Dataset on the compounds from the leaves of Vietnamese Machilus thunbergii and their anti-inflammatory activity

Machilus thunbergii has a history of traditional applications including treating dyspepsia, apoplexy, headaches, abdominal pain, abdominal distension, and leg edema [1]. It is also employed for alleviating allergies, inflammation, pain relief, promoting blood circulation, addressing costal chondritis, and sinusitis [2]. Research into the chemical composition of M. thunbergii has revealed the presence of lignans, flavonoids, lactones, and essential oils [1,[3], [4], [5]. While some investigations have explored the inhibitory effects of extracts and lignan compounds from this species on NO production [6], [7], [8], there has been no research into the flavonoids isolated from this plant and their potential for inhibiting NO production, given our reachable referencing. The ethyl acetate (EtOAc) soluble fraction of M. thunbergii leaves was subjected to column chromatography (CC) using silica gel and Sephadex LH-20 for compound isolation. Nuclear magnetic resonance (NMR) data primarily facilitated the determination of isolated compound structures. Anti-inflammatory activity was evaluated against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophage RAW264.7 cells. Anti-inflammatory activity-guided fractionation led to the isolation of twelve secondary metabolites (1−12). The compounds were identified as quercetin (1), kaempferol (2), rhamnetin (3), quercitrin (4), hyperoside (5), reynoutrin (6), guaijaverin (7), afzelin (8), astragalin (9), rutin (10), kaempferol-3-O-rutinoside (11), and rhamnetin-3-O-rutinoside (12). Compounds 3, 5, 6, 9, 11, and 12 were isolated from M. thunbergii for the first time. Evaluation against LPS-induced NO production in macrophage RAW264.7 cells showed that 1−3 exhibited potent inhibitory activity with IC50 values of 15.45, 25.44, and 19.82 µM, respectively. Compounds 4−9 demonstrated IC50 values ranging from 42.15 to 67.42 µM, while 10−12 exhibited inactivity (IC50 > 100 µM).


a b s t r a c t
Machilus thunbergii has a history of traditional applications including treating dyspepsia, apoplexy, headaches, abdominal pain, abdominal distension, and leg edema [1] .It is also employed for alleviating allergies, inflammation, pain relief, promoting blood circulation, addressing costal chondritis, and sinusitis [2] .Research into the chemical composition of M. thunbergii has revealed the presence of lignans, flavonoids, lactones, and essential oils [1 , 3-5] .While some investigations have explored the inhibitory effects of extracts and lignan compounds from this species on NO production [6][7][8] , there has been no research into the flavonoids isolated from this plant and their potential for inhibiting NO production, given our reachable referencing.The ethyl acetate (EtOAc) soluble fraction of M. thunbergii leaves was subjected to column chromatography (CC) using silica gel and Specifications Table Subject Chemistry, Biochemistry Specific subject area Isolating and elucidating the structures of the isolated compounds, along with the assessment of their ability to inhibit NO production Data format Raw, Analyzed Type of data

Table, Figure Data collection
The leaves of M. thunbergii were gathered from Hoa Binh province, Vietnam, in February 2021.Compounds were isolated by using column chromatography (CC), the following materials were utilized: silica gel (Si 60 F254, 40-63 mesh, Merck, St. Louis, MO, USA), YMCGEL (ODS-A, 12 nm S-150 μm, YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (Sigma-Aldrich, MO, USA).The chemical structure of isolated compounds was determined by nuclear magnetic resonance (NMR) data primarily.The cytotoxic assay was conducted by MTS assay [9] .The inhibition of NO production assay was determined using the Griess reaction [10] .Data source location • Institution: Phenikaa University Nano Institute (PHENA), Phenikaa University • This study also presented detailed data for cytotoxic and anti-inflammatory activities.
• The findings imply that M. thunbergii and its natural secondary metabolites could offer anti-inflammatory effects through NO inhibition.

Data Description
The dataset in this article contains full extraction and isolation and NMR data of compounds isolated from M. thunbergii as tabulated in Fig. 1 and Supplementary data file.Furthermore, this article also identified the chemical stucture of isolated compounds from M. thunbergii , Fig. 1 , and Supplementary data file.Effect on cell viability by LPS stimulation in the presence of compounds was determined by MTS assay and expressed as a percentage of the control without the addition of indicated compounds, Fig. 2 .To assess the inhibitory activity on NO production, RAW 264.7 cells were exposed to varying concentrations (1-30 μM) of the isolated compounds, and the level of NO production was determined using the Griess reaction as illustrated in Table 1 .Inhibitory effect of com pounds 1 and 3 on the LPS-induced NO production in RAW264.7 cells.RAW264.7 cells were pre-treated with various concentrations (1, 3, 10, and 30 μM) of the tested compounds for 1 hour, followed by treatment with LPS (1 μg/mL), and then incubated for 24 hours.Control values were obtained in the absence of both LPS and the compounds, Fig. 3 .

