Anti-Inflammatory Flavonoids from Agrimonia pilosa Ledeb: Focusing on Activity-Guided Isolation

To elucidate the anti-inflammatory properties and constituents of Agrimonia pilosa Ledeb. (A. pilosa), a comprehensive investigation was conducted employing activity-guided isolation. The anti-inflammatory effects were evaluated through an in vitro nitric oxide (NO) assay on lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. Seven bio-active compounds with anti-inflammatory properties were successfully isolated from the butanol fraction and identified as follows: quercetin-7-O-β-d-rhamnoside (1), apigenin-7-O-β-d-glucopyranoside (2), kaempferol-7-O-β-d-glucopyranoside (3), quercetin (4), kaempferol (5), apigenin (6), and apigenin-7-O-β-d-glucuronide-6″-butylester (7). All isolated compounds showed strong NO inhibitory activity with IC50 values ranging from 1.4 to 31 µM. Compound 6 demonstrated the most potent NO inhibition. Compound 7, a rare flavonoid, was discerned as a novel anti-inflammatory agent, ascertained through its inaugural demonstration of nitric oxide inhibition. Subsequently, a comprehensive structure-activity relationship (SAR) analysis was conducted employing eight flavonoids derived from A. pilosa. The outcomes elucidated that flavones exhibit superior NO inhibitory effects compared to flavonols, and the aglycone form manifests greater potency in NO inhibition than the glycone counterpart. These results highlight A. pilosa as a promising source of effective anti-inflammatory agents and indicate its potential as a health-beneficial dietary supplement and therapeutic material.


Introduction
Agrimonia pilosa (A.pilosa), also known as hairy agrimony, is an herbaceous flowering plant belonging to the family Rosaceae, order Rosales.The aerial parts of A. pilosa have been used in traditional medicine in Korea, Japan, and China [1], and the plant is native to East Asia [2]. A. pilosa contains diverse chemical components such as flavonoids [3], isocoumarins [4,5], tannins [6], and terpenoids [7].Recent studies reported that A. pilosa extracts demonstrated broad physiological activities, such as antioxidant, anti-inflammatory, anti-cancer, nitric oxide scavenging, and α-glucosidase inhibitory activities [8][9][10][11][12].In a previous study, five phenolic compounds, namely aromadendrin, dihydrokaempferol 3-O-β-D-glucoside, quercitrin, aglimonolide-6-O-α-D-glucoside, and loliolide, were found to be NO scavengers in this plant [12].In addition, the ethanol fraction from A. pilosa extract showed excellent anti-inflammatory activity, including NO scavenging, inflammatory cytokine suppression, and inflammatory enzyme repression activity [13].As part of our comprehensive investigation of anti-inflammatory compounds from A. pilosa, the constituents of A. pilosa were investigated.The macrophage-derived NO that is present under treatment with cytotoxic molecules such as LPS, is considered to play a key role in inflammation and immune response [14].Thus, the inhibition of NO production can be used as a criterion for the evaluation of anti-inflammatory effects.The aim of this study is to investigate the anti-inflammatory compounds present in the butanol fraction of A. pilosa (APB) that exhibit the most potent anti-inflammatory effects and to elucidate their chemical structures using various spectroscopic techniques such as UV spectroscopy, mass spectrometry (MS), and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy.Furthermore, this study aims to evaluate and compare the anti-inflammatory activities of these compounds by measuring their inhibitory impact on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, thereby providing valuable insights into the potential therapeutic applications of A. pilosa and their active compounds in conditions associated with inflammation and immune response.

