Chemical characterization of balloon flower (Platycodon grandiflorum) sprout extracts and their regulation of inflammatory activity in lipopolysaccharide‐stimulated RAW 264.7 murine macrophage cells

Abstract The balloon flower (BF) is a potent natural source of phytochemical compounds and is associated with our health. The sprouting process is accompanied by significant changes in phytochemical compounds in comparison with their original plants. Even though many studies are conducted with BF, there are not yet reports of BF sprouts. In the present study, we determined the chemical composition and biological activity of BF sprouts that had been cultivated for 50 days. Kaempferol‐3‐O‐galactoside and 1‐O‐caffeoylquinic acid were identified as major components of whole BF sprouts. The leaves/stems of the sprouts had higher total phenolic and flavonoid contents and lower IC50 values in DPPH• and ABTS•+ scavenging assays than whole sprouts or roots. The roots of the sprouts had the highest polygalacin D content (1.44 mg/g). We also determined the effects of different parts of BF sprouts on RAW 264.7 macrophage cells. When these cells were stimulated with lipopolysaccharide (LPS), their nitrite and pro‐inflammatory cytokine production increased. BF sprouts suppressed the LPS‐induced production of nitrite, tumor necrosis factor‐α, and interleukin‐6 in a concentration‐dependent manner without causing any cytotoxic effects. Nitrite and pro‐inflammatory cytokine production were significantly inhibited by the roots and leaves/stems, respectively. The inhibitory effects of BF sprouts on LPS‐stimulated inflammatory responses in RAW 264.7 macrophage cells were associated with suppressed NF‐κB activation. These findings suggest that BF sprouts could be a valuable source of bioactive compounds and exert anti‐inflammatory effects due to their polygalacin D, deapi‐platycodin D3, and polyphenol content.

shown diverse pharmacological activities and strongly suppress inflammatory responses by blocking the generation of pro-inflammatory mediators (Elijah et al., 2014;. Choi et al., 2001 reported that 22-year-old BRs proved beneficial to adult patients with diabetes and lung cancer, and aqueous extracts from BRs that had been cultivated for more than 20 years inhibited the growth of tumors in mice. BRs are commonly harvested for their functional effects after quite a long duration (e.g., 4 years) of growth (Elijah et al., 2014;Park, Kim, Lee, Kim, & Baik, 2012).
Even though many studies are conducted with BRs, there are not yet reports describing the chemical composition and functionality of BF sprouts. Therefore, in this study, flavonoids from whole BF sprouts, and saponins from different parts of BF sprouts (i.e., whole sprouts, roots, and leaves/stems) were analyzed. We then studied the effects of BF sprout extracts in an in vitro model of inflammation and found that these extracts could ameliorate inflammatory factors by inhibiting nuclear factor kappa-B (NF-κB) activation and regulating cytokine production in RAW 264.7 macrophages.

| Preparation of balloon flower sprout extracts
BF sprouts were collected from a farm in Hoengseong-gun, Gangwon Province, South Korea in 2018. BF seeds were germinated, and sprouts were cultivated for 50 days before being collected. In a total of 100 samples, the average size of a whole BF sprout was about 19 cm (leaf and stem, 11 cm; root, 8 cm) and the average weight was about 46 g. Two-year-old BRs (Etteum) were collected from a farm in Yeongdong-gun, Chungbuk Province, South Korea in 2018. The BF sprouts and BRs were washed. The leaves (including the stems) were separated from the roots of BF sprouts. The raw sprouts and BRs were freeze-dried (chamber pressure of 20 mTorr, shelf temperature of −40°C, cold trap temperature of −70°C; Bondiro, programmable freeze-dryer, Ilshin Co. Ltd.), pulverized and stored at −70°C prior to LC analysis. For experiments other than LC analysis, 40 ml of 70% EtOH was added to 2 g of the freeze-dried powder and subjected to 30 min of ultrasonic extraction for extract preparation. The extract was then centrifuged at 1,500 x g at 4°C for 10 min, concentrated by evaporation of the EtOH in a rotary vacuum evaporator (Eyela), freeze-dried and stored at −70°C in an airtight polythene container.