Chemical structure identifications
The MeOH extract was partitioned with n -hexane and EtOAc to yield n -hexane and EtOAcsoluble fractions.This fraction was subsequently subjected to CC using silica gel and Sephadex LH-20 to isolate twelve secondary metabolites ( 1 -12 ) ( Fig. 1 and Supplementary data).
Compounds 10 -12 were isolated as yellow amorphous powder.The presence of an olefinic group [ δ C 158.6-159.6 (C-2) and δ C 158.6 135.6-135.8(C-3)], along with a ketone carbon at C-4 ( δ C 158.6 179.5-179.6) in the 1 H-and 13 C-NMR spectra indicated that compounds 10 -12 were flavonols [11] .The 1 H-and 13 C-NMR spectra of compound 10 were similar to those of compound 1 , except for the substitution of the hydroxyl group at C-3 in 1 with a rutinose unit Likewise, the 1 H-and 13 C-NMR spectra of compound 11 were akin to those of compound 2 , but with the replacement of the hydroxyl group at C-3 in compound 2 by a rutinose unit in compound 11 ( Fig. 1 and Supplementary Materials).On the other hand, the 1 H-and 13 C-NMR spectra of compound 12 were also reminiscent of those of compound 3 , except for the substitution of the hydroxyl group at C-3 in compound 3 by a rutinose unit in compound 12 ( Fig. 1 and Supplementary data).Consequently, compounds 10 -12 were identified as rutin ( 10 ) [13] , kaempferol-3-O-rutinoside ( 11 ) [13] , and rhamnetin-3-O-rutinoside ( 12 ) [16] , respectively.This study marks the first identification of kaempferol-3-Orutinoside ( 11) and rhamnetin-3-O-rutinoside ( 12 ) from M. thunbergii as well.

Anti-inflammatory activity
In the initial experiment, a cytotoxic assay was conducted to establish the safe and non-toxic concentrations of the isolated compounds ( 1 -12 ) for the subsequent assays.The non-toxic nature of the isolated compounds was demonstrated by the maintenance of over 90% cell viability as determined by the MTS assay [9] .Notably, the isolated compounds exhibited toxicity to RAW 264.7 cells at a concentration of 100 μM.Consequently, this concentration was excluded from the treatment range, and concentrations ranging from 1 to 30 μM were selected for further investigation, Fig. 2 .
To assess the inhibitory activity on NO production, RAW 264.7 cells were exposed to varying concentrations (1-30 μM) of the isolated compounds, and the level of NO production was determined using the Griess reaction [10] .As illustrated in Table 1 , compounds 1 -3 displayed the most potent inhibitory activity against LPS-induced NO production, showcasing IC 50 values of 15.45, 25.44, and 19.82 μM, respectively.Following this, compounds 4 -9 exhibited IC 50 values ranging from 43.87 to 67.42 μM, respectively, while compounds 10 -12 demonstrated inactivity (IC 50 > 100 μM).As a reference, sappanone A was employed as a positive inhibitor and notably suppressed LPS-induced NO production with an IC 50 value of 8.32 μM.
The control group did not receive either LPS or the samples.Consequently, the observed inhibitory effects of these compounds on NO production were not influenced by any cytotoxic impact.Upon stimulation with LPS (1 μg/mL), the control group exhibited an approximately 11fold increase in NO production after 24 h.In contrast, compounds 1 and 3 demonstrated a dosedependent reduction in NO production 24 h following LPS stimulation, Fig. 3 .
Macrophages, known for their role in inflammatory pathways, release various mediators such as proinflammatory cytokines, hydrolytic enzymes, growth factors, cytotoxic cytokines, and nitric oxide (NO) [17] .The transcription of activated macrophages induces the expression of inducible nitric oxide synthase (iNOS), responsible for generating NO from L-arginine.An excessive output of NO by iNOS can lead to detrimental effects like septic shock and inflammatory disorders.Therefore, assessing NO production induced by lipopolysaccharide (LPS) through iNOS inhibition provides insight into the inflammatory process's modulation [18] .In our study, compound 1 , the flavanonol quercetin, exhibited the strongest inhibition of NO production (IC 50 = 15.45 μM) among the isolated compounds.This effect could be attributed to the presence of 3,4hydroxylation in the benzene ring, similar to previous reports [19 , 20] .Compounds 2 and 3 showed reduced inhibitory activities (IC 50 values of 25.44 and 19.82 μM, respectively), likely due to the absence of 3-hydroxylation (compound 2 ) or the presence of methoxy groups (compound 3 ) in the benzene ring [10 , 20] .Compounds 4 −7 displayed further reduced inhibitory activities (IC 50 values ranging from 42.15 to 46.56 μM) compared to compounds 1 −3 , possibly due to the sugar units (rhamnose, galactose, xylose, and arabinose) despite the presence of 3,4hydroxylation in the benzene ring.Compounds 8 and 9 exhibited even greater reduction in inhibitory activities (IC 50 values of 64.28 and 67.42 μM) due to the combination of sugar units (rhamnose and glucose) and the lack of 3-hydroxylation in the benzene ring.Lastly, compounds 10 −12 were inactive (IC 50 > 100 μM) due to the presence of multiple sugar units (rutinose unit) combined with the absence of 3-hydroxylation (compound 11 ) or the presence of methoxy groups (compound 12 ) in the benzene ring [10 , 20] .

