Variations in the Antioxidant, Anticancer, and Anti-Inﬂammatory Properties of Different Rosa rugosa Organ Extracts

: Rosa rugosa is widely used as a health food and medicine due to its broad pharmacological properties. Although the bioactivities obtained from plant materials is related to the type and amount of phytochemicals in each extract, no systematic information is available on the organ-dependent bioactivities of R . rugosa . Here, the antioxidant, anticancer, and anti-inﬂammatory activities of R . rugosa stem, leaf, ﬂower, and fruit ethanol extracts were evaluated. Overall, the stem extract exhibited the highest levels of DPPH radical scavenging activity, ferric reducing power, and oxygen radical antioxidant capacity compared with other organ extracts, whereas leaves contained potent anticancer compounds that were particularly effective against A549 cells. Additionally, the leaf extract inhibited the MEK/ERK signaling pathway, resulting in the transcriptional repression of pro-inﬂammatory mediators in LPS-stimulated RAW 264.7 cells. Furthermore, signiﬁcant correlation between phytochemical content and bioactivities indicated that phenolic compounds play as a major antioxidant compound of R . rugosa . Taken together, these ﬁndings suggested that the spatial distribution of the phytochemicals contributed to the biological activities of R . rugosa . Given that R . rugosa fruits and ﬂowers are already being used in health foods and medicine, these results indicate that the leaves and stems of R . rugosa should also be included and used as natural sources of antioxidant, anticancer, and anti-inﬂammatory agents.


Introduction
Phytochemicals, defined as naturally occurring compounds in plants, have recently garnered increasing worldwide interest due to their therapeutic effects and their potential as a primary source for synthesizing and developing new drugs [1].In higher plants, phytochemicals accumulate separately in specific organs, because they play individual roles in physiological processes [2].The spatial distribution of phytochemicals is mainly due to the organ-specific expression of various biosynthesis-related genes and/or longdistance transport [3] and is the major cause of variation in biological activities among different plant organs.
Rosa rugosa Thunb., a deciduous shrub of the genus Rosa, is widely distributed across East Asia, including Korea, China, and Japan, and has been used in traditional East Asian herbal medicine to treat various illnesses [4].The flowers of R. rugosa have long been used in traditional Chinese medicine for the treatment of diarrhea, bleeding, and menstrual pain [5].The therapeutic properties of R. rugosa flowers have been largely attributed to volatile compounds including linalool, phenylethyl alcohol, citronellol, α-bisabolol, anthocyanins, procyanidins, and proanthocyanidins [6,7], whereas triterpenoid saponins (pulsatilloside F, hederacolchiside F, patrinia saponin H3, hederasaponin B, and cussosaponin C) in R. rugosa are also known as potential agents for the treatment of diabetes mellitus [8].In the cosmetic and food industries, the fruits of R. rugosa are valued for their bioactive compounds, including carotenoids, flavonoids, polysaccharides, and essential oils [9].
Therefore, in this study, ethanol extracts of stem, leaf, flower, and fruit were submitted to investigate variation in biological activates of R. rugosa organs.In addition, the mitogen-activated protein kinase (MAPK) signaling pathway and the expression of pro-inflammatory mediators in LPS-stimulated RAW 264.7 cells (a murine macrophage cell line) were analyzed to determine the possible anti-inflammatory mechanisms of the leaf extract.These findings thus provide insights into the properties of different R. rugosa organs, including lesser utilized structures such as the stem and leaf, and will help to motivate further interest in the use of R. rugosa as a regular source in the cosmetic and pharmaceutical industries.

Plant Materials and Extraction
R. rugosa samples (leaves; CBNU-Rr-1, stems; CBNU-Rr-2, flowers; CBNU-Rr-3 and fruits; CBNU-Rr-4, Figure 1A) were harvested from the research forest at Chungbuk National University.EtOH extract has been used to identify the active compounds in R. rugosa [10].Thus, R. rugosa samples were soaked in absolute EtOH for 24 h at room temperature.After filtration, the EtOH extracts were evaporated using a rotary vacuum evaporator and kept at −20 • C until required.