| Total phenolic content
The total phenolic content (TPC) was estimated by the F-C method, as described by Elizabeth and Gillespie (Elizabeth & Gillespie, 2007), with slight modifications. Briefly, 100 μl of each extract in ethanol was mixed with 200 μl of 10% F-C reagent and the mixture was kept at room temperature (RT) for 5 min. Then, 800 μl of 700 mM Na 2 CO 3 was added to each tube. Subsequently, 200 μl of the mixture was transferred to 96-well plate, and the absorbance was measured at 765 nm on a multimode microplate reader (Infinite M200 Pro; TECAN). The TPC was expressed as mg of gallic acid equivalents per g of freeze-dried extract (GAE mg/g). All experiments were run in triplicate.

| Total flavonoid content
The total flavonoid content (TFC) was determined by the AlCl 3 method, and quercetin was used a reference compound (Anna & Andlauer, 2011). A volume of 100 μl of the ethanolic extract was mixed with 5 μl of 10% AlCl 3 and 5 μl of a 1 M CH 3 COOK solution in a microplate. The final volume of the mixture was adjusted to 250 μl with distilled water. The mixture was allowed to stand for 30 min at RT. Then, the absorbance was measured at 415 nm. The TFC was expressed as mg of quercetin equivalents per g of freeze-dried extract (QE mg/g). All experiments were run in triplicate.

| Antioxidant activity
The free radical scavenging capacities of samples compared with ascorbic acid were determined by the DPPH • and ABTS• + scavenging assays. Extracts from different parts of the sprouts or from BRs were evaluated for their ability to scavenge DPPH radicals by the method of Blois (Blois, 1958) with suitable modifications.
Briefly, 40 μl of ethanolic extracts at different concentrations were placed in the wells of a microplate. Subsequently, 160 μl of 0.15 mM DPPH in EtOH was added and the mixture was kept at RT for 30 min in the dark. The control was treated with 95% EtOH. A multimode microplate reader was used to measure of the absorbance at 517 nm.
For the production of ABTS •+ , 7.4 mM ABTS was reacted with 2.6 mM K 2 S 2 O 8 and stored in the dark at RT for 12 hr. The ABTS •+ solution was diluted to yield an absorbance of 1.2-1.5 at 734 nm.
Then, 15 μl of ethanolic extracts at different concentrations were placed in the wells of a microplate. Subsequently, 285 μl of the ABTS •+ solution was added, and the absorbance was read at 734 nm after 5 min.
The percentage of scavenging was calculated from the obtained absorbance by the equation: % scavenging = ((Abs control-Abs sample)/Abs control) × 100. Then, curves were constructed which the percentage of scavenging was plotted against the sample concentration in mg/ml (DPPH • ) or µg/ml (ABTS •+ ). The equation of this curve was used to calculate the IC 50 , corresponding to the sample concentration that reduced the initial DPPH • or ABTS •+ absorbance by 50%. A smaller IC 50 value corresponds to higher antioxidant activity. All analyses were conducted in triplicate.

| UPLC-DAD-QTOF/MS analysis
Each freeze-dried powdered sample (0.1 g) was suspended in 10 ml of a 50% EtOH-water solution (v/v) containing the internal standard (100 ppm of galangin and 2,4,5-trimethoxycinnamic acid). The contents were sonicated at RT for 30 min in an ultrasonic bath (power, 300 W; frequency, 40 Hz). Then, the mixture was centrifuged at 1,500 x g at 4°C for 15 min. The supernatant was filtered through a 0.2-µm PVDF syringe filter (Whatman, Kent). The analysis was performed on an ultra-performance liquid chromatograph

| HPLC-UV/ELSD analysis
Each freeze-dried powdered sample (0.1 g) was placed in 10 ml of 50% EtOH, and saponins were extracted in an ultrasonic bath at RT for 30 min as described above. Then, the extract was centrifuged at 1,500 x g for 10 min at 4°C. The residue was re-extracted as mentioned above. The combined extract was completely evaporated and then diluted to 2 ml in a 50% EtOH-water solution.
High-performance liquid chromatography-ultraviolet analysis was performed, and the results were quantified with a light-scattering detector (e2695, Waters Co). For this purpose, 20 μl of the filtrate was injected into the HPLC system. Compounds were separated on a Kinetex XB-C 18 column (250 mm × 4.6 mm, 5 μm,

