Constituents of Chimaphila japonica and Their Diuretic Activity

Three new phenols (1–3), one new cyclohexanol (4), two known phenols (5–6), and six known flavonoids (7–12) were isolated from the n-butanol of the 75% ethanol extract of all plants of Chimaphila japonica Miq. Among them, compound 5 was named and described in its entirety for the first time, and compounds 9 and 10 were reported in C. japonica for the first time. The structures of all compounds were confirmed using a comprehensive analysis of 1D and 2D NMR and HRESIMS data. Biological results show that compounds 4, 7, and 11 exhibited potent diuretic activity. The modes of interaction between the selected compounds and the target diuretic-related WNK1 kinase were investigated in a preliminary molecular docking study. These results provided insight into the chemodiversity and potential diuretic activities of metabolites in C. japonica.


Introduction
Diuretics play a crucial role in enhancing urine production and facilitating the excretion of water and electrolytes from the body, making them indispensable for the management of a wide spectrum of medical conditions, such as hypertension, congestive heart failure, kidney disorders, and certain edematous states [1,2].Diuretics are classified into different types, such as loop, thiazide, potassium-sparing, and carbonic anhydrase inhibitors, based on their site of action in the kidneys [3].Nevertheless, the utilization of these pharmacological agents is coupled with potential side effects or adverse consequences.Improper or excessive use of diuretics can lead to electrolyte and fluid loss, triggering compensatory mechanisms such as the renin-angiotensin system (RAS), which increases renal sodium retention throughout the nephron [4,5].Therefore, there is an urgent need to develop alternative drugs that are more effective and have fewer side effects.With the growing understanding of the physiology of renal salt, water reabsorption, and their regulation, new possibilities have been spawned for diuretic development.With-No-Lysine kinase 1 (WNK1), a member of the serine/threonine kinase family, was first identified in 2000.It is characterized by the unusual location of lysine in kinase subdomain I, as opposed to subdomain II [6].To date, WNK1 has been found to be involved in a wide range of physiological and pathological processes, particularly in the control of ion transport and electrolyte balance in the kidney [7,8].The diuretic impact of WNK1 inhibitors such as WNK 463 has been verified in vivo, which makes WNK1 kinase the emerging target for screening novel diuretics [9].
Natural products have a long history of being used as medicines to treat a variety of human diseases and are a valuable source of safe and extremely effective diuretics.The discovery and development of novel diuretic agents from natural products represent an attractive avenue [10].The genus Chimaphila is a typical member of the Ericaceae family, which grows naturally in Bhutan, China, Japan, Korea, and Russia; it comprises about five species around the world, of which three species (one of which is endemic) can be found in China.Chimaphila japonica Miq. is a perennial herbaceous plant that has diuretic, astringent, analgesic, and other effects; and it can treat various conditions such as edema, hydrops, etc. [11].At present, little research has been carried out on the chemical composition of the plant; the biological activity is mainly directed towards crude extracts, and the pharmacodynamic material basis is unclear.To date, only a few terpenoids, flavonoids, sterols, quinoids, and phenolic glycosides have been reported [12,13].Therefore, an indepth study of the active ingredients of C. japonica is essential.In our continuing search for potent diuretic agents from medicinal plants, petroleum ether (PE), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH) soluble fractions from the 75% ethanol extract of whole-plant C. japonica were evaluated.Herein, the diuretic bioguided isolation of the active n-BuOH constituents of the soluble fraction, together with the diuretic activity of some of the isolated compounds, is evaluated and the possible diuretic mechanisms of the active compounds are investigated.

In Vitro Cytotoxicity
First, we used the MTT method to examine the cytotoxicity of the compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) on MDCK cells at a concentration of 100 µmol/L, and the results are shown in Table 5.Based on the experimental results, all compounds, with the exception of 2 and 3, showed no or minor toxicity relative to MDCK cells and could be used for subsequent activity testing.