Determination of Total Phenolic, Flavonoid, Carotenoid, and Saponin Contents
Total phenolic contents (TPC), total flavonoid contents (TFC), and total carotenoid contents (TCC) were determined as described by Jin et al. [11].The TPC and TFC in each extract were calculated in terms of micrograms of gallic acid equivalents (µg GAE/mg of extract) and quercetin equivalents (µg QE/mg of extract), respectively.Additionally, total saponin content (TSC) was analyzed using the vanillin-sulphuric acid assay as described by Le et al. [12].The TSC in each extract was expressed in micrograms of diosgenin equivalents (µg DE/mg of extract).

Analysis of the Antioxidant Capacities of R. rugosa Extracts
The free radical scavenging activity of each extract was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals as described by Kwak et al. [13], and the results were expressed as the concentration required to reduce half of the DPPH free radicals (RC50).
To analyze the ferric reducing power of each sample, various concentrations of extracts (100, 200, and 300 µg/mL) were mixed with 0.2 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 0.2 mL of 1% potassium ferricyanide as described by Kwak et al. [13].After incubation at 50 • C for 20 min, 10% trichloroacetic acid and 0.1% ferric chloride were added to the mixture.The absorbance was measured at 750 nm, and higher absorbance of the reaction mixture indicated a higher reducing power.Ascorbic acid (AsA) was used as a positive control to analyze free radical scavenging activity and reducing power.
The oxygen radical antioxidant capacity (ORAC) assay was performed as described by Ju et al. [14].Fluorescence intensity was monitored using a SpectraMax Gemini EM microplate reader (Molecular Devices, Pleasanton, CA, USA) with fluorescent filters (485 nm excitation and 530 nm emission).The area under the curve was calculated for each sample by integrating the relative fluorescence curve.ORAC values were expressed as µM of Trolox equivalents (µM TE).

Determination of Cell Viability, NO Production, and Caspase Activity
Cell viability was determined using an MTT solution as described by Yoo et al. [15] and the absorbance was measured at 520 nm.NO production in LPS-stimulated RAW264.7 cells was determined using the Griess reagent system (Promega Co., Ltd., Madison, WI, USA) according to the manufacturer's instructions.NO concentrations were calculated using a nitrite standard curve.Caspase-3 activity and caspase-9 activity were analyzed using the Caspase-3/CPP32 Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) and the Caspase-9 Fluorometric Assay Kit (BioVision, Milpitas, CA, USA), respectively, according to the manufacturer's instructions.

Protein Extraction and Western Blotting
Proteins were extracted from cells using RIPA buffer as described by Yoo et al. [16], after which the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for protein quantification.Next, 15 µg protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane.The membranes were then blocked using a 5% nonfat dried milk solution, after which they were incubated with specific antibodies.The protein band signals were visualized and detected using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a chemiluminescence system (Azure Biosystems, Inc., Dublin, CA, USA).

Determination of Gene Expression Levels Using Quantitative Real-Time PCR (qRT-PCR)
Total RNA from extract-treated RAW 264.7 cells was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA).After synthesizing cDNA using ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan), the expression patterns of each gene (iNOS, COX-2, IL-1α, IL-1β, and IL-6) were analyzed via qRT-PCR, as described by Ju et al. [14].The expression levels of each gene were normalized to actin.Table S1 summarizes all qRT-PCR primer sequences.

Statistical Analysis
All experiments were performed in triplicate.Significant differences between the groups were determined via Duncan's multiple range test.The p-values < 0.05 were considered statistically significant.Correlations between physicochemical content and bioactivities were determined using Pearson's correlation coefficient.