RAW 264.7 mouse macrophage cells (TIB-71, American Type
Culture Collection) were maintained in DMEM supplemented with 10% FBS and 100 U/ml Pen/Strep at 37°C in a humidified 5% CO 2 incubator. For all experiments, cells were grown to 80%-90% confluence and subjected to no more than 10 passages. Cells were seeded in 96-well plate (5 × 10 5 cells/well) and allowed to adhere.
Then, the cells were treated for 24 hr with different concentrations of the samples (0-500 μg/ml); untreated cells were used as controls.
The medium was replaced with fresh medium containing EZ-Cytox solution (0.01 ml/well), and the cells were incubated at 37°C for 2 hr. The absorbance was then measured at 450 nm on a microplate reader. Cell viability (%) was calculated by the following equation: (absorbance of the sample/mean absorbance of the control) × 100.

| Quantification of nitrite and cytokine production
The concentrations of nitrite, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the cell culture supernatants were measured with the Griess reagent for nitrite and with ELISA kits for TNF-α and IL-6 (Alpco). RAW 264.7 cells were plated in a 12-well culture plate at a density of 2.4 × 10 6 cells/well. The cells were pretreated with the samples for 2 hr prior to 24 hr treatment with 1 μg/ml LPS. The supernatants of cells cultured with or without the samples were then analyzed.
For the measurement of nitrite levels (Yook, Kim, Kim, & Cha, 2017), equal volumes of the culture medium and Griess reagent were mixed and incubated at 37°C for 15 min. The resultant absorbance at 530 nm was measured on a microplate reader. The nitrite release of the treated cells was expressed as a percentage of that of untreated control cells.
The concentrations of TNF-α and IL-6 were calculated from standard curves developed with known concentrations of recombinant TNF-α and IL-6 according to the manufacturer's protocols.

| NF-κB reporter gene assay
For the investigation of NF-κB activation, RAW 264.7 cells stably transfected with an NF-κB luciferase reporter were cultured by GBSA Bio center. The cells were seeded in 384-well white plates (2 × 10 4 cells/well) and allowed to adhere for 24 hr. Then, the cells were pretreated with the samples (100 μg/ml) or Bay11-7082 (10 μM) for 30 min and were stimulated with LPS (10 ng/ml) for 3 hr.
Untreated cells were used as controls, and Bay11-7082 (an inhibitor of cytokine-induced IκB-α phosphorylation) was used for the positive control group. After that, 20 μg/ml Bright-Glo (Bright-Glo luciferase assay system, Promega) was applied to the cells through a JANUS MDT multiple pipetting system (Perkin-Elmer, Inc.), and the cells were incubated for 2 min. Luciferase activation was measured with an EnVision Xcite Multilabel reader (Perkin-Elmer, Inc., Ultrasensitive Luminescence mode).

| Statistical analysis
All the data in this study were processed by SPSS (software version 20.0; IBM Corp.). Comparisons were made by one-way analysis of variance (ANOVA) and Duncan's multiple range tests. An independent t test was used to examine nitrite and cytokines levels. A p-value <.05 was considered to denote statistical significance.

| Antioxidant content and activity of balloon flower sprout extracts
Phenolic compounds including phenolic acids and flavonoids are secondary plant metabolites (Sadegh et al., 2014). Due to the antioxidant properties of these compounds, there is a growing interest in using them for disease prevention (Elijah et al., 2014). We found higher TPCs and TFCs in whole BF sprouts (83.37 GAE mg/g and 46.21 QE mg/g) than in BRs (22.33 GAE mg/g and 2.06 QE mg/g) (  Another study suggested that the free radical scavenging activity of Arctium lappa could be attributed to caffeoylquinic acid derivatives (Maruta, Kawabata, & Niki, 1995). Arctium lappa, commonly known as burdock, is used for its root and has long been cultivated for food and folk medicine, similar to BFs (Predes, Ruiz, Carvalho, Foglio, & Dolder, 2011). Caffeoylquinic acids were recently identified as major constituents of the aerial parts of Chrysanthemum coronarium L. (Wan, Li, Liu, Chen, & Fan, 2017

