate the effect of the samples on cell viability of RAW264.7 cells, the cells were treated using a 10% Ez-Cytox Cell Viability assay kit in the culture medium for 1 h. Cell viability was evaluated by measuring the OD at 450 nm using a microplate reader (Bito-Tek Instruments, Winooski, VT, USA). To assess the inhibition of NO secretion by the samples, an NO assay was conducted. After 2 h of treatment, the cells were stimulated with 50 or 100ng/mL LPS. After 20 h of stimulation, the supernatant of the cultured cells was incubated with the same amount of Griess reagent. The NO concentration was evaluated by measuring the OD at 550 nm using a microplate reader (Bito- Tek Instruments, Winooski, VT, USA).
α-Glucosidase inhibitory assay − First, 10 µL of the α- glucosidase enzyme (4 U/mL in 50 mM phosphate buffer, pH 6.8) was mixed with 10 µL of various concentrations of samples (four times the expected concentrations) and incubated for 5 min at 37°C. Then, 20 µL of 1 mM p-nitrophenyl glucopyranose in 50 mM phosphate buffer (pH 6.8) was added to initiate the reaction. After 20 min of incubation at 37°C, 50 µL of 1 mM sodium carbonate was added to terminate the reaction. α-Glucosidase inhibitory activity was measured at 405 nm. α-Glucosidase inhibitory activity was calculated using the following formula:11)
\(\begin{aligned} \alpha \text {-Glucosidase Inhibition }(\%)=& \frac{\text { OD control }-\text { OD sample }}{\text { OD control }} \\ & \times 100 \end{aligned}\)
OD control: absorbance (405 nm) of 100% enzyme activity (only solvent with enzyme).
OD sample: absorbance of the test samples (405 nm) (with enzyme).
Stock solutions and HPLC conditions − The experimental stock solution was prepared by dissolving 20 mg of each tree sprout extract in MeOH under sonication for 20 min and subsequent filtration using a 0.45-μm PVDF membrane. Quantitative analyses of chlorogenic (1), caffeic (2), and pcoumaric acids (3) were performed in a gradient elution HPLC system using a reverse-phase YMC-Pack Pro C18 column (4.6 × 250 mm, 5 μm). The column temperature was maintained at 25°C. The injection volume was 10 μL, and the flow rate was set to 1 mL/min. UV detection was performed at 325 nm. The mobile phase consisted of 0.5 % acetic acid in water (A) and acetonitrile (B); the composition of the gradient elution system was: 95% A at 0 min, 80% A at 25 min, 60% A at 30 min, 100% B at 35 min, 100% B at 40 min, 95% A at 45 min, and 95% A at 55 min. All injections were performed three times.
Calibration curves − Standard stock solutions of chlorogenic (1), caffeic (2), and p-coumaric acids (3) were prepared by dissolving these compounds in MeOH (1 mg/mL) under sonication for 20 min and subsequent filtration using a 0.45- μm PVDF membrane. The working solutions used to construct the calibration curve were prepared by serially diluting the stock solutions. The calibration functions (Table I) of chlorogenic (1), caffeic (2), and p-coumaric acids (3) were calculated using the peak area (Y) and concentration (X, mg/mL) and represented as the mean value ± standard deviation (SD) (n=3).
Table I. Linearity of chlorogenic (1), caffeic (2), and p-coumaric acids (3)
aY=peak area, X=concentration of standards (mg/mL)
br2 =correlation coefficient based on five data points in the calibration curves
Statistical analyses − All experiments were independently performed at least in triplicate. Data are presented as mean ± SD. The Tukey’s method for one-way analysis of variance was applied to evaluate the difference in the mean values between groups using R statistical software (version 3.3.3)12). A p value less than 0.05 was considered statistically significant.
Results and Discussion
Cytotoxicity of the extracts on the human gastric adenocarcinoma AGS cell line − Screening the cytotoxic effect of the 12 species of tree sprout extracts on the AGS cell line revealed that the EtOH extract of RVF exhibited the most potential in suppressing the proliferation of AGS cells with the half-maximal inhibitory concentration (IC50) of 7.14 µg/mL (Fig. 2). By contrast, the other samples moderately inhibited the growth of AGS cells. Cisplatin was used as a reference drug in this study with an IC50 of 42.52 µM. RVF is used as a medicinal herb in East Asia, including China, Korea, and Japan.13) It has demonstrated various pharmacological effects, such as anti-bacterial, anti-cancer, anti-inflam- matory, anti-oxidant, anti-viral, and anti-diabetic activities, in pre-clinical and clinical trials.14) In a clinical study, RVF was shown to exert considerable therapeutic effects in patients with metastatic colon,15) advanced or metastatic pancreatic,16) and advanced non-small cell lung cancer.17) The anti-cancer activity of RVF was established in case studies of patients with gastric,18) liver,19) renal tumor,20) and pulmonary cancer.21) In this study, RVF was the strongest inhibitor of AGS cell proliferation with an IC50 of 7.14 µg/mL. This is in accordance with the results of a study that reported that RVF inhibited the growth of AGS cells at an IC50 of 50 µg/mL.22) This finding indicated that the EtOH extract from RVF sporouts affected AGS cells at a lower concentration than the extract from grown trees.