Antioxidant Activities of Different Organ Extracts of R. rugosa
Antioxidants prevent the damage caused by unstable forms of oxygen, known as reactive oxygen species (ROS), which have been linked to various diseases that include inflammation, cancer, and various cardiopathies [17].Although synthetic antioxidants are commonly used as additives in the food, cosmetic, and pharmaceutical industries, consumer demands and lobbying from health organizations have led to a general trend towards replacing synthetic antioxidants with plant-derived antioxidants [18,19].Plant extracts are among the most valued and promising antioxidant ingredients in the pharmaceutical industry and could thus become the basis for novel treatments against oxidative stress-induced human diseases.To investigate the antioxidant activity of R. rugosa, the DPPH-radical scavenging activities of different plant organ extracts were analyzed.As shown in Figure 1B, the stem (SE, RC50 = 45.3 ± 1.20 µg/mL) and leaf (LE, RC50 = 66.1 ± 4.64 µg/mL) exhibited much stronger DPPH-radical scavenging activities compared to the flower (FlE, RC50 = 146.5 ± 8.31 µg/mL) and fruit (FrE, RC50 = 224.3± 36.89 µg/mL).Additionally, the Fe 3+ -Fe 2+ transformation capacity of each extract was quantified using the reducing power assay, a commonly used single electron transfer (SET)-based assay [15].This finding indicated that the reducing power of each extract and ascorbic acid (positive control) on Fe 3+ was concentration-dependent. FrE exhibited the lowest antioxidant activity, whereas other extracts had a relatively higher reducing power of 300 µg/mL compared to the 30 µg/mL of ascorbic acid (Figure 1C).Furthermore, the antioxidant capacities of these extracts were analyzed using the ORAC assay, which is a hydrogen atom transfer (HAT)based assay [15].As shown in Figure 1D, SE at 10 µg/mL exhibited the highest ORAC values of 160.47 ± 24.71 µM TE.Taken together, these findings indicate that SE possesses strong antioxidant properties in both the HAT and SET mechanisms.

Effects of R. rugosa Organ Extracts on Cancer Cell Proliferation
Although several studies have reported on the significant role of natural products as a source of novel drugs [20], only approximately 5% to 15% of higher plants have been chemically and pharmacologically investigated for use as therapeutic agents [21].In this context, the effect of R. rugosa organ extracts on the proliferation of human cancer cells was determined.As shown in Figure 2A, treatment with 100 µg SE or LE significantly inhibited A549 cell viability (37% and 45% inhibitions, respectively), whereas FlE did not exhibit cytotoxic activity against any of the tested cell lines.Additionally, FrE exhibited cytotoxicity against SK-MEL-2 cells (15%).LE, which showed the highest cytotoxicity, exhibited a doseand time-dependent inhibitory effect on the growth of A549 lung cancer cells (Figure 2B).
Caspase-dependent apoptosis is among the most important pathways for the inhibition of tumor growth and metastasis [22].Caspase-3 is among the major effectors of apoptosis and is activated either by extrinsic pathways via caspase-8 or by intrinsic pathways via caspase-9 [23].When A549 cells were treated with 50 µg/mL or 100 µg/mL LE, the activity of caspase-3, but not caspase-9, was strongly induced (Figure 2C), indicating that LE induced cell death via caspase-3, which in turn was activated by the extrinsic caspase-8 apoptotic pathway.Nevertheless, further studies are required to confirm the anticancer properties and safety of LE.These findings suggested that LE is a promising anticancer agent, as demonstrated by its potent inhibitory effects on lung adenocarcinoma epithelial cell proliferation through the induction of caspase-3 activity.

