Synergistic Anti-Inflammatory Effects of Ethanol Extracts from Chrysanthemum zawadskii Flower and Cudrania tricuspidata Fruit Occur via Inhibition of the NF-κB Signaling Pathway

Chrysanthemum zawadskii (CZ) and Cudrania tricuspidata (CT) are both traditional Korea herbal medicines, which is widely used to treat fever, cough, gastritis, and women's diseases that may be linked to inflammatory response. Although it has been used to treat diseases related to inflammation, there has been no case of the synergistic anti-inflammatory properties of both extracts. Our data revealed that ethanol extracts of dried whole CZ exhibited free radical-scavenging capacity in vitro, reduced LPS-induced intracellular reactive oxygen species, and decreased the LPS-induced upregulations of the mRNAs encoding iNOS, COX-2, and IL-6 in RAW 264.7 cells, without significant cytotoxicity. This anti-inflammatory effect was most evident from flower extracts: ethanol extracts from flowers significantly reduced the LPS-induced upregulations of iNOS and COX-2 at a concentration of 100 μg/ml. An ethanol extract of the fruit from CT also exerted a radical scavenging capacity and suppressed LPS-induced proinflammatory gene expression: 5.5 μg/ml of the ethanol extract significantly reduced the ability of LPS to induce the mRNA expression levels of iNOS and IL-6 without apparent cytotoxicity. Furthermore, as little as 1.0 μg/ml of the combined ethanol extracts of CZ flower and CT fruit reduced the LPS-induced changes monitored herein, decreasing the upregulations of iNOS and IL-6, and decreasing the nuclear localization of NF-κB p65. These results suggest that the observed synergistic anti-inflammatory effects may be mediated via inhibition of NF-κB signaling. Taken together, these data suggest that ethanol extracts from CZ flowers and CT fruits have synergistic anti-inflammatory effects and that a combination of the two extracts could prove useful for the treatment of inflammation-related diseases.


Introduction
Infammation is part of the complex biological response that a body uses to mitigate injury or harmful stimuli, such as pathogens, damaged cells, or irritation.During the infammatory response, various cells (known as infammatory cells) release specialized substances, including vasoactive amines, vasoactive peptides, and proinfammatory cytokines.Tese infammatory mediators are benefcial for host defense [1], but their excessive and chronic productions are also related to various diseases, such as diabetes, cancer, and infectious diseases [2].For example, infammation can lead to increases in reactive oxygen species (ROS) and free radicals, which damage cell structures, including DNA and surrounding tissues.Hence, natural active ingredients that act as antioxidants by scavenging free radicals and/or inhibiting the production of intracellular ROS (e.g., polyphenols, vitamins, and minerals) can prevent disease and reduce infammation.Recently, a great deal of research efort has been devoted towards discovering anti-infammatory compounds of plant origin as potential natural and safe medicines with no harmful side efects.
A variety of plants have traditionally been used in human medicine in Korea, and studies have shown that several Korean herbal medicines have anti-infammatory efects.For instance, water extracts from Stauntonia hexaphylla fruit [3], ginseng root [4], and Vaccinium oldhamii leaves [5] are known to suppress the ability of lipopolysaccharide (LPS, an infammatory stimulator) to induce the production of proinfammatory cytokines and mediators.
Chrysanthemum zawadskii (CZ) is a traditional Korean medicinal plant used to treat fever, cough, gastritis, and women's diseases that may be linked to infammatory response.Several reports have shown that CZ extracts have anti-infammatory efects.For example, Han et al. reported that linarin, a major physiological active compound from CZ, inhibited the LPS-induced productions of nitric oxide (NO) and cytokines in RAW 264.7 cells [6].Methanol extracts from dried CZ leaf suppressed LPS-induced inducible nitric oxide synthase (iNOS) expression and NO production [7], and hexane and ethanol extracts of whole CZ plants inhibited LPS-induced interleukin-(IL-) 1β and cyclooxygenase-(COX-) 2 gene expression, and NO production [8].
Although nonsteroidal anti-infammatory drugs (NSAIDs) are well known to reduce pain and exert excellent anti-infammatory efects, they can cause multiple adverse efects [9], including gastrointestinal, cardiovascular, and renal complications [10].Terefore, researchers are urgently seeking natural products that show anti-infammatory effects and/or increase anti-infammatory efects and without side efects.We herein investigated the anti-infammatory efects of CZ extracts and their potential synergistic antiinfammatory efects with extracts from Cudrania tricuspidata (CT), which is another Korean natural product.