Activity-Guided Isolation
In our preliminary screening, the methanol extract from A. pilosa exhibited excellent anti-inflammatory (AIN) activities.While previous studies have reported the antiinflammatory activities of agrimonolide and tiliroside derived from A. pilosa [12,13], an exhaustive investigation on anti-inflammatory activity-guided isolation is currently lacking in the literature.Consequently, to elucidate the anti-inflammatory principle underlying A. pilosa, an anti-inflammatory activity-guided isolation was performed.
Anti-inflammatory activity was evaluated by nitric oxide production in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage.In order to elucidate the anti-inflammatory compounds of A. pilosa, the methanol extract of AP (APM) was obtained and fractionated with hexane, ethylacetate, and butanol, successively.At non-cytotoxic concentrations (0-40 µg/mL) with or without the presence of 1 µg/mL LPS, the inhibitory effects of the A. pilosa extract and solvent fractions on macrophage activation were investigated.The butanol fraction of A. pilosa (APB) showed the strongest inhibitory effect on LPS-induced NO production.The anti-inflammatory activity of APB was further studied.The APB (83.4 g), the highest active fraction, was subjected to C 18 column chromatography (CC) to yield fourteen fractions (Figure 1).The anti-inflammatory activities of fourteen fractions, F1~F14, were measured through nitric oxide (NO) production in lipopolysaccharide (LPS)stimulated RAW 264.7 macrophages (Table 1).Among fourteen fractions, F6, F7, and F10 showed strong NO inhibitory effects.Three active fractions from the butanol fraction of A. pilosa (APB) were fractionated with various separation techniques, successively.Fraction F6 was subjected to C 18 column chromatography (CC), and twelve fractions were obtained from F6. Active fractions, F6-IX (7.4 g) and F6-XI (5.1 g) from F6 were subjected to silica gel CC to yield compounds 4 (74.1 mg) and 5 (54.0 mg), respectively.Fraction F7 was subjected to C 18 CC to yield seven fractions, F7-I~F7-VII.Among the fractions from F7, F7-II, the most active fraction, was rechromatographed using C 18 CC to yield fifteen fractions, F7-II 1 ~F7-II 15 .Compounds 1 and 2 were derived from F7-II 4 , while compounds 3, 4 (15.2 mg), and 5 (13.2 mg) originated from F7-II 5 , using silica gel CC.Compounds 4 and 5 were isolated from F6 and F7.Additionally, compound 6 was successfully isolated from F7-II 11 using silica gel CC.Compound 7 was obtained from Fraction F10 subjected to silica gel column chromatography.Subsequently, quantitatively, the isolated amounts of anti-inflammatory compounds from these highly active fractions were determined as follows: compound 1 (23 mg), compound 2 (8 mg), compound 3 (197 mg), compound 4 (88 mg), compound 5 (67 mg), compound 6 (8 mg), and compound 7 (28 mg).This precise quantification provides quantitative insights into the distribution of these bioactive entities within the most potent fraction, thereby establishing a foundation for further detailed analyses of their therapeutic potential and mechanisms of action.Compounds 3, 4, and 5 were the primary anti-inflammatory compounds within the butanol fraction of A. pilosa.
analyses of their therapeutic potential and mechanisms of action.Compounds 3, 4, and 5 were the primary anti-inflammatory compounds within the butanol fraction of A. pilosa.

Identification of Bio-Active Compounds
Following a rigorous bioactivity-guided isolation strategy, seven distinct anti-inflammatory compounds were successfully isolated (Figure 2).The purity of all isolated compounds was determined by HPLC analysis.When the purity of compounds 1-7 was greater than 95%, structural characteristic analysis was performed.In order to identify the structural characteristics, the set of spectroscopic data, including retention times of HPLC profiles, mass spectral data, and NMR chromatograms, was estimated.The structure of the compounds was also confirmed using comprehensive spectroscopic analysis data by

Identification of Bio-Active Compounds
Following a rigorous bioactivity-guided isolation strategy, seven distinct anti-inflammatory compounds were successfully isolated (Figure 2).The purity of all isolated compounds was determined by HPLC analysis.When the purity of compounds 1-7 was greater than 95%, structural characteristic analysis was performed.In order to identify the structural characteristics, the set of spectroscopic data, including retention times of HPLC profiles, mass spectral data, and NMR chromatograms, was estimated.The structure of the compounds was also confirmed using comprehensive spectroscopic analysis data by comparison of the literature [15][16][17][18][19][20][21][22][23][24] and their 1H-NMR and MS data.Their analytical data were as follows.