Anti-Inflammatory Effect of R. rugosa Organ Extracts
Innate immune cells including macrophages produce and respond to nitric oxide (NO), which is a signaling molecule involved in several physiological and pathological processes [24].However, overproduction of NO due to the activation of inducible nitric oxide synthase (iNOS) under different stress conditions has been linked to numerous human diseases such as arthritis, asthma, diabetes, inflammation, and septic shock [24].Therefore, the inhibition of NO production under inflammatory stimuli is a major target for the discovery of anti-inflammatory agents [25].The ability of R. rugosa organ extracts to modulate the production of NO was evaluated in RAW 264.7 macrophages via co-treatment of extracts with lipopolysaccharide (LPS).As shown in Figure 3A, SE and LE inhibited LPS-induced NO production in a dose-dependent manner.Particularly, 100 µg/mL of SE and LE reduced NO production by more than 60% compared to a mock control.To investigate whether this effect was mediated by cytotoxic mechanisms, the cytotoxicity of organ extracts in LPS-stimulated RAW 264.7 cells was analyzed using the MTT assay.This finding suggested that LE did not affect cell viability regardless of the presence of LPS, whereas SE slightly reduced cell viability (Figure 3B), indicating that the inhibitory effect of LE on LPS-induced NO production was not attributed to cytotoxicity.
Pro-inflammatory mediators such as cytokines (interleukin (IL)-1α, -1β, and -6), NO, and prostaglandin E2 (PGE2) are known initiators and mediators of the inflammatory response [26].Therefore, reducing the production of pro-inflammatory mediators has been suggested as an important underlying mechanism of anti-inflammatory agents.To investigate the effect of LE on the production of these pro-inflammatory mediators, the expression levels of pro-inflammatory genes including iNOS and cyclooxygenase-2 (COX-2), which encode enzymes for NO and PGE2 synthesis, respectively, were analyzed using qRT-PCR.As shown in Figure 4A, the LPS-induced expression of IL-6, IL-1α, IL-1β, iNOS, and COX-2 was significantly reduced by LE treatment, indicating that the anti-inflammatory effect of LE is mediated by the inhibition of pro-inflammatory mediators in LPS-stimulated RAW 264.7 cells.In R. rugosa extract, rugosic acid A, oleanolic acid acetate, and ursolic acid were identified as anti-inflammatory compounds [10], suggesting that different amounts of these compounds are the major cause of variation in anti-inflammatory activities among different organ extracts.In LPS-stimulated macrophage cells, toll-like receptor-4 (TLR4) activates mitogenactivated protein kinase (MAPK) cascades, including the MEK/ERK signaling pathway, which plays an essential role in the production of pro-inflammatory mediators [27].Therefore, the MEK/ERK signaling pathway has been proposed as a potential therapeutic target for controlling inflammation [28,29].Next, the activation of MEK1/2 and ERK1/2 was analyzed to investigate the inhibitory effect of LE on MEK/ERK signaling in LPS-stimulated RAW 264.7 cells.As shown in Figure 4B, LPS strongly induced the activation of the MEK/ERK pathway, whereas LE inhibited the LPS-induced phosphorylation of MEK1/2 and ERK1/2 in a dose-dependent manner.This demonstrated that the transcriptional repression of pro-inflammatory mediators was due to the inhibition of the MEK/ERK signaling pathway by LE.

Correlation between the Phytochemical Content and Biological Activities of R. rugosa Extracts
Herbal medicines often exhibit synergistic interactions, and therefore their therapeutic effects can be potentiated when delivered in mixtures [30,31].Among various active ingredients, phenolic compounds, flavonoids, carotenoids, and saponins are responsible for a multitude of biological functions including antioxidant, anticancer, antimicrobial, and anti-inflammatory activities [32,33].Therefore, these compounds are largely responsible for the nutritional and health benefits of medicinal plants.In R. rugosa organ extracts, the TPC and TSC were highest in SE (TPC, 213.20 ± 10.33 µg GAE/mg of extract; TSC, 662.85 ± 41.21 µg DE/g of extract) and lowest in FrE (TPC, 47.71 ± 0.24 µg GAE/mg of extract; TSC, 84.04 ± 31.06 µg DE/g of extract).Additionally, LE contained the highest TFC (23.78 ± 4.62 µg QE/mg of extract), and TCC (7.07 ± 4.62 µg/mg of extract) compared with other extracts (Table 1).Pearson's correlation analysis was conducted to determine how biological activities are related to the phytochemical contents in different organ extracts.In terms of the relationships between phytochemical content and antioxidant activities, the most significant positive correlation was identified between TPC and reducing power (r 2 = 0.991), followed by the correlations between TPC and ORAC (r 2 = 0.953).TSC also exhibited good correlation with reducing power (r 2 = 0.899) and ORAC (r 2 = 0.902).Furthermore, although TFC showed no correlation with any antioxidant activity test, correlations were identified between TFC and anticancer activity (A549 MTT assay, r 2 = −0.864),as well as between TFC and anti-inflammatory activity (NO assay, r 2 = −0.745)(Figure 5).Leaves and stems of several rose species were previously reported as a rich source of phenolic acids, which are known as antioxidant compounds [9].In addition, LC-MS/MS-MRM analysis indicated that LE contained a higher amount of phenolic acids (e.g., ferulic acid, isoferulic acid, caffeic acid, gallic acid, p-coumaric acid) compared with FrE [9].In the case of flavonoids, high amounts of chlorogenic acid, rutin, protocatechuic acid, and hydroxybenzoic acid in the immature fruit extract of R. rugosa were observed compared to other extracts, whereas rutin (quercetin 3-O-β-rutinoside) was found as the major flavonoid in the roots [34].In leaf extract of R. rugosa, more flavonoids, such as apigenin, tiliroside, apigenin-7-O-glucoside, kaempferol-3-O-rutinoside, kaempferol and related ester compounds also have been detected [34].These findings suggest that the variations in the biological activities of R. rugosa organ extracts should be mediated by the spatial distribution of active compounds.Different letters indicate significant differences between groups (p < 0.05).