Preparation of Extracts. Chrysanthemum zawadskii
(CZ) and Cudrania tricuspidata (CT) fruit harvested in 2019 were purchased from a local store (j-dream, Jeongeup, South Korea).Before extraction, whole plants, portions of the plant, or fruit were ground in an electric grinder.Dried CZ (50 g), CZ fower (50 g), or CT fruit (100 g) were extracted with 1,000, 500, and 500 mL of ethanol, respectively, at 60 °C under an ultrasonic condition for 2 h.For water extract preparation, dried CZ (50 g) and CT fruits (100 g) were extracted with 500 mL of water under pressure at 121 °C for 1 h.Te ethanol and water extracts were then fltered, and concentrates were dissolved in dimethyl sulfoxide (DMSO) or distilled water, respectively.All extract stock solutions were stored at −20 °C until use.

DPPH and ABTS Assay.
Antioxidant capacity was analyzed using the DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assays.Samples were diluted appropriately with DMSO, and ascorbic acid was used as an antioxidant standard.Te DPPH assay was performed as described in a previous study [11,12].A solution of 0.2 mM DPPH in 80% (v/v) methanol was stirred for 1 h, and the absorbance of the solution was adjusted to 0.650 ± 0.020 at 517 nm using fresh 80% (v/v) methanol.Ten, 10 μL of sample was mixed with 90 μL of DPPH solution and incubated at 25 °C for 10 min in the dark (covered with aluminum foil).Absorbance was assessed at 517 nm.For the ABTS assay, 7 mM of ABTS was mixed at a 1 : 1 ratio with 2.45 mM of potassium persulfate and stirred at 25 °C for 16 h.Te mixture was diluted with fresh PBS bufer until the absorbance at 734 nm reached a value of 1. Ten, 10 μL of sample was mixed with 90 μL of the ABTS solution and incubated at 25 °C for 10 min in the dark (covered with aluminum foil).Absorbance was assessed at 734 nm.Standard curves for both assays were obtained by measuring the DPPH and ABTS scavenging activities of 2, 4, 10, 20, and 140 μg vitamin C/ml.All reactions were performed in triplicate.Te following equation was used: DPPH or ABTS radical scavenger (%) � (1 − sample absorbance/control absorbance) × 100.

Cell Viability
Test.RAW 264.7 cells were plated to 24well plates at 2 × 10 6 cells/well.After 24 h, cells were treated with various concentration of extracts and incubated for a further 24 h.MTT solution was added to cells, and the plates were incubated for 1 h at 37 °C.Formazan crystals were dissolved with a 1 : 1 solution of DMSO : EtOH, and absorbance at 570 nm was read on a microplate reader.

Extracts from Chrysanthemum zawadskii Reduce LPS-Induced Infammatory Responses.
To investigate the antiinfammatory efects of CZ, we frst extracted natural ingredients from dried whole CZ specimens comprising stem, leaf, and fower.Water and ethanol extractions produced yields of 0.13% and 0.404%, respectively (Figure 1(a)).We then tested the antioxidant potentials of the water-and ethanol-soluble extracts using the DPPH and ABTS assays, which are typical in vitro assays for free radical-scavenging ability.As shown in Figure 1(b), both extracts enhanced free radical scavenging in a dose-dependent manner.Under our experimental conditions, 500 μg/ml of water-soluble extract did not exhibit free radical scavenging, whereas the same concentration of ethanol-soluble extract exhibited good free radical-scavenging activity (82.8% in the ABTS assay and 67.23% in the DPPH assay).Tus, ethanol-soluble extracts appeared to have a better antioxidant efect.Next, to investigate the anti-infammatory efect of CZ, we established a cellular model by applying lipopolysaccharide (LPS) to the murine macrophage cell line, RAW 264.7.As shown in Figure 1(b), ethanol extracts showed anti-infammatory efects.Our assays revealed that LPS (0.1 μg/ml) induced the mRNA expression levels of iNOS, COX-2, and IL-6 in RAW 264.7 cells at 24 h after stimulation.Te ethanol extract (100 μg/ml) reduced the LPS-induced upregulations of these genes by 28%, 38%, and 30%, respectively, but such efects were not seen in cultures treated with the water extract (Figure 1(c)).Te ethanol-soluble extracts were also found to reduce LPS-induced intracellular ROS production in RAW 264.7 cells (Figure 1(d)) but did not negatively afect cell viability; rather, the treatment somewhat increased the proliferation of RAW 264.7 cells (Figure 1(e)).Together, these data suggest that a whole-plant ethanol extract of CZ exhibits considerable anti-infammatory potential.