| Saponin content of extracts from different parts of balloon flower sprouts
BRs are known for their high saponin and triterpenoid contents (Lee,  Various platycodon saponins from BRs are known to exhibit anti-inflammatory activity by blocking the generation of pro-inflammatory mediators (Elijah et al., 2014;. In this study, we analyzed different parts of BF sprouts (including whole sprouts) for eight kinds of saponins, but interestingly, only polygalacin D and deapi-platycodin D 3 were detected (Figure 2). The polygalacin D content was the highest in the roots (roots 1.44 ± 0.02 mg/g, whole sprouts 1.15 ± 0.01 mg/g, leaves/stems 0.97 ± 0.01 mg/g). On the other hand, the deapi-platycodin D 3 was the highest in the leaves/stems (roots 0.37 ± 0.00 mg/g, whole sprouts 0.58 ± 0.03 mg/g, leaves/stems 0.88 ± 0.04 mg/g). Ahn et al., 2005 investigated various saponins and found that polygalacin D was a potential inhibitor of LPS-induced nitrite production. The authors suggested that the single glucosyl group in the R1 position of the polygalacin D triterpenoid saponins is responsible for their anti-inflammatory properties (Ahn et al., 2005). In addition, phenolic compounds and flavonoids are well known as antioxidants with immune-system-promoting and anti-inflammatory effects (Tungmunnithum, Thongboonyou, Pholboon, & Yangsabai, 2018).
Therefore, we hypothesized that BF sprouts could down-regulate inflammation due to their flavonoid and polygalacin D content.

| BF sprouts reduce inflammation-related factors in LPS-stimulated RAW 264.7 cells
Certain dietary components are thought to ameliorate low-grade inflammation that can eventually result in chronic diseases such as cardiovascular disease, cancer, and Alzheimer's disease (Kong, Lee, & Wei, 2019). Thus, there is widespread interest in identifying additional dietary compound with anti-inflammatory activity. BRs have been widely used in medicine as folk remedies for chronic inflammatory diseases (Ahn et al., 2005). To the best of our knowledge, this is the first study to evaluate the anti-inflammatory properties of BF sprouts. As a first step, we conducted a cytotoxicity assay to evaluate the effects of BF sprout extracts on the viability of RAW 264.7 cells (Table 3). Concentrations of BF sprout extracts and BR extracts from 5 to 400 μg/ml were regarded to be in an appropriate range for further studies, as they did not exhibit any cytotoxic effects in RAW 264.7 cells (cell viabilities were over 94% relative to the untreated control group).
To examine the anti-inflammatory activity of BF sprouts, we stimulated RAW 264.7 cells with LPS and then treated them with varying extract concentrations. We then measured the extracellular levels of nitrite ( Figure 3) and pro-inflammatory cytokines such as TNF-α and IL-6 ( Figure 4). The most prominent phenomenon in the process of inflammation is the increase in nitrite production (Jung, Kim, Park, Jeong, & Ko, 2018). Nitrite overproduction is one of the early signs of acute or chronic inflammation, and nitrite is released by various immune cells, including macrophages (Delgerzul, Muhammad, Im, Nisar, & Hwang, 2018). As shown in Figure 3a, LPS treatment triggered significant nitrite production, but this increase was effectively and concentration-dependently inhibited by extracts from different parts of BF sprouts and BRs. BF sprout root extracts and whole sprout extracts inhibited nitrite production to the greatest extent relative to the LPStreated control (72% and 65%, respectively, at 400 μg/ml, Figure 3b).
LPS substantially induces macrophages to release pro-inflammatory cytokines that upregulate inflammatory reactions (Delgerzul et al., 2018). BF sprout extracts significantly inhibited the LPS-induced release of TNFα and IL-6 ( Figure 4a,b). Leaf and stem extract particularly reduced TNF-α and IL-6 levels, and whole sprout extracts also significantly reduced IL-6 levels ( Figure 4b). Note: Cell viability was measured by cytotoxicity assays and calculated relative to the untreated control value (0 μg/ml). Data are expressed as means ± SDs. Means with different superscripts in the same column differ significant ( *** p < .001).
TA B L E 3 Effects of extracts from different parts of balloon flower sprouts on cell viability in RAW 264.7 macrophages IL-6 is produced by macrophages at inflammatory sites (Delgerzul et al., 2018). Compared with other cytokines released during the inflammatory response, IL-6 is a key stimulator of the acute inflammatory response (Gabay, 2006). TNF-α plays various roles in inflammation and the immune system (Zelova & Hosek, 2013) and is known to induce the production of primary pro-inflammatory mediators such as nitrite (Ren & Torres, 2009). TNF-α is also known to activate NF-κB, a transcriptional activator of NOD-like receptor P3 (Liu, Zhang, Joo, & Sun, 2017). NF-κB is a key transcription factor in macrophages and is required for the induction of a large number of inflammatory genes, including TNF-α, IL-1β, and IL-6, which are ultimately secreted as pro-inflammatory cytokines . The NF-κB reporter gene was upregulated in the LPS group compared with the untreated control group ( Figure 5).  (Jang et al., 2013;. A recent study suggested that NF-κB was modulated by the interaction of polyphenols, phenolic acids, saponins, and triterpenoids (Serafini & Peluso, 2016).
According to these results, BF sprouts, which contain flavonoids and polygalacin D in their roots, leaves and stems, should be highly regarded for regulating inflammatory progression. However, further studies are required to elucidate the molecular mechanisms underlying the anti-inflammatory effects of BF sprouts.