Fig. 2. AGS cell viability after 24 h treatment with various concentrations of the extracts or cisplatin. Cell viability was determined using the Ez-cytox assay kit. Data are presented as the mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001 compared with untreated group.
In addition, RVF had the highest caffeic acid (2) content (1.70 mg/g extract) among the tree sprout extracts studied(Table II). However, chlorogenic (1), caffeic (2), and p-coumaric acids (3) were not the most abundant compounds in RVF (Fig. 2). Therefore, the cytotoxic effects of RVF cannot be solely attributed to these compounds. As mentioned earlier, many studies have indicated the anti-cancer effects of RVF in vitro experiments, animal models, and clinical trials. RVF has active compounds such as fustin,23) gallic acid, and fisetin. Especially butein, and sulfuretin from heartwood of RVF has been reported to have anti-cancer effects.24, 25) However, the underlying molecular mechanism is not clearly known. In addition, there are little information about effects or active compounds of RVF sprouts, so further studies are needed. This study suggests tree sprouts as a new and potential target for the extraction of biologically active compounds from RVF. It also contributed to the preliminary assessment of the effect EtOH extract from tree sprouts on AGS cell proliferation.
Table II. The contents of chlorogenic (1), caffeic (2), and p-coumaric acids (3) in the tree sprout extracts
Tree sprout extracts inhibited NO secretion of RAW264.7 cells − The Ez-cytox assay kit was used to measure the effectiveness of the extracts on the proliferation of RAW264.7 cells. Four concentrations of the extracts were evaluated (12.5, 25, 50, and 100 µg/mL) to treat RAW264.7 cells without LPS stimulation. The results showed that except for RVF extract, the RAW264.7 cell proliferation was not decreased after treatment as compared to that of the control group, which demonstrated that these extracts did not have a significant effect on the cell viability of RAW264.7 cells up to 100 µg/mL (Fig. 3). RVF inhibited the growth of RAW264.7 cells above 12.5 µg/mL.
Fig. 3. Effect of the different concentrations of the extracts on macrophage cell viability using the Ez-cytox assay kit. Cell viability (%)=(ODextract/ODcontrol)×100. Data are presented as the mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001 compared with control (CTL).
To evaluate the effect of the extracts on NO secretion in LPS-stimulated RAW264.7 macrophages, an NO assay was performed. As shown in Fig. 4, compared with the control group, NO secretion in LPS-treated groups was significantly increased after stimulation with LPS. NO secretion was inhibited the most by MAB at 100 µg/mL with an IC50 of 83.44 µg/mL, and it did not affect the proliferation of RAW264.7 cells. In addition, the EtOH extract from SST inhibited NO secretion with the lowest IC50 of 54.42 µg/mL. RVF exerted the cytotoxicity from the concentration of 12.5 µg/mL, thus its inhibition on the NO secretion was owing to the toxicity. Other extracts had a moderate anti-inflammatory effect on RAW264-7 cells, including AAG, SBD, and SST, with an IC50 of more than 100 µg/mL. Dexamethasone, an indicator of the anti-inflammatory effect,26) was used as a reference drug in this assay with an IC50 of 24.16 µM (Fig. 4).
Fig.4.Inhibitory effect of the extracts on NO secretion in macrophages stimulated by LPS. Data are presented as the mean ± SD (n=3). #p<0.001 compared with control (CTL), *p<0.05, **p<0.01, ***p<0.001 compared with the LPS group.
MAB has been used as a traditional phytotherapy to treat various conditions, including wound healing, asthma, cough, edema, diabetes, eye infection, and nosebleed.27) It has also been reported to have pharmacological activities, such as anti-diabetic,28) anti-cancer,29) anti-microbial,30) immunomodulatory,31) nephroprotective,32) and hepatoprotective33) effects. MAB inhibited airway inflammation induced by LPS in mice.34) In the present study, its EtOH extract suppressed NO secretion up to 31% compared to that in the LPS-treated group at a concentration of 100 µg/mL, whereas the MeOH extract significantly inhibited NO production in LPS-stimulated RAW264.7 cells at a concentration of 5 µg/mL.35) The content of chlorogenic acid (1) was the highest in MAB (Table II). Fur- thermore, chlorogenic (1) and p-coumaric acids (3) were two of the most abundant compounds in the EtOH extract from MAB. Therefore, the cytotoxicity on AGS cells and anti inflammatory effect on RAW264.7 cells of the EtOH extract of MAB sprout may be related to the abundance of these phenolic acids. This study suggests that the EtOH extract of MAB sprouts is a potential cytotoxic agent for AGS cells and anti-inflammatory compound for RAW264.7 cells.
α-Glucosidase inhibitory effect of the extracts − To evaluate the α-glucosidase inhibitory activity of the extracts, an in vitro α-glucosidase inhibitory test was carried out using a colorimetric procedure. Only the EtOH extract from FMS significantly inhibited α-glucosidase activity at a concentration of 100 µg/mL, with an inhibition of 38.37% (Fig. 5). Acarbose was chosen as a positive control for this study, with inhibitory activities of 61.16%, 66.79%, and 69.89% at concentrations of 25, 50, and 100 mM, respectively. A previous study showed the anti-obesity effect of FMS seed extract in a high-fat diet-fed mouse model.36) Furthermore, the content of chlorogenic acid (1) in FMS was 6.578 mg/g extract, which was the third highest among the tree sprouts. Chlorogenic acid (1) has been reported to exert anti-obesity effects and improve lipid metabolism in mice with high-fat diet-induced obesity.37) This evidence indicates that chlorogenic acid (1) may play a major role in the α-glucosidase inhibition of FMS. Therefore, analysis of the content of chlorogenic acid (1) in FMS requires further assessment.
Fig. 5. α-Glucosidase inhibitory activity of the extracts and acarbose. α-GlucosidaseInhibition(%)= \(\begin{aligned} & \frac{\text { OD control }-\text { OD sample }}{\text { OD control }} \\ \end{aligned}\)× 100, Data are presented as the mean±SD (n=3). **p<0.01, ***p<0.001 compared with control
Phenolic acid contents−HPLC-UV analysis of chlorogenic (1), caffeic (2), and p-coumaric acids (3) was performed for quantitative assessment. The HPLC-UV method showed good separation, and the retention times of chlorogenic (1), caffeic (2), and p-coumaric acids (3) were 19.4, 22.4, and 28.6 min, respectively (Fig. 6). A 325 nm wavelength was effective for the detection of phenolic acids. The standard calibration curves for chlorogenic (1), caffeic (2), and p-coumaric acids (3) are listed in Table I. The calibration curves were determined by linearly plotting the peak area against the prepared concentrations and were analyzed using linear regression analysis. Good regression coefficients (r2) for chlorogenic (1), caffeic (2), and p-coumaric acids (3) were determined to be 1.0000, 0.9994, and 0.9994, respectively. The peaks of chlorogenic (1), caffeic (2), and p-coumaric acids (3) were identified in the HPLC-UV chromatograms of the tree sprout extracts. Furthermore, the content of each phenolic acid in the sprouts was calculated. The phenolic acid contents in the tree sprout extracts are shown in Table II. In general, the content of chlorogenic acid (1) was higher than that of caffeic (2) and p-coumaric acids (3) in the tree sprout extract (Fig. 7). The highest total phenolic acid content was found in MAB (14.63 mg/g ext.).
Fig. 6. HPLC chromatogram of chlorogenic acid (1), caffeic acid (2), and p-coumaric acid (3).
Fig. 7. HPLC chromatograms of the EtOH extract of MAB (A), RVF (B), and SST (C).
Conclusion
Our findings provided further information regarding the bioactivities, namely cytotoxicity in AGS cells, inhibition of NO production in LPS-stimulated RAW264-7 cells, and α- glucosidase inhibition, of EtOH tree sprout extracts. Preliminary analysis of phenolic acids—chlorogenic (1), caffeic (2), and p-coumaric acids (3)—was performed using HPLC- UV. In conclusion, the beneficial activities are generally high in the tree sprout extracts with high phenolic acid content. This study suggests that the bioactivities of these extracts may be related to the contents of the mentioned phenolic acids and provides an approach for the development of supplements or functional foods that have health benefits.
Acknowledgments
This work was supported by the Research Program for Forest Science & Technology Development of the National Institute of Forest Science (Project No. FG0403-2018-03).
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