Anti-Inflammatory Effects of Isolated Compounds
Nitric oxide (NO) serves as a signaling molecule pivotal in the pathogenesis of inflammation.Under normal physiological conditions, it exerts an anti-inflammatory effect.Conversely, in abnormal situations characterized by overproduction, NO is regarded as a pro-inflammatory mediator that instigates inflammation [13].Macrophage-derived nitric oxide (NO) generated during treatment with cytotoxic molecules like LPS is recognized to play an important role in inflammation and immune responses [14].Consequently, the inhibition of NO production stands as a valuable criterion for assessing anti-inflammatory effects.Compounds 1-7 were tested for anti-inflammatory activity through NO inhibition in LPS-treated RAW 264.7 macrophages at non-cytotoxic concentration ranges (0-20 µM).When the isolated compounds were treated in the NO production system, NO accumulation was significantly inhibited in a dose-dependent manner.The highest NO inhibitory ability was obtained from compound 6 (apigenin), with an IC 50 value of 1.43 µM, followed by compounds 5 > 2 > 7 > 4 > 3 > 1 (IC 50 values of 5.75, 8.03, 14.68, 19.51, 22.24, and 31.26µM, respectively (Table 3)).Eighteen anti-inflammatory compounds including ten flavonoids, five terpenoids, and three isocoumarins from A. pilosa were reported [25].Their nitric oxide (NO) inhibitory effects demonstrated a range of 38 to 177 µM for flavonoids, 125-132 µM for terpenoids, and 25-76 µM for isocoumarins.Within the pool of 58 flavonoids isolated from A. pilosa, ten specific flavonoids, including tiliroside, catechin, pilosanidin A, B, pilosanol A, B, C, N, naringin, and dihydrokaempferol glucopyranoside, were reported as antiinflammatory compounds, exerting their effects through NO inhibition.Among the known anti-inflammatory compounds in A. pilosa, pilosanidin A demonstrated the highest NO inhibitory ability with an IC 50 value of 38 µM [25].Notably, the seven flavons and flavonols isolated in this study exhibited superior anti-inflammatory activity compared to pilosanidin A (38 µM), underscoring the efficacy of the activity-guided isolation methodology in identifying potent bioactive compounds.Seven compounds isolated in this study were first reported as NO inhibitors in this plant.Compound 7, identified as a rare flavonoid, emerged as a novel anti-inflammatory agent, as evidenced by its inaugural demonstration of nitric oxide inhibition.The study highlighted the significance of this compound, given its rarity and substantial anti-inflammatory properties.Additionally, it is worth mentioning that the methyl ester of apigenin-7-O-β-D-glucuronide, previously isolated from the ethyl acetate leaf extract of Manilkara zapota, exhibited noteworthy anti-cancer efficacy against breast cancer.This comprehensive research not only sheds light on the anti-inflammatory potential of specific compounds in A. pilosa but also suggests avenues for further exploration.The in-depth investigation of compound 7 is deemed indispensable, as it not only serves as a foundational element but also lays the groundwork for unraveling the diverse spectrum of biological activities associated with this botanical entity.

Structure-Activity Relationship of Flavones and Flavonols from A. pilosa
Eight compounds from A. pilosa were selected for a structure-activity relationship study.The structural determinants influencing the inhibitory activity of flavonoid aglycone and its glucosylated derivatives from Agrimonia pilosa on nitric oxide (NO) were systematically investigated and summarized in Table 4.This investigation aimed to elucidate the intricate connections between the structural characteristics of flavonoids and their anti-inflammatory properties, providing a nuanced understanding of the molecular determinants governing the observed pharmacological effects.The presence of a hydroxyl group at position 3 ′ on the B ring of the flavone moiety influenced NO inhibitory activity, with a decrease observed as follows: apigenin (IC 50 = 3.69 µM) demonstrating greater efficacy compared to luteolin (IC 50 = 4.62 µM).Additionally, the hydroxyl group at position 3 on the C ring emerged as a critical factor for NO inhibitory activity, with the flavonol moiety featuring a hydroxyl group at this position leading to a diminished NO inhibitory effect.The descending order of NO inhibitory activity was observed as follows: apigenin (IC 50 = 3.69 µM) > luteolin (IC 50 = 4.62 µM) > kaempferol (IC 50 = 14.43 µM) > quercetin (IC 50 = 19.50 ± 1.71 µM).The examination of NO inhibitory activities of flavones' and flavonols' glycosides revealed a decreasing trend according to the order of Apigenin-Glc (IC 50 = 8.03 µM) > Luteoline-Glc (IC 50 = 14.37 µM) > Kaempferol-Glc (IC 50 = 22.24 µM) > Quercetin-Glc (IC 50 = 31.27µM).Increased -OH groups and glycosylation correlated with diminished NO inhibitory effects, suggesting that the number of -OH groups and glycosylation of flavones are crucial factors in NO inhibitory activity.The effects of hydroxyl group and glycosylation on activity were reported in a study comparing the flavone and flavone-Glc structure and antioxidant activity [26].The bioactivity-structure relationship for the antioxidant activity of flavonoids from A. pilosa showed that the glycosidation of flavonoids at C-6 in the A-ring enhanced the stability of free radicals, thereby increasing antioxidant capacity [27].Meanwhile, glycosidation at C-7 in the A-ring and the hydroxyl group at C-3 in the C-ring significantly decreased NO inhibitory activity in the present study.