Conclusions
The present study investigated the variations in the biological activities of R. rugosa organ extracts, and suggested that the antioxidant potential of SE was higher than that of other organ extracts.Furthermore, LE exhibited the strongest anticancer activity by stimulating the activation of caspase-3, in addition to possessing strong anti-inflammatory effects through the inhibition of the MEK/ERK signaling pathway in LPS-treated RAW 264.7 cells.Moreover, variations in the biological activities among different plant organs were mediated by the spatial distribution of phytochemicals, including phenolic compounds, flavonoids, carotenoids, and saponins.Further studies on the isolation and safety of the bioactive compounds will be needed to motivate further interest in the use of this plant.These results provide valuable information on R. rugosa stems and leaves as the choice of plant materials to be used as a potential source in the cosmetic and pharmaceutical industries.

Figure 1 .
Figure 1.Morphology of R. rugosa and antioxidant activity of its organ extracts.Morphology of R. rugosa stems, leaves, flowers, and fruits (A).The antioxidant activities of R. rugosa organs were measured by DPPH-radical scavenging (B), reducing power (C), and ORAC (D) assays.The DPPHradical scavenging activity was calculated and reported as RC50.The ORAC values of each organ extract were expressed as Trolox equivalents (µM TE).Different letters indicate that the means were significantly different at p < 0.05 (means ± SE of three independent experiments).Ascorbic acid, AsA; Stem extract, SE; leaf extract, LE; flower extract, FlE; fruit extract, FrE.

Figure 2 .
Figure 2. Anticancer effects of R. rugosa organ extracts on human cancer cell lines.(A) Effect of R. rugosa organ extracts on human cancer cell viability, as determined by the MTT assay.(B) Timeand dose-dependent effect of LE on the viability of A549 cells.(C) Effect of LE on caspase-3 or caspase-9 activity in A549 cells.DMSO-treated samples were used as mock controls.The plots represent the mean ± SE of three independent experiments.Different letters indicate significant differences between groups (p < 0.05).Stem extract, SE; leaf extract, LE; flower extract, FlE; fruit extract, FrE.

Figure 3 .
Figure 3. Anti-inflammatory effects of R. rugosa organ extracts on LPS-stimulated RAW 264.7 cells.Effects of each extract on NO production (A) and cell viability (B) in LPS-stimulated RAW 264.7 cells.DMSO-treated samples were used as mock controls.The values represent the mean ± SE of three independent experiments.Different letters indicate significant differences (p < 0.05), as determined by Duncan's multiple range test.Stem extract, SE; leaf extract, LE; flower extract, FlE; fruit extract, FrE.

Figure 4 .
Figure 4. Molecular mechanisms of leaf extract (LE) in LPS-stimulated RAW 264.7 cells.(A) Effect of LE on the expression of LPS-induced pro-inflammatory mediators (iNOS, COX-2, IL-1α, IL-1β, and IL-6).The expression levels of each gene were normalized to actin.(B) Effect of LE on the LPS-induced MEK/ERK signaling pathway.Each protein was immunoblotted three times using independently prepared lysates.All values are reported as the mean ± SE.DMSO-treated samples were used as a mock control.Different letters indicate significant differences between groups (p < 0.05).