Plant material
The leaves of M. thunbergii were gathered from Hoa Binh province, Vietnam, in February 2021.Botanical identification was expertly conducted by Nguyen Quoc Binh, Ph.D., of the Vietnam National Museum of Nature, Vietnam Academy of Science and Technology (VAST).To ensure traceability, a voucher specimen (MT-L-1610) was meticulously archived at the Natural Product Research and Development Lab, Phenikaa University, Vietnam.

Cell culture
RAW264.7 cells were purchased from the American Type Culture Collection (Manssas, VA, USA) and were maintained in Dulbecco's Modified Essential.These cells were grown at 37 °C in DMEM supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% heatinactivated fetal bovine serum (FBS, Cambrex, Charles City, IA, USA).The cells were maintained in a humidified 5% CO 2 and atmosphere at 37 °C.Cells were counted with a hemocytometer and the number of viable cells was determined by trypan blue dye exclusion.

Determination of NO production
The level of NO production was determined by measuring the amount of nitrite from the cell culture supernatants as described previously [10] .The RAW 264.7 cells were seeded at a density of 1 × 10 5 cells/well in 24 well plates and incubated for 12 h at 37 °C and 5% CO 2 .Then media of each well were aspirated and fresh FBS (Fetal Bovine Serum)-free DMEM (Dulbecco's Modified Eagle Medium) media were replaced.Different concentrations of isolated compounds (1, 3, 10 and 30 μM) were prepared in FBS-free DMEM to give a total volume of 500 μL in each well of a microtiter plate.After 1 h treatment, cells were stimulated with or without 1 μg/ml of LPS for 24 h.The quantity of nitrite in the culture medium was measured as an indicator of NO production.Amounts of nitrite, a stable metabolite of NO, were measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid).Briefly, 100 μL of cell culture medium was mixed with 100 μL of Griess reagent.Subsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader (Biotek, Winooski, VT, USA).Fresh culture medium was used as a blank in every experiment.The quantity of nitrite was determined from a sodium nitrite (NaNO 2 ) standard curve.For this experiment, Sappanone A was used as a positive control.

Statistical analysis
Data are presented as the mean ± standard deviation (SD).Graphs were generated, and statistical analyses were conducted using SigmaPlot 6.0 and SigmaStat 3.1 (Systat Software, San Jose, CA, USA).For the analysis, we employed ANOVA followed by the Tukey's test on prevalidated data.Alternatively, Prism (GraphPad Software, San Diego, CA, USA) was used for comparisons between two groups, utilizing an unpaired Student's t-test.For comparisons involving more than two groups, we used one-way nonparametric ANOVA with Tukey's test followed by Bonferroni post hoc analysis or correlation analysis, as appropriate.Statistical significance levels were set at * * p < 0.05; * p < 0.1.

Limitations
Although our study focused solely on the inhibitory activity of NO production, it did not evaluate other inflammatory agents such as prostaglandin E2 (PGE2) or proinflammatory cy-tokines like ILs, TNF-α, and IFN-γ .Furthermore, we did not investigate inhibitory mechanisms at a molecular level through techniques like western blotting to assess iNOS and cyclooxygenase-2 (COX-2) inhibition, or docking studies due to resource limitations.Despite these limitations, our study yielded positive outcomes, including the isolation of compounds 3, 5, 6, 9, 11, and 12 for the first time and the evaluation of their NO inhibitory activity from Vietnamese M. thunbergii .These novel findings will be the foundation for more comprehensive studies in the future.

Ethics Statement
There were no ethical requirements for the collection and analysis of the data.All software used for the curation and analysis of the dataset were open source.

Fig. 2 .
Fig. 2. Effect on cell viability by LPS stimulation in the presence of compounds 1 −12 .Cell viability was determined by MTS assay and expressed as a percentage of the control without the addition of indicated compounds 1 −12 .

Fig. 3 .
Fig. 3. Inhibitory effect of compounds 1 and 3 on the LPS-induced NO production in RAW264.7 cells.RAW264.7 cells were pre-treated with various concentrations (1, 3, 10, and 30 μM) of the tested compounds for 1 hour, followed by treatment with LPS (1 μg/mL), and then incubated for 24 hours.Control values were obtained in the absence of both LPS and the compound.The blank group was used as 0.1% DMSO-treated cells.Data are presented as the mean ± SD of results from three independent experiments ( * p < 0.01; * * p < 0.05).
a The inhibitory effects are represented as the molar concentration (μM) giving 50% inhibition (IC 50 ) relative to the vehicle control.Values are mean ± S.D ( n = 3); b Positive control.