In Vitro Diuretic Activity
Generally, the transport of Na + and Cl − plays an essential role in glomerular filtration and tubular reabsorption.In this study, a Transwell chamber seeded with MDCK cells was used to simulate the renal tubules and investigate the inhibitory effect of compounds (1,(4)(5)(6)(7)(8)(9)(10)(11)(12) on NaCl transport in the renal tubules at 100 µmol/L.As shown in Table 6, all compounds, except compound 10, exhibited highly inhibitory activity on Na + transport (p < 0.0001) compared to the blank group.As for the transport of Cl − , compounds 1, 4, 6-7, and 9-11 exhibited extremely inhibitory activity (p < 0.0001), compound 5 exhibited good inhibitory activity (p < 0.001), and compound 12 has a general inhibitory activity (p < 0.5).The results showed that some compounds exhibited excellent inhibitory activity on Na + transport, particularly 4, 7, and 11, with inhibition rates higher than 20%.On the other hand, compounds 7 and 11 exhibited a strong inhibitory effect on Cl − transport (20.72% and 27.68%), which was significantly higher than or close to the positive control hydrochlorothiazide (23.42%).In order to explore the relationship between the Na + and Cl − transport inhibition activity of potential compounds and time, the inhibitory activities of compounds with inhibition rates that were higher than 20% were further evaluated at 1, 2, and 3 h.It can be observed in the data shown in Figure 3 that the transport inhibition rates of these compounds reached a peak (greater than 20%) during the second hour.The inhibition rate of compound 7 on Na + transport inhibition was more than 30.24% at 1 h, which was better than that of positive control hydrochlorothiazide (27.91%).The suppression rate of Na + transport inhibitory activity in compound 11 exceeds that of hydrochlorothiazide at 3 h, and the suppression rate may be more stable.As shown in Figure 4, the inhibition rates of compounds 7 and 11 on Cl − transport reached a peak (greater than 20%) during the second hour, and the inhibitory rate of compound 11 for Cl − transport inhibition activity exceeded 27.68%, which exceeded the positive control (hydrochlorothiazide, 23.42%).Unfortunately, the stability of these compounds is relatively weak.In short, the preliminary test results demonstrated that compounds 4, 7, and 11 have potential for application in diuretic activity.

Molecular Docking
Based on the results obtained in previous in vitro experiments, compounds 4, 7, and 11, which have better inhibitory activity on Na + and Cl − transports, were selected for molecular docking studies to further explore the diuretic mechanisms of the selected compounds.
WNK1 kinase was employed to evaluate the diuretic effects of selected compounds by docking them into the active site of the WNK1 kinase domain (PDB ID 5DRB) [24].WNK463 was re-docked to the active site to validate docking reliability.The results indicated the binding mode of co-crystallized and re-docked WNK463 was almost the same in the active site of the WNK1 kinase domain in Figure 5A (binding energy: −7.47 kcal/mol).
Compared with WNK463, compound 4 mainly formed two hydrogen bonds with Asp368 and Thr301, which were key for Na + and Cl − transport.In addition, the hydrophobic interaction with Phe356 was essential for the binding of compound 4 and the WNK1 kinase domain, with a binding energy of −6.12 kcal/mol (Figure 5B).In this study, compound 7 formed two hydrogen bonds with Asp368 and Met304, and hydrophobic interactions with Val235, Ala248, and Phe356, in Figure 5C (binding energy: -5.87 kcal/mol).Detailed interaction analyses of compound 11 revealed hydrogen bond interactions with Met304, Val281, and Cys250, and hydrophobic interactions with the Ala248, Thr301, and Val281 of WNK1, with a binding energy of −6.34 kcal/mol (Figure 5D).
It is commonly believed that the lower the binding energy, the stronger the binding force of the two molecules.In addition, if the binding energy is below -5 kcal/mol, the two molecules are considered to be strongly bound [25].Molecular docking results showed that the selected compounds have a certain binding capacity.Thus, it can be observed that the diuresis potential of the active compounds can be realized by inhibiting the activity of the WNK1 kinase domain.Their binding driving forces comprise hydrophobic and hydrogen bond interactions.

