Iridoid Glycosides and Coumarin Glycoside Derivatives from the Roots of Nymphoides peltata and Their In Vitro Wound Healing Properties

Nymphoides peltata has been used as a medicinal herb in traditional medicines to treat strangury, polyuria, and swelling. The phytochemical investigation of the MeOH extract of N. peltata roots led to the isolation of three iridoid glycosides and three coumarin glycoside derivatives, which were characterized as menthiafolin (1), threoninosecologanin (2), callicoside C (3), and scopolin (4), as well as two undescribed peltatamarins A (5) and B (6). The chemical structures of the undescribed compounds were determined by analyzing their 1 dimensional (D) and 2D nuclear magnetic resonance (NMR) spectra and using high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS), along with the chemical reaction of acid hydrolysis. The wound healing activities of the isolated compounds 1–6 were evaluated using a HaCaT cell scratch test. Among the isolates, scopolin (4) and peltatamarin A (5) promoted HaCaT cell migration over scratch wounds, and compound 5 was the most effective. Furthermore, compound 5 significantly promoted cell migration without adversely affecting cell proliferation, even when treated at a high dose (100 μM). Our results demonstrate that peltatamarin A (5), isolated from N. peltata roots, has the potential for wound healing effects.


Introduction
The skin is the body ′ s outermost organ and its first line of defense against external factors [1].However, the structural integrity of the skin can be compromised by physical and chemical factors and result in functional impairments [2].Wound healing is a complex process that involves the repair of damaged tissue and typically occurs in four distinct phases: hemostasis, inflammation, proliferation, and remodeling [3,4].In particular, the inflammatory phase is a key determinant of whether the wound healing process is delayed or accelerated [5].During this phase, inflammatory cells such as neutrophils, monocytes, macrophages, T-lymphocytes, and mast cells are recruited to wounds by vasodilation [6].Neutrophils are the first inflammatory cells to arrive at wounds and are responsible for protection from bacterial infection and perpetuating the early inflammatory stage by secreting pro-inflammatory cytokines [7].Simultaneously, monocytes from blood are attracted to wounds and differentiate into macrophages, which produce an array of growth factors and cytokines and facilitate re-epithelialization and angiogenesis [8].During the later inflammatory stage, lymphocytes and mast cells participate in the remodeling phase and accelerate wound healing [9,10].Recently, various studies have been conducted to identify plant secondary metabolites with therapeutic or cosmetic potential with limited side effects compared to commercial drugs that promote skin regeneration [11][12][13].
Menyanthaceae is a family of perennial aquatic plants that consists of about 60 species in six genera [14].The genus Nymphoides is the largest in Menyanthaceae and contains about 55 species [15].Plants of this species are native to temperate and subtropical regions and are endemic in Korea, China, and Japan [16,17].Phytochemical studies on the Nymphoides genus have revealed that they contain polyphenols, flavonoids, triterpenes, and ferulic acid as major compounds, and that their pharmacological activities include anticonvulsant, antioxidant, and anti-diabetic effects [18][19][20][21].The species Nymphoides peltata, also known as the yellow floating heart, is a medicinal herb used in traditional Chinese medicines, such as Ben Cao Gang Mu, and in traditional Indian medicines, such as Ayurveda, and is used to treat heat strangury, polyuria, diuretic, antipyretic, and swelling [18].In a recent pharmacological study, an MTT assay revealed a 95% ethanol extract of N. peltata exhibited significant antitumor activity against prostate cancer (PC3) and osteosarcoma cells (U2OS) [22].Also, our recent studies showed that a 95% ethanol (EtOH) extract of N. peltata root inhibited IL-4 expression in PMA/ionomycin-induced RBL-2H3 cells and had anti-atopic effects in oxazolone-and 2,4-dinitrochlorobenzene (DNCB)-induced mouse models [23].Although many studies have reported the pharmacological activity of N. peltata extract, little research has been conducted on its phytochemical composition at the species level.In this study, we isolated and identified three iridoid glycosides (1-3) and three coumarin glycoside derivatives (4-6), including two new coumarin glycosides (5 and 6) from the MeOH extract of N. peltata root.The chemical structures of the new compounds were determined by analyzing their 1 dimensional (D) and 2D nuclear magnetic resonance (NMR)spectra and using high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS), along with the chemical reaction of acid hydrolysis.Herein, we have described the isolation and structural characterization of the compounds (1)(2)(3)(4)(5)(6) and the evaluation of their potential wound healing effects using an in vitro human keratinocyte scratch model.

