Histone Deacetylase Inhibitors and Antioxidants From the Root of Gluta usitata

Abstract

A new glycoside, glutacoside (1), as well as 6 known compounds was isolated and identified from the root of Gluta usitata. Their structures were determined by Infrared spectroscopy, Mass spectroscopy, and 1-Dimensional and 2-dimensional nuclear magnetic resonance spectroscopy data. The histone deacetylase (HDAC) inhibitory and antioxidant activities of the obtained compounds were evaluated. Molecular docking experiments of the selected compound with representatives of class I (HDAC2 and HDAC8) and class II (HDAC4 and HDAC7) HDAC isoforms displayed potential isoform-selective HDAC inhibitors. Molecular docking data showed consistent results to the in vitro experiments with the highest potency against HDAC8. The docking studies suggested that the phenolic and carbonyl group can be favorably accommodated at the gorge region of the binding site. Furthermore, the phenolic groups also acted as major roles for antioxidant activities.

The genus Gluta (Anacardiaceae) comprises approximately 42 species and is widely distributed throughout the tropical forests of Southeast Asia. Gluta usitata (wall) Ding Hou (previously known as Melanorrhoea usitata), locally known as Namkliang or Thai lac tree, is a flowering plant, medium to large tree with straight clean cylindrical trunks and dark green leaves.¹ Gluta usitata is well known as a major source of lacquer. The lacquer contains various compounds including urushiol, laccol, thitsiol, resorsinol, and phenol derivatives.^2
-4 Some urushiol derivatives have been evaluated as histone deacetylase 8 (HDAC8) inhibitors by molecular docking study.⁵ Recently, we have reported the chemical constituents from the twigs and the flowers of this plant.^6,7 To the best of our knowledge, no compounds from the root of this plant have been reported.

Acetylation and deacetylation of histone protiens play an important role in the epigenetic regulation of transcription in cell. Acetylation is carried out by histone acetyl transferases (HATs) and deacetylation is controlled by histone deacetylases (HDACs). The balance between the action of HDACs and HATs defines the level of acetylation of the histones and the effect involved in the control of the cell cycle progression, differentiation, apoptosis, and tumorigenesis. Therefore, HDACs have been validated as prominent therapeutic target for a broad range of human disorders such as cancers, diabetes, and brain disorders.^8

-11 In humans, HDACs are grouped into 4 classes based on their homology to yeast models, subcellular localization, and enzymatic activities. Eighteen mammalian isoforms of HDACs have been reported.¹² Class I (HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and 10), and class IV (HDAC11) HDACs are zinc-dependent metalloproteins. Class III (SIRT1-7) HDACs are NAD⁺-dependent proteins. Different HDAC isoforms have been associated with different types of diseases and cancers. For example, class I, HDAC1, 2, and 3, has been associated with ovarian, pancreatic, lung, and gastric cancers, whereas HDAC2 has been detected in neurodegenerative diseases as well.^13

-18 Class IIa, HDAