Cellular Anti-Inflammatory and Antioxidant Activities of Bamboo Sasa albomarginata Leaf Extract and Its Constituent Coumaric Acid Methyl Ester

Background Hot water extract of Sasa albomarginata (Kumazasa) leaves is commercially available and used as a dietary supplement or skincare cream. The extract possesses anti-inflammatory activity on the mouse atopic dermatitis model. To elucidate the mechanism of in vivo activity, we have studied the cellular anti-inflammatory and antioxidant activities of the extract and its constituents. Methods Secretion of mouse and human IL-6 was measured by ELISA. ROS production was measured by a fluorescent reagent. Ultrahigh performance liquid chromatography (UHPLC)/MS was used for the ingredient analysis. Results The Sasa albomarginata extract inhibited inflammatory mediators such as LPS-induced NO, IL-6, and ROS production in mouse monocyte leukemia RAW264.7 cells. It also inhibited iNOS, IL-6, and IL-1β expressions. Moreover, it inhibited LPS-induced IL-6 expression and production in human monocyte leukemia THP-1 cells differentiated into macrophages. The HPLC analysis of the extract revealed the existence of coumaric acid, ferulic acid, and coumaric acid methyl ester. Coumaric acid methyl ester but not coumaric acid or ferulic acid inhibited LPS-induced NO, IL-6, and ROS production in RAW264.7 cells and IL-6 production in differentiated THP-1 cells. Conclusion The hot water extract of Sasa albomarginata leaves and one of its constituents possess cellular anti-inflammatory and antioxidant activities.


Introduction
e moisture cream is widely used to protect skin from dryness and cracking. To provide moisture to skin, glycerin, mineral oil, hyaluronic acid, and so on are used. If antiinflammatory compounds are added to these constituents, the cream should be more effective to protect the skin even from cracking and inflammation. Especially, pressure ulcers are often a serious problem in most nursing homes. For this purpose, natural materials such as edible plant extracts would be more advantageous than chemicals because of ease of development. Sasa albomarginata is a bamboo called Kumazasa often found in Hokkaido, Japan, and Sichuan, China. Latin names of Kumazasa include Sasa albomarginata, Sasa veitchii, and Sasa senanensis. e leaves are well known as food for pandas. e extract was reported to possess antibacterial activity. e organic solvent extract of Sasa albomarginata leaves was reported to show antimicrobial activity against bacteria, fungi, and yeast. Both acidic and phenolic fractions of the extract showed antimicrobial activity [1]. Cytomegalovirus is one of the herpesviruses. e hot water-soluble fraction of Sasa albomarginata inhibited the cellular infection of human cytomegalovirus [2]. ey isolated five compounds from the extract, and among them, tricin showed antiviral activity in a plaque assay with a human embryonic fibroblast cell line. e antiviral activity of hot water extract was also shown in the plaque assay for the pseudorabies virus, a kind of herpesvirus [3]. Antitumor activity was reported for an alkaline extract of Sasa senanensis known as Sasa Health [4]. e extract was added to the drinking water and inhibited both the onset and growth of spontaneous mammary tumors in a high mammary tumor strain of SHN virgin mice. Sasa Health also inhibited spontaneous mammary tumorigenesis in another mouse model with human breast cancer [5]. Oral administration of Sasa Health delayed the development of tumors. It also inhibited mammary duct branching and side bud development with reduced angiogenesis. Kumazasa leaves are known to possess anti-inflammatory activity. Oral administration of a hot water extract of Sasa albomarginata leaves significantly reduced the incidence of stress-, ethanol-induced and indomethacin-induced gastric ulcers in rats [6]. e extract also inhibited a release of histamine from rat mast cells [6]. e methanol extract of Sasa veitchii did not inhibit NF-κB activation but inhibited cyclooxygenase activity [7]. Moreover, the hot water extract of Sasa albomarginata was reported to ameliorate a mouse model of atopic dermatitis by topical administration [8]. e study employed DS-Nh mice in which atopic dermatitislike lesions were spontaneously developed with an increase of serum IgE. e extract ameliorated the skin inflammation score and decreased serum IgE. Since the hot water extract of Sasa albomarginata leaves possesses anti-inflammatory activity on skin inflammation, skin cream containing the extract has been developed. Meanwhile, coumaric acid, ferulic acid, and caffeic acid were found to exist in the steam or hot water extract of Sasa albomarginata leaves [9]. ese three compounds possess related cinnamic acid structures [10]. In the present research, we first studied the cellular anti-inflammatory and antioxidant activities on hot water extract of Sasa albomarginata leaves. en, we tried to find the chemicals contained in Sasa albomarginata extract that possess anti-inflammatory and antioxidant activities.

Materials.
e hot water extract of Sasa albomarginata leaves (Kumazasa extract) was purchased from Toyo Ink Co., Ltd. (Tokyo, Japan). p-Coumaric acid, caffeic acid, and ferulic acid were purchased from Sigma-Aldrich (St. Louis, MO). Coumaric acid methyl ester was purchased from Tokyo Chemical Industry (Tokyo, Japan). e test chemicals were added to each well and further incubated for 24 h. Next, MTT solution was added to each well and incubated for 2 h. en, the supernatant was replaced by 100 µl DMSO and pipetted to dissolve formazan crystals. Absorbance was determined on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA) at 570 nm.

Nitric Monoxide
Production. RAW264.7 cells at a density of 3 × 10 5 cells/ml were seeded onto 96-well plates and incubated for 1 h. e test chemicals were added to each well and further incubated for 1 h and then exposed to 100 ng/ml LPS for 24 h at 37°C. e supernatant was harvested and then 50 μl aliquots were mixed with an equal volume of Griess reagent in a 96-well plate. Absorbance was then determined on a microplate reader (Bio-Rad, Hercules, CA) at 570 nm.

Measurement of Mouse IL-6
Production. RAW264.7 cells at a density of 3 × 10 5 cells/ml were seeded onto 96-well plates and incubated for 1 h. e test compound solutions at different concentrations were added to each well and further incubated for 1 h and then exposed to 100 ng/ml LPS for 24 h at 37°C. IL-6 concentration in culture supernatants was measured using Mouse IL-6 ELISA kit (R&D Systems, Minneapolis, MN). Briefly, the assay diluent and the supernatants were added to the well and incubated at room temperature for 2 h. en, the plate was washed 5 times, and Mouse IL-6 conjugate was added to the well for 2 h. e plates were washed 5 times and freshly mixed substrate solution was added to each well and incubated for 30 min at room temperature in the dark. en, diluted hydrochloric acid was added to stop the reaction. e absorbance was measured at 450 nm by the microplate reader (Bio-Rad, Hercules, CA).

Measurement of Human IL-6
Production. THP-1 cells at a density of 1.5 × 10 5 cells/ml were seeded onto 96-well plates and differentiated into macrophagelike cells using PMA. Differentiated THP-1 cells were washed with PBS and fresh medium and incubated for 24 h. e test chemical solutions were added to each well and further incubated for 1 h and then exposed to 100 ng/ml LPS for 24 h at 37°C. IL-6 concentration in culture supernatants was measured using Human IL-6 ELISA kit (R&D Systems, Minneapolis, MN). Briefly, the assay diluent and the supernatants were added to the well and incubated at room temperature for 2 h. en, the plate was washed 4 times, Human IL-6 conjugate was added to the well and incubated at room temperature for 2 h. e Scientific World Journal e plates were washed 4 times, and freshly mixed substrate solution was added to each well and incubated for 20 min at room temperature in the dark. en, 2 N Sulfuric Acid was added to stop the reaction. e absorbance was measured at 450 nm by the microplate reader (Bio-Rad, Hercules, CA).