Te Chrysanthemum zawadskii Flower Endows Anti-Infammatory Efects.
Assuming that the fower was the more potent source of active ingredients, we further investigated the anti-infammatory efects of ethanol extracts from the CZ fower (Figure 2).We obtained the natural ingredients at a yield of around 0.27% for both fresh and dried fowers (Figure 2 In particular, 100 μg/ml of the ethanol extract from dried fowers reduced the LPS-induced upregulations of the transcripts for iNOS and COX-2, whereas the extract from fresh fowers mildly decreased these parameters (Figure 2(b)), suggesting anti-infammatory activities of ethanol extracts from both dried and fesh fowers.Our MTT data further revealed that ethanol extracts from fresh fowers showed no cytotoxicity up to 100 μg/ml in RAW 264.7 cells, whereas ethanol extracts from dried fowers exhibited some slight cytotoxicity against RAW 264.7 cells at this concentration (Figure 2(d)), supporting our contention that CZ fower extracts exhibit anti-infammatory efects with low potential for side efects.Since LPS is known to stimulate infammatory gene expression through the activation of nuclear factor-kappa B (NF-κB) [13,14], we further examined the efect of the extract on NF-κB (Figure 3).Immunofuorescence staining showed that both dried and fresh fower extracts dose-dependently reduced the LPS-induced nuclear translocation of NF-κB subunit p65 (Figure 3).Together, these results indicate that ethanol extracts from both dried and fresh fowers have anti-infammatory activities.

Cudrania tricuspidata Fruit Extract Reduces LPS-Induced
Free Radical Production and Upregulation of Proinfammatory Gene Expression.We then searched for a natural Evidence-Based Complementary and Alternative Medicine product that might potentiate the anti-infammatory efects of CZ.CT has been used in traditional fermented jams, juices, and alcoholic beverages in Korea.In terms of active ingredients, the fruit of CT has been shown to contain prenylated isofavonoids, benylated favonoids, and 5,7,3′,4′tetrahydroxy-6,8-diprenylisofavone [15].Since CT is known to exhibit anti-infammatory efects [16] and protect cells against oxidative stress-induced neurotoxicity [17], we examined the anti-infammatory efect of CT (Figure 4).We extracted natural ingredients from CT fruit with either water or ethanol (Figure 4(a)).Ethanol extraction showed a high yield of 0.6%, whereas room temperature water and hot water both showed low yields of ∼0.1% (Figure 4(a)).Similar to our fndings with CZ, the ethanol extracts showed much higher antioxidant and anti-infammatory activities (Figures 4(b) and 4(d)).At 550 μg/ml, the ethanol extract showed free radical-scavenging activity (81.0% in the ABTS assay and 62.0% in the DPPH assay) similar to that obtained with the CZ extract; however, only very weak activities were seen for both water-soluble extracts (Figure 4(b)).Te ethanol extract showed no cytotoxicity up to a dose of 5.5 μg/ ml (Figure 4(c)).At this nontoxic concentration, the ethanol extract completely abolished the LPS-induced upregulations of iNOS and IL-6 gene expression (Figure 4(d)).Tese data suggest that ethanol extracts of CT fruit have antiinfammatory efects.