Molecular Docking
Based on the results obtained in previous in vitro experiments, compounds 4, 7, and 11, which have better inhibitory activity on Na + and Cl − transports, were selected for molecular docking studies to further explore the diuretic mechanisms of the selected compounds.
WNK1 kinase was employed to evaluate the diuretic effects of selected compounds by docking them into the active site of the WNK1 kinase domain (PDB ID 5DRB) [24].WNK463 was re-docked to the active site to validate docking reliability.The results indicated the binding mode of co-crystallized and re-docked WNK463 was almost the same in the active site of the WNK1 kinase domain in Figure 5A (binding energy: −7.47 kcal/mol).
Compared with WNK463, compound 4 mainly formed two hydrogen bonds with Asp368 and Thr301, which were key for Na + and Cl − transport.In addition, the hydrophobic interaction with Phe356 was essential for the binding of compound 4 and the WNK1 kinase domain, with a binding energy of −6.12 kcal/mol (Figure 5B).In this study, compound 7 formed two hydrogen bonds with Asp368 and Met304, and hydrophobic interactions with Val235, Ala248, and Phe356, in Figure 5C (binding energy: −5.87 kcal/mol).Detailed interaction analyses of compound 11 revealed hydrogen bond interactions with Met304, Val281, and Cys250, and hydrophobic interactions with the Ala248, Thr301, and Val281 of WNK1, with a binding energy of −6.34 kcal/mol (Figure 5D).
It is commonly believed that the lower the binding energy, the stronger the binding force of the two molecules.In addition, if the binding energy is below −5 kcal/mol, the two molecules are considered to be strongly bound [25].Molecular docking results showed that the selected compounds have a certain binding capacity.Thus, it can be observed that the diuresis potential of the active compounds can be realized by inhibiting the activity of the WNK1 kinase domain.Their binding driving forces comprise hydrophobic and hydrogen bond interactions.
All reagents solvents were of reagent grade or purified according to standard methods before use.

Plant Material and Identification
The C. japonica was harvested from the Changbai Mountain area, Jilin Province, China, in July 2018, and it was identified by Prof. Ming-shan Zheng (School of Pharmaceutical Sciences, Yanbian University, China).A voucher specimen (20180705-XDC) was deposited at the Department of Pharmacognosy, School of Pharmaceutical Sciences, Yanbian University, China.

Extraction and Isolation
The air-dried whole plants of C. japonica (3.3 kg) were extracted with 75% ethanol (3 × 40 L) via reflux.The extract was freed from the solvent using a rotavapor to yield 642.7 g of EtOH extract.Part of the crude extract (627.2 g) was suspended with distilled water and successively partitioned into PE (3 × 1.4 L), EtOAc (3 × 1.4 L), and n-BuOH (3 × 1.4 L) sequentially to obtain PE, EtOAc, n-BuOH, and aqueous fractions.

In Vitro Cytotoxicity Assays
Each compound was evaluated for cytotoxicity on DMCK cells using MTT assays.A 96-well plate containing 1 × 10 4 cells per well was seeded with logarithmic growth-phase DMCK cells, which were then cultured for 24 h in a cell culture incubator.After that, the culture medium was changed to one that included medication (100 µmol/L for the treatment groups, 100 µmol/L for the control group, or 100 µmol/L for the positive control group).In total, 100 µL of the appropriate solution was added to each well, and they were then incubated for a further 24 h.After that, culture media were taken out and changed for a DMEM solution that included 20 µL of MTT (5 mg/mL).After incubating for 4 h, the liquid in each well was removed and replaced with 150 µL of DMSO, which was shaken for 10 min to ensure thorough mixing.The absorbance at 490 nm was measured to determine the optical density (OD) of each well.The cell growth inhibition rate was calculated as follows: cell growth inhibition rate (%) = (OD Blank − OD Experimental )/OD Blank × 100%.