General Experimental Procedure
The UV spectra were obtained from UV spectrometer (Power Wave XS, Bio-Tek In-

Plant Materials
Dried aerial parts of Agrimonia pilosa (A.pilosa) were procured from Yangnyeong market in Daegu, Korea, with a verified origin in Korea.Prof. Kang S.H., affiliated with the Department of Natural Medicine Resources at Semyung University in Jecheon, Republic of Korea, conducted the botanical identification.A voucher specimen (NP-116) was deposited at the herbarium of Kyungpook National University (Daegu, Republic of Korea).Plant material was chopped and stored at room temperature until the commencement of experiments.

LC-ESI-MS Analysis
LC-MS were carried out on a Shimadzu LCMS-8050 mass spectrometer (Shimadzu, Tokyo, Japan) and on a Waters-Micromass quadrupole-time of flight Ultima TM Global instrument (Waters, Manchester, UK) spectrometer equipped with a DAD detector (set at 280 nm and 360 nm) and MS via an ESI interface in positive and negative ion scan mode.Chromatographic separations for crude extract and isolates were performed using a Shim-pack GIS-ODS column (4.6 × 250 mm, 5.0 µm, YMC, Japan).The mobile phase consisted of 0.5% formic acid in water (solvent A) and methanol (solvent B).The sample was eluted with the following linear gradient: starting from 20% B to 30% B in 5 min, to 50% B in 10 min, to 70% B in 15 min, to 100% B in 20 min, and to 20% B in 35 min.Flow rate was set 0.8 mL/min and the injection volume was set at 10 µL.The column temperature was maintained at 35 • C. The MS parameters were as follows: Q3 scan mode, capillary temperature of 250 • C, sheath gas (N2) flow rate of 3 L/min, aux gas (N2) flow rate of 10 L/min, ion spray voltage of 2.5 kV.Full MS scans in the FT cell were acquired in the range of m/z 100-975.The isolation window was set at m/z 1.0, and the stepped collision energy was used at 20, 40, and 60 eV.Xcalibur (Ver.4.0) was employed for data collection and analysis.

Cell Viability and NO Inhibitory Activity
To evaluate the NO inhibitory activity of samples at non-cytotoxic concentrations, the cell cytotoxicity of compounds 1-7 was estimated by MTT assay [28] with some modifications.Briefly, RAW 264.7 cells were seeded in 96-well plates (2 × 10 5 cells/ well) and stabilized for 24 h.The attached cell was pre-treated with different concentrations (0-40 µM) of sample or DMSO (blank) for 20 h.For MTT assay, MTT solution (5 mg/mL) was added to the well plate and reacted for 4 h at 37 • C, and then cells were lysed with 100 µL DMSO.The MTT formazan in each well was used to measure the absorbance at 550 nm using a UV/Vis spectrophotometer (BioTek, Power Wave XS, Hampton, NH, USA).The level of NO produced in the LPS-treated RAW 264.7 cells treated with the samples was measured according to previously reported method [29] with some modifications.Briefly, RAW 264.7 cells were seeded in 96-well plates (2 × 10 5 cells/well) and stabilized

Figure 1 .
Figure 1.Activity-guided isolation of seven compounds from the butanol fraction of A. pilosa.

Figure 1 .
Figure 1.Activity-guided isolation of seven compounds from the butanol fraction of A. pilosa.

1 .
General Experimental ProcedureThe UV spectra were obtained from UV spectrometer (Power Wave XS, Bio-Tek In-obtained from UV spectrometer (Power Wave XS, Bio-Tek In-T, USA).Thin-layer chromatography (TLC) was carried out on pre-Quercetin 19.50 ± 1.71 Molecules 2024, 29, x FOR PEER REVIEW 8 of 12

Table 1 .
NO inhibitory activities in LPS-treated RAW 264.7 macrophage cells of the fractions from A. pilosa L.

Table 1 .
NO inhibitory activities in LPS-treated RAW 264.7 macrophage cells of the fractions from A. pilosa L.

Table 3 .
Comparison of NO inhibitory effects on LPS-treated RAW 264.7 cells of compounds (1-7) from A. polisa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.

Table 4 .
The structure-activity relationship of flavonoids from A. pilosa.