| CON CLUS IONS
BRs have been used as food and employed in traditional herbal medicine, but years of growth are commonly needed to exploit their functional effects. On the other hand, sprouts are relatively easy to use because they can be cultivated in a short period of time, and beneficial compounds can be obtained both their roots and their leaves. However, research on BF sprouts has not yet been reported. Therefore, the results of this work are noteworthy, not only in demonstrating that BF sprouts exert antioxidant activity, but also in revealing that they contain a variety of phenolic acids and flavonoids. The antioxidant activity F I G U R E 3 Effects of extracts from different parts of balloon flower sprouts on (a) the nitrite concentration and (b) the %inhibition of nitrite production in RAW 264.7 macrophages. BR, balloon flower root. Data are expressed as means ± SDs. The LPS-stimulated group without sample treatment was compared with each indicated group by an independent t test ( *, #, †, ‡ p < .05, **, ##, † †, ‡ ‡ p < .01, and ***, ###, † † †, ‡ ‡ ‡ p < .001). *, whole sprouts; #, roots of sprouts; †, leaves and stems of sprouts; ‡, BRs of BF sprout extracts was attributed to these phenolic compounds, particularly kaempferol-3-O-galactoside and 1-O-caffeoylquinic acid.
Furthermore, the inhibitory effects of BF sprout extracts on LPSstimulated inflammatory responses in RAW 264.7 macrophage cells were associated with the suppression of NF-κB activation. BF sprout extracts inhibited the production of nitrite, TNF-α, and IL-6 without causing any cytotoxic effects in macrophage cells. Consequently, our results indicated that BF sprouts are a valuable and effective source of bioactive compounds and may regulate inflammatory activity due to their polygalacin D and polyphenolic content.
*, whole sprouts; #, roots of sprouts; †, leaves and stems of sprouts; ‡, BRs F I G U R E 5 Inhibition of NF-κB activity by extracts from different parts of balloon flower sprouts. BR, balloon flower root; Bay11, Bay11-7082 (an inhibitor of cytokine-induced IκB-α phosphorylation). Data are expressed as means ± SDs. Different letters represent significant differences between groups according to one-way analysis of variance followed by Duncan's multiple range test (p < .001).
The LPS-stimulated group without sample treatment was compared with each indicated group by independent t test ( * p < .05, ** p < .01 and *** p < .001)

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
Neither animal nor human testing was involved in this study.