Structural Elucidation of the New Compounds
Compound 5 was isolated as a pale brown solid.Its molecular formula was determined as C25H24O12 from the proton-adducted molecular ion at m/z 539.1138 [M + Na] + (calculated for C25H24O12Na, 539.1165) in the positive-ion mode of HR-ESI-MS data (Figure S5).The IR spectrum exhibited absorptions corresponding to hydroxyl (3385 cm −1 ) and carbonyl (1705 cm −1 ) functionalities.The 1 H NMR (Table 1) spectrum of compound 5 exhibited the presence of proton signals attributable to four olefinic double bonds at δH 6.22
Compound 6 was isolated as a pale brown solid.The molecular formula was confirmed to be C 26 H 26 O 12 from the molecular ion peak at m/z 553.1324 [M + Na] + (calculated for C 26 H 26 O 12 Na, 553.1322) using positive-ion HR-ESI-MS (Figure S12).The 1 H and 13 C NMR (Table 1 and Figures S13 and S14) of 6, obtained with the assistance of HSQC (Figure S16) and HMBC spectra (Figure S17), were almost identical to those of 6, except for the chemical shifts of an additional methoxy group at δ H 3.81 (3H, s) and δ C 56.5).The locations of the two methoxy groups in compound 6 were clearly assigned at C-6 and C-3 ′′ by the HMBC experiment, where the HMBC correlations between methoxy group at δ H 3.81 and C-6 (δ C 146.2) and between methoxy group at δ H 3.79 and C-3 ′′ (δ C 148.5) provided critical information for the locations of methoxy groups (Figure 2).Finally, the gross structure of compound 6 was unambiguously confirmed by the analysis of the 1 H-1 H COSY (Figure S15) and HMBC correlations (Figure 2).Therefore, the chemical structure of 6 was elucidated to be 6-methoxy-coumarin-7-O-(6 ′ -O-feruloyl)-β-D-glucopyranoside, as shown in Figure 2, and named peltatamarin B.