Synergistic Anti-Infammatory Efects of Ethanol Extracts from Chrysanthemum zawadskii Flower and Cudrania tricuspidata
Fruit.Finally, we examined the combinatorial anti-infammatory efect of the ethanol extracts from CZ fower and CT fruit (Figure 5).Toward this end, we combined ethanol extracts from CZ fower and CT fruit at concentrations that did not exert cytotoxicity under our experimental condition, as verifed by MTT assays of RAW 264.7 cells treated with either extract alone or cotreated with both extracts together (Figure 5(a)).Interestingly, although monotreatment with 10 μg/ml of ethanol extract from dried fowers of CZ or 0.1 μg/ml of ethanol extract from CT fruits failed to alter the LPS-induced upregulations of iNOS, COX-2, and IL-6, cotreatment of these concentrations together signifcantly reduced the LPS-induced upregulations of iNOS and IL-6 (Figure 5(a)).Furthermore, cotreatment of ethanol extracts from fresh fowers of CZ and CT fruit showed better anti-infammatory efects, with only 1.0 μg/ml   .Interestingly, cotreatment of these extracts containing extracts from dried CZ fowers showed a synergistic restoration of IκBα levels after 30 min, but this efect was observed with the extract from fresh CZ fowers of CZ after 5 min (Figure 6(c)), supporting better anti-infammatory efects of fresh fowers.Taken together, these data indicate that ethanol extracts from CZ fowers and CT fruits have a synergistic antiinfammatory efect.

Discussion
CZ has been used as an herbal medicine in Korea for various diseases, such as gastroenteric diseases, bladder-related diseases, and uterine diseases including menstrual disorders and infertility [18,19].Te benefcial efects are thought to be associated with anti-infammatory efects.Consistent with previous studies, our data confrmed that CZ was effective as an antioxidant and anti-infammatory agents.In particular, ethanol extracts from fresh fowers showed better efectiveness than those of dried fowers or dried whole CZ (Figures 1 and 2).Interestingly, the CZ fower ethanol extracts showed better anti-infammatory activity than the whole dried CZ at the dose of 100 μg/ml (Figures 1 and 2).CT fruit has also been used as an herbal remedy in Korea.A previous study showed that an active compound isolated from CT fruit exerted antiatherosclerotic and neuroprotective activities [20].CT extracts were found to inhibit the LPS-induced upregulations of iNOS expression, COX-2 expression, and NO production [21,22] and to suppress  Dermatophagoides farinae-induced atopic dermatitis [23].Consistently, the ethanol extracts of CT fruit showed antiinfammatory activities similar to those of CZ fowers (Figure 4).When we tested various combinations of the two extracts, 10 μg/ml of ethanol extract from fresh fowers of CZ and 0.1 μg/ml of ethanol extract from CT fruits completely inhibited the LPS-induced upregulations of iNOS and IL-6 (Figure 5(b)).Since the combination of ethanol extract from fresh fowers of CZ and ethanol extract from fruits of CT exerted synergistic inhibitory efects on multiple proinfammatory biomarkers, including iNOS and IL-6 (Figure 5), we investigated the underlying molecular mechanisms.Te NF-κB signaling pathway is known to be essential for regulating infammation [13,14].Consistent with this, our results demonstrated that ethanol extracts of both fresh fowers of CZ decreased the LPS-stimulated nuclear localization of p65 (Figure 3), and combination treatment with extracts from both fresh fowers of CZ and fruits of CT showed more potent inhibition than either monotreatment, at lower doses (Figure 6).Whereas 100 μg/ml of the ethanol extract from dried fower of CZ signifcantly reduced the ability of LPS to upregulate the mRNAs encoding iNOS and IL-6 (Figure 2) and decreased the LPS-induced nuclear localization of p65 (Figure 3), cotreatment of ethanol extracts from fresh fowers of CZ and fruits of CT showed better anti-infammatory efects, with only 1.0 μg/ml of each extract needed to signifcantly reduce the LPSinduced upregulations of iNOS and IL-6 (Figure 5).
Here, we show that cotreatment with low doses of each extract reduced the LPS-induced nuclear localization of p65 (Figure 6).Tese fndings suggest that the observed synergistic anti-infammatory efects occur via inhibition of the NF-κB signaling pathway.In summary, since the corresponding monotreatments had only mild efects, we conclude that these extracts together have a synergistic anti-infammatory efect.Tis synergism strongly suggests that the cotreatment of these compounds could replace the natural material whose use is limited due to its toxicity despite its anti-infammatory properties.Also, given that these plants have long been used as folk remedies, they are expected to have high safety when coapplied to treat infammation and infammationrelated diseases.