In Vitro Diuretic Activity Assay
Log-phase MDCK cells were seeded in the upper chambers of Transwell plates (4 × 10 4 cells/well), and 800 µL of complete culture medium was added to the lower chambers.After 24 h of incubation at 37 °C, the electrical resistance of the upper chamber cells was measured one by one (R = (R cell − R blank ) × 0.04π).When the electrical resistance of the upper chamber cells reached ≥ 300 Ω cm 2 , the upper chamber medium was removed and replaced with the drug solution.The blank group (given normal saline), experimental group (1, 4-12, 100 µmol/L), and hydrochlorothiazide group (200 µmol/L) were set, and each well was treated with 200 µL of the corresponding solution.The cells were then further incubated for 24 h.After that, the upper and lower chamber fluids were removed, and 200 µL of NaCl solution (21 mg/mL) was added to the upper chamber, while 800 µL of DMEM was added to the lower chamber for continued incubation.At 1, 2, and 3 h, 50 µL of the lower chamber fluid was taken, and the OD values were measured using Na + and Cl − detection kits.The transport inhibition rate (%) was calculated as follows: transport inhibition rate (%) = (OD blank − OD experimental )/OD blank × 100%.

Molecular Docking Study
To explore the interaction between different compounds and the WNK1 kinase, the crystal structure of the WNK1 kinase domain in complex with WNK463 (PDB ID 5DRB) was selected to perform molecular docking studies [24].Using the PubChem database (http://pubchem.ncbi.nlm.nih.gov/,accessed on 5 January 2024) for the 2D structure of small molecule ligands, the 2D structures were fed into Chem Office 2022 software to produce their 3D structures.Then, the RCSB PDB database (http://www.rcsb.org/,accessed on 5 January 2024) was used to screen the protein targets, and the crystal structure with high resolution was used as the molecular docking receptor.The PyMOLWin 2.6 software was used to dewater and dephosphate the protein.The Molecular Operating Environment 2019 software was used to minimize the energy of the compounds, pretreat the target proteins, and find the active pockets.Finally, MOE 2019 was run for molecular docking.The results were visualized using PyMOL and Discovery Studio 2019 software.

Statistical Analyses
Statistical analyses were performed by Graphpad Prism 6.0.Data were expressed as mean ± standard deviation (SD) based on at least three independent experiments.And differences between groups were analyzed by one-way analysis of variance and Student's t-test.A p < 0.05 was considered to be statistically significant, p < 0.01 was considered as a significant difference, p < 0.0001 considered as an extremely significant differences.

Conclusions
In summary, to identify compounds with potent diuretic activity, the components of C. japonica were separated, resulting in the identification of 12 compounds (1-12), including three previously undescribed phenols (1-3) and one new cyclohexanol (4).Bioassays demonstrated that compounds 4, 7, and 11 possess potent diuretic activity.The docking study further revealed that the diuresis potential of the active compounds could be realized by the WNK1 kinase domain.Their binding driving forces comprise hydrophobic and hydrogen bond interactions.However, in order to improve the bioavailability of active compounds, further structural modification and mechanism analysis are necessary in the future.

a
Values are the mean ± SD of three replicates.b Hyd (hydrochlorothiazide) was used as the positive control.c * p < 0.05 vs. blank group.d ** p < 0.01 vs. blank group.e **** p < 0.0001 vs. blank group.
Compound 4 was obtained as colorless oil.Its molecular formula was determined as C 13 H 24 O 7 via HR-ESI-MS at m/z 293.1596 [M + H] + (calcd.for 293.1595).The 1 H NMR spectrum (Table

Growth Inhibition Rate (%) a Compound Growth Inhibition Rate (%) a
a Values are the mean ± SD of three replicates.b No inhibition action.c Hyd represents hydrochlorothiazide.
a Values are the mean ± SD of three replicates.b Hyd (hydrochlorothiazide) was used as the positive control.c * p < 0.05 vs. blank group.d ** p < 0.01 vs. blank group.e **** p < 0.0001 vs. blank group.
The suppression rate of Na + transport inhibitory activity in compound 11 exceeds that of hydrochlorothiazide at 3 h, and the suppression rate may be more stable.As shown in Figure4, the inhibition rates of compounds 7 and 11 on Cl − transport reached a peak (greater than 20%) during the second hour, and the inhibitory rate of compound 11 for Cl − transport inhibition activity exceeded 27.68%, which exceeded the positive control (hydrochlorothiazide, 23.42%).Unfortunately, the stability of these compounds is relatively weak.In short, the preliminary test results demonstrated that compounds 4, 7, and 11 have potential for application in diuretic activity.