Evaluation of Biological Activity of the Isolated Compounds
Keratinocytes are constituents of the epidermis and play a pivotal role in the regeneration of the epidermis during wound healing (hemostasis, inflammation, proliferation, and remodeling) [29,30].The wound healing activities of compounds 1-6 from N. peltata were evaluated using a HaCaT cell scratch test.This in vitro test is useful for evaluating cell migration from wound edges over scratches [31].Treatment with 2-bromo-palmitate (2BP, the negative control) reduced cell migration and increased wound area to 127.31%.Lysophosphatidic acid (LPA), which contributes to epidermal regeneration by modulating cellular responses [32], was used as the positive control and the reduced wound area to 25.32%.Furthermore, our results revealed that HaCaT cells showed increased wound healing in the presence of scopolin (4) (to 37.46%) or peltatamarin A (5) (to 38.67%) groups (Figure 3 and Figure S19).
Keratinocytes are constituents of the epidermis and play a pivotal role in the regeneration of the epidermis during wound healing (hemostasis, inflammation, proliferation, and remodeling) [29,30].The wound healing activities of compounds 1-6 from N. peltata were evaluated using a HaCaT cell scratch test.This in vitro test is useful for evaluating cell migration from wound edges over scratches [31].Treatment with 2-bromo-palmitate (2BP, the negative control) reduced cell migration and increased wound area to 127.31%.Lysophosphatidic acid (LPA), which contributes to epidermal regeneration by modulating cellular responses [32], was used as the positive control and the reduced wound area to 25.32%.Furthermore, our results revealed that HaCaT cells showed increased wound healing in the presence of scopolin (4) (to 37.46%) or peltatamarin A (5) (to 38.67%) groups (Figures 3 and S19).Previous studies have shown that the bioactivities of various coumarins are influenced by the types of substituents at C-6 and/or C-7 [33][34][35].6,7-OH coumarin had better anti-inflammatory and antioxidant activities than 6-OCH3 and 7-OH coumarin [33,34].Previous studies have shown that the bioactivities of various coumarins are influenced by the types of substituents at C-6 and/or C-7 [33][34][35].6,7-OH coumarin had better anti-inflammatory and antioxidant activities than 6-OCH 3 and 7-OH coumarin [33,34].Moreover, a coumarin glycoside with a 6-OH group on the coumarin scaffold had a more potent anti-diabetic effect than the glycoside with a 6-OCH 3 substitution [36].These results improved our understanding of the structure-activity relationships of compound 5 compared to compound 6.In the present study, compound 5 showed a two-fold increase in wound healing versus compound 6.
The effects of compound 5 on HaCaT cell proliferation and migration were also investigated at various concentrations (1, 3, 10, 30, or 100 µM).Interestingly, compound 5 at 1, 3, 10, 30, or 100 µM had no significant effect on cell proliferation as determined by an MTT assay.On the other hand, the wound healing analysis showed treating HaCaT cells with 10 µM LPA (the positive control) reduced the wound area to 24.60% versus the non-treated controls (100%) (Figure 4A).However, treatment with compound 5 at 3, 10, or 30 µM reduced wound areas to about 50%, and at 100 µM, it reduced wound area to 29.17% (Figure 4B,C).Skin damage induces the migration of immune cells to the site of injury, and these cells then secrete inflammatory species and growth factors [29,37].
Furthermore, this process, is facilitated by inflammatory cells and growth factors and plays an important role in wound healing by increasing cell proliferation and restoring damaged tissues [37,38].Therefore, our results suggest that compound 5 has potential use as a wound healing promoter and that it does so by enhancing cell migration but not cell proliferation.
MTT assay.On the other hand, the wound healing analysis showed treating HaCaT cells with 10 µM LPA (the positive control) reduced the wound area to 24.60% versus the nontreated controls (100%) (Figure 4A).However, treatment with compound 5 at 3, 10, or 30 µM reduced wound areas to about 50%, and at 100 µM, it reduced wound area to 29.17% (Figure 4B,C).Skin damage induces the migration of immune cells to the site of injury, and these cells then secrete inflammatory species and growth factors [29,37].Furthermore, this process, is facilitated by inflammatory cells and growth factors and plays an important role in wound healing by increasing cell proliferation and restoring damaged tissues [37,38].Therefore, our results suggest that compound 5 has potential use as a wound healing promoter and that it does so by enhancing cell migration but not cell proliferation.

Plant Material
The root parts of N. peltata were collected in the Hantaek Botanical Garden Foundation, Yongin-si, Gyeonggi-do, Republic of Korea, in June 2021.The plants were authenticated by Dr. Jung Hwa Kang.A voucher specimen (PNU-0040) was deposited in the Medicinal Herb Garden, Pusan National University.

Acid Hydrolysis and Absolute Configuration Determination of Sugar Moieties
The absolute configuration of the sugar moieties was determined using an HPLC-UVbased method [28].Compounds 5 (1.5 mg) and 6 (1.5 mg) were hydrolyzed in the presence of 1N HCl at 80 • C for 2 h, and EtOAc was used for the extraction.The aqueous layer was neutralized with repeated evaporation under a vacuum evaporator and dissolved in anhydrous pyridine (0.5 mL) with the addition of L-cysteine methyl ester hydrochloride (1.0 mg).After the reaction mixture was heated at 60 • C for 1 h, o-tolylisothiocyanate (50 µL) was added and the mixture was kept at 60 • C for 1 h.The reaction product was evaporated under a vacuum evaporator and dissolved in MeOH.Next, the dissolved reaction product was directly analyzed by a LC/MS (MeOH/H 2 O, 1:9 → 7:3 gradient system (0-20 min), 100% MeOH (21-31 min), 0% MeOH (32-42 min); flow rate of 0.3 mL/min) using an analytical Kinetex C 18 100 Å column (100 × 2.1 mm i.d., 5 µm).The sugar moiety of compounds 5 and 6 was identified as β-glucopyranoside, based on the comparison with the standard using LC/MS analysis.