Measurement of Reactive Oxygen Species (ROS)
Production. RAW264.7 cells at a density of 3 × 10 5 cells/ml were seeded onto 96-well plates or 24-well plates incubated overnight. e test compound solutions were added to each well and further incubated for 1 h and then exposed to 100 ng/ml LPS for 24 h at 37°C. e production of ROS was determined using DCFH-DA (Sigma, St. Louis, MO), an oxidant-sensitive fluorescent probe. e medium was removed from each well, and the cells were washed with Ca 2+ , Mg 2+ -free PBS (PBS − ) twice. After being incubated for 30 min with 20 µM DCFH-DA, the supernatant was removed. Cells were then washed with PBS − twice and added by 200 µl PBS − . ereafter, the fluorescence was read with excitation at 485 nm and emission at 535 nm on a fluorescence plate reader, and the cells were visualized by a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan).

Measurement of the DPPH-Radical Scavenging Ratio.
e 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging effect was measured using DPPH Antioxidant Assay kit (Dojindo, Kumamoto, Japan). Briefly, sample solution (20 µl) was added to each well in 96-well microplate, followed by 80 µL assay buffer and 100 µl DPPH working solution. en, the plate was incubated for 30 min at room temperature in the dark. e absorbance was measured at 517 nm by the microplate reader.

Ultrahigh Performance Liquid Chromatography (UHPLC) Analysis.
e UHPLC analysis was performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA) to determine the amount of coumaric acid and ferulic acid. An Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 µm; Waters Corp.) was used at 40°C for the separation. e mobile phase consisted of water with 0.1% formic acid  e Scientific World Journal (Solvent A) and acetonitrile with 0.1% formic acid (Solvent B). e mobile phase flow rate was 0.2 ml/min. e gradient program was as follows: 10% Solvent B at 0-0.5 min; 10-50% at 0.5-8 min; 50-60% at 8-8.1 min; 60% at 8.1-9 min; 60-10% at 9-9.1 min; 10% at 9.1-10 min. e total run was 10 min. e autosampler was set at 15°C and the injection volume of each sample was 2 µl. e signals were detected at 325 nm.

UHPLC-MS Analysis.
To quantify the concentration of coumaric acid methyl ester, mass spectrometry was performed on an LCT premier XE (Waters Corp.). e separations were performed on the same UPLC system, column, temperature of the autosampler, injection volume of samples, and mobile phase at UHPLC analysis. e column oven was maintained at 30°C. e mobile phase gradient condition was as follows: 5% Solvent B at 0-0.5 min; 5-80% at 0.5-8 min; 80% at 8-9 min; 80-5% at 9-9.1 min; 5% at 9.1-10 min. e total run was 10 min. e mass spectrometer analysis was operated in the electrospray ionization negative mode. MS source conditions were set as follows: capillary voltage, 2.8 kV; source temperature, 120°C; desolvation temperature, 400°C; desolvation gas flow, 800 l/h; cone gas flow, 50 l/h; V mode.

Statistical Analysis.
All results were shown as mean-± standard deviation (S.D.). Student's t-test was used to analyze differences between two groups. e differences over two groups were detected by one-way analysis of variance (ANOVA) followed by Dunnett's posthoc test.

Inhibition of Inflammatory Mediator and ROS Production by Sasa albomarginata Extract in RAW264.7 Cells.
e extract was dissolved directly in the medium. Sasa albomarginata extract did not show cytotoxicity below 3 mg/ml in RAW264.7 cells (Figure 1(a)). e extract inhibited LPSinduced NO production (Figure 1(b)) and IL-6 secretion (Figure 1(c)) at 3 mg/ml or lower concentrations. In Figure 1(b), a high concentration of the extract might influence the colorimetric assay. LPS induced ROS production in RAW264.7 cells, and the Sasa albomarginata extract inhibited ROS production at 1 and 3 mg/ml (Figures 1(d) and 1(e)). e fluorescence was measured directly (Figure 1(d)) or by counting the fluorescent cells ( Figure 1(e)). e extract showed in vitro antioxidant activity in the assay using DPPH (Figure 1(f )).