Figure 1 :
Figure 1: Antioxidant and anti-infammatory efects of ethanol extracts from dried Chrysanthemum zawadskii.(a) Extraction yield of dried Chrysanthemum zawadskii (CZ) and (b) antioxidant activity was determined using the DPPH assay (top panel) or the ABTS assay (bottom panel).Te indicated concentration of water (left panel) or ethanol extract (right panel) from dried CZ was mixed with assay bufer, and the free radical-scavenging ability was analyzed as described in the Materials and Methods.(c) RAW 264.7 cells were treated with the indicated amount of the extract with or without 0.1 μg/ml of LPS for 24 h.Te mRNA levels of iNOS, COX-2, and IL-6 were analyzed using RT-PCR.β-Actin was detected as a control.(d) RAW 264.7 cells were incubated with the extract (100 μg/ml) in the presence or absence of 0.1 μg/ml of LPS for 24 h.Intracellular ROS levels were measured by DCA-DA assay.(e) RAW 264.7 cells were treated with the indicated amount of extract for the indicated period.Te MTT assay was performed, and the results are presented as the percentage of untreated control.Data were represented as mean ± standard deviation (SD).* P < 0.05 vs. control.# P < 0.05 vs. LPS treatment.

Figure 2 :Figure 3 :
Figure 2: Anti-infammatory efects of ethanol extracts from Chrysanthemum zawadskii fowers.(a) Fresh and dried fowers of CZ were extracted using 99% ethanol (top panel) and percentage of yield extract (bottom panel) as indicated.(b) RAW 264.7 cells were treated with the indicated amount of extract with or without 0.1 μg/ml of LPS for 24 h.Te expression levels of mRNAs were analyzed using RT-PCR.β-Actin was detected as a control.(c) RAW 264.7 cells were incubated with extract in the presence or absence of 0.1 μg/ml of LPS for 24 h.Intracellular ROS levels were measured by DCA-DA assay.(d) RAW 264.7 cells were treated with the indicated amount of extract for 24 h, and MTT assay was performed.Results are presented as a percentage of the untreated control.Data were represented as mean ± standard deviation (SD).* P < 0.05 vs. control.# P < 0.05 vs. LPS treatment.

Figure 4 :
Figure 4: Anti-infammatory efects of Cudrania tricuspidata fruit extracts.(a) Cudrania tricuspidata (CT) fruit was extracted using 99% ethanol, room temperature water or hot water (top panel).Extraction yield of CT fruit (bottom panel).(b) Te indicated concentration of extract from CT fruit was mixed with assay bufer and analyzed for the free radical-scavenging ability.(c) RAW 264.7 cells were treated with the indicated amount of extract for 24 h, and the MTT assay was performed.Results are presented as a percentage of the untreated control and represent the mean ± SEM.(d) RAW 264.7 cells were treated with the indicated amount of extract with or without 0.1 μg/ml of LPS for 24 h.Te expression levels of mRNAs were analyzed using RT-PCR.β-Actin was detected as a control.Data were represented as mean ± standard deviation (SD).* P < 0.05 vs. control.

Figure 5 :Figure 6 :
Figure 5: Synergistic anti-infammatory efect of extracts from Chrysanthemum zawadskii fower and Cudrania tricuspidata fruit.RAW 264.7 cells were treated with each extract alone or cotreated with both extracts.Te ethanol extracts of CZ were from either dried fower (a) or fresh fower (b).Cell viability was determined using the MTT assay (top panel).Te mRNA levels of iNOS, COX-2, and IL-6 were analyzed using RT-PCR (bottom panel).(c) RAW 264.7 cells were treated with each extract alone or cotreated with both extracts as indicated.Te mRNA levels of TNF-α and IL-6 were analyzed using RT-PCR.Data were represented as mean ± standard deviation (SD).*P < 0.05 vs. LPS treatment.
, and DCF fuorescence was detected at absorbance Ex/Em � 485/535 nm using a fuorescence microplate reader (SpectraMax i3; Molecular Devices, USA).2.8.ImmunofuorescenceStaining.RAW 264.7 cells on cover glasses were treated with or without the indicated concentrations of extract with 0.1 μg/ml LPS for 0.5 h.Te cells were then fxed with 3.5% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 15 min.Te slides were incubated with 0.5% BSA blocking bufer and probed with anti-p65 antibody for overnight at 4 °C.After being washed with PBS, the slides were incubated with FITC-conjugated anti-rabbit antibody (Life Technologies, Eugene, OR) at room temperature for 1 h.Each cover glass was mounted on a glass slide with mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI) and visualized by confocal microscopy (Carl Zeiss, Gottingen, Germany).