Wound Healing Assay
The wound healing assay was performed as previously described with some modification [31].HaCaT cells were seeded in 24-well plates at a density of 2 × 10 5 cells per well and allowed to attach for one day.Scratches were then created in each well using a 200 µL pipette tip.After washing with PBS, cells were treated with 2-bromo-palmitate (2BP) or lysophosphatidic acid (LPA) as negative and positive controls, respectively, at 10 µM or with compounds 1~6 at 10 µM.In addition, samples were cultured in a growth medium containing 1% FBS and 1% penicillin-streptomycin for 48 h to minimize the effects of growth factors.Wound images were captured immediately after wounding and after 24 or 48 h of culture using a microscope camera HK5.1 CMOS (Koptic, Yongin-si, Republic of Korea).Captured images were quantified using Image J software 1.52a (National Institutes of Health, Bethesda, MD, USA).

MTT Assay
HaCaT cells were seeded in 96-well plates at a density of 2 × 10 4 cells per well in 100 µL.After washing the cells with DPBS (Dulbecco's Phosphate-buffered Saline), cells were treated with 2BP or LPA as negative and positive controls, respectively, at 10 µM or with compound 5 at 1, 3, 10, 30, or 100 µM, and samples were cultured in DMEM containing 1% FBS for 24 h.After removing the supernatant, 100 µL of EZ-cytox assay reagent (Dogenbio, Seoul, Republic of Korea) was added, and the cells were incubated for 30 min.Subsequently, absorbance at 450 nm was measured using the Infinite M1000 microplate reader (Tecan, Mannedorf, Zürich, Switzerland) to assess cell viability.

Statistics Analysis
Data are expressed as the mean ± standard deviation (SD), and analysis was performed using GraphPad Prism software version 5.04 (GraphPad Software Inc., San Diego, CA, USA).The difference between the experimental groups was analyzed through the ANOVA test.Values of p < 0.05 were considered statistically significant.

Figure 3 .
Figure 3.Effect of compounds 1-6 on HaCaT cell wound healing assay results.Scratched HaCaT monolayers were treated with 10 µM of 2BP, LPA, or compounds 1-6, and cell migration was observed under a microscope after 48 h of culture (A).Photographs of a wounded area of compounds 4 and 5 were captured using a 4× objective lens for a microscope camera (scale bar = 100 µm) (B).Each bar is presented as mean ± SD of three independent experiments.# p < 0.05 and ### p < 0.001 vs. CON group; ** p < 0.01 and *** p < 0.001 vs. 2BP group.

Figure 3 .
Figure 3.Effect of compounds 1-6 on HaCaT cell wound healing assay results.Scratched HaCaT monolayers were treated with 10 µM of 2BP, LPA, or compounds 1-6, and cell migration was observed under a microscope after 48 h of culture (A).Photographs of a wounded area of compounds 4 and 5 were captured using a 4× objective lens for a microscope camera (scale bar = 100 µm) (B).Each bar is presented as mean ± SD of three independent experiments.# p < 0.05 and ### p < 0.001 vs. CON group; ** p < 0.01 and *** p < 0.001 vs. 2BP group.

Figure 4 .
Figure 4. Effects of compound 5 on HaCaT cells as determined by MTT and wound healing assays.Scratched HaCaT cell monolayers were treated with 10 µM of 2BP; 10 µM of LPA; or 1, 3, 10, 30, or 100 µM of compound 5 for 48 h.Cell proliferation was evaluated using an MTT assay (A), and cell migration was evaluated using the wound healing assay (B).Photographs of a wounded area were captured using a 4× objective lens for a microscope camera (scale bar = 100 µm) (C).Each bar is presented as mean ± SD of three independent experiments.# p < 0.05 and ### p < 0.001 vs. CON group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 2BP group.

Figure 4 .
Figure 4. Effects of compound 5 on HaCaT cells as determined by MTT and wound healing assays.Scratched HaCaT cell monolayers were treated with 10 µM of 2BP; 10 µM of LPA; or 1, 3, 10, 30, or 100 µM of compound 5 for 48 h.Cell proliferation was evaluated using an MTT assay (A), and cell migration was evaluated using the wound healing assay (B).Photographs of a wounded area were captured using a 4× objective lens for a microscope camera (scale bar = 100 µm) (C).Each bar is presented as mean ± SD of three independent experiments.# p < 0.05 and ### p < 0.001 vs. CON group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 2BP group.