Inhibition of iNOS, IL-6 and IL-1β Expressions by Sasa albomarginata Extract in RAW264.7 Cells. NO is produced by inducible NO synthase (iNOS).
We have studied the effect of Sasa albomarginata extract on LPS-induced iNOS, IL-6, and IL-1β expressions using real-time PCR. e incubation time was 4 h, and the extract was not toxic below 10 mg/ml (Figure 2(a)). It inhibited iNOS (Figure 2(b)), IL-6 ( Figure 2(c)) and IL-1β (Figure 2(d)) expressions at the nontoxic concentrations. e Sasa albomarginata extract was not prominently toxic at 3 mg/ml (Figure 3(a)) in THP-1 cells. It also inhibited the LPS-induced IL-6 secretion in THP-1 cells at nontoxic concentrations (Figure 3(b)). We have also studied the effect of Sasa albomarginata extract on IL-6 expression using real-time PCR. e incubation time e Scientific World Journal 7 was 4 h, and the extract was not prominently toxic even at 10 mg/ml (Figure 3(c)). It also inhibited the LPS-induced IL-6 expression in THP-1 cells at nontoxic concentrations ( Figure 3(d)).

HPLC Analysis of Sasa albomarginata Extract.
Since coumaric acid, caffeic acid, and ferulic acid were reported to exist in the extract, we measured the content of these acids. As a result, large amounts of coumaric acid and ferulic acid but not caffeic acid were found in the extract. en, we analyzed the cellular anti-inflammatory activity of these acids by LPS-induced NO production, but they showed no activity. Since acidic compounds may not pass though the cell membrane, we tested the anti-inflammatory activity of each ester. ese methyl esters inhibited NO production. en, we measured the content of these esters by HPLC. We found the existence of coumaric acid methyl ester as shown in Figure 4.

Inhibition of Inflammatory Mediator and ROS Production by Coumaric Acid Methyl Ester in RAW264.7 Cells.
Coumaric acid methyl ester did not show cytotoxicity below 10 μg/ml in RAW264.7 cells (Figure 5(a)). Coumaric acid methyl ester inhibited LPS-induced NO production ( Figure 5(b)) and IL-6 secretion ( Figure 5(c)) at 10 μg/ml or lower concentrations. Coumaric acid methyl ester also inhibited the LPS-induced ROS production at 3 and 10 μg/ ml (Figures 5(d) and 5(e)). Fluorescence was measured directly ( Figure 5(d)) or by counting the fluorescent cells ( Figure 5(e)). Unlike the Sasa albomarginata extract (Figure 1(f )), the chemical did not show antioxidant activity in the same assay system.

Discussion
Hot water extract of Kumazasa (Sasa albomarginata) inhibited LPS-induced cellular inflammation. e extract was found to contain comparatively large amounts of coumaric acid and ferulic acid, although they showed no anti-inflammatory activity. However, the methyl ester of coumaric acid contained in the extract showed cellular anti-inflammatory activity. Both coumaric acid and coumaric acid methyl ester contained in Lavatera trimestris flowers were reported to show in vitro radical scavenging activity [11]. Methanolic extracts from long English cucumber leaves were fractionated and tested for their antifungal activity against Cladosporium cucumerinum. After isolation and purification, the active principle was found to be coumaric acid methyl ester [12]. Coumaric acid methyl ester is one of the bioactive components of the plant Costus speciosus.
is plant is traditionally used in Asia to treat catarrhal fevers and skin diseases. e coumaric acid methyl ester was found to inhibit migration and tube formation in human umbilical vein endothelial cells (HUVEC) possibly via inhibition of the VEGF-VEGFR2 pathway [13]. It also inhibited physiological and tumor angiogenesis in zebrafish [13]. Caffeic acid and ferulic acid structures are close to that of coumaric acid. Caffeic acid methyl ester but not caffeic acid was reported to show cellular and in vivo anti-inflammatory activity [14]. erefore, it may be possible that the Sasa albomarginata extract and coumaric acid methyl ester would show in vivo anti-inflammatory activity. Coumaric acid methyl ester showed antioxidant activity in our study. Ferulic acid methyl ester also shows  e Scientific World Journal antioxidant activity, and the mechanism has been studied. Using ethyl linoleate as the oxidation substrate, the antioxidation reaction of ferulic acid methyl ester produced a dimer of ferulic acid methyl ester having a dihydrobenzofuran moiety [15]. A para-hydroxy group is necessary for the formation of dihydrobenzofuran moiety in this reaction. Since coumaric acid has a para-hydroxy group, the reported mechanism may be applicable to the anti-oxidant reaction of this compound. A large amount of coumaric acid and ferulic acid exists in the water extract of Kumazasa leaves. But they did not inhibit LPS-induced NO production at the non-toxic concentrations. Possibly, these compounds are too polar to penetrate the cell membrane. On the other hand, we have found coumaric acid methyl ester contained in the extract (Figure 4), inhibited LPS-downstream signaling as shown in Figures 5 and 6. It may contribute to the anti-inflammatory activity of the extract. However, since the content is comparatively little, the contribution is likely to be limited. It is possible that coumaric acid and ferulic acid are endogenously esterified by lipase [16]. Song et al. has studied permeability and antimelanogenic effects of coumaric acid and coumaric acid methyl ester [17]. In an ex vivo skin permeation assay, topically applied coumaric acid rather than the methyl ester in the cream could more effectively diffuse into the aqueous medium underneath the skin. us, in our case, coumaric acid and ferulic acid themselves are likely to show anti-inflammatory activity in vivo with or without esterification. is time we employed hot water extract of Sasa albomarginata leaves because of the reported in vivo anti-inflammatory activity. However, it is likely that alcoholic extraction may provide a more potent extract because of the effective isolation of esterified compounds. e plant extract showed potent antioxidant activity in vitro, while coumaric acid methyl ester did not inhibit in vitro. It is possible that the chemical modifies NF-κB related signaling to inhibit the production of ROS [18]. Plant extracts are generally considered to be safe and they often show anti-inflammatory or antimicrobial activity. Launaea sarmentosa extract inhibited LPS-induced cellular inflammation via suppression of NF-κB/ MAPK signaling with Nrf2 activation [19]. Cannabis extract and its ingredient cannabidiol inhibited the productions of cellular mediators of skin inflammation [20]. Smilax guianensis extract prevented LPS-induced cellular inflammation by inhibiting the NF-κB pathway in RAW264.7 cells [21]. Methanol extract from Saussurea involucrata inhibited the LPS-stimulated inflammatory responses also in cultured RAW264.7 cells [22]. Azadirachta indica bark extract restricted coronaviral infection and replication model [23]. Kumaizasa (Sasa senanensis) leaf extract was reported to inhibit LPS-induced IL-1β and TNF-α expression in macrophages but increased IL-8 production in HEK293 cells [24]. Alkaline extract of Sasa sp. inhibited IL-1β-induced prostaglandin E2 in human gingival fibroblasts [25]. Kim et al. prepared a cellular inflammatory bowel disease (IBD) model with coculture of human intestinal epithelial Caco-2 cells and RAW264.7 cells [26]. Sasa quelpaertensis leaf extract suppressed LPSinduced inflammatory mediator expressions in this system.

Conclusion
e hot water extract of Sasa albomarginata leaves inhibited LPS-induced inflammatory responses and ROS production at the nontoxic concentrations in macrophage-like cells. e extract was shown to contain coumaric acid, ferulic acid, and small amount of coumaric acid methyl ester. Since coumaric acid methyl ester showed anti-inflammatory and antioxidant activities, the active principles may include coumaric acid and related compounds.

Data Availability
e data used to support the findings of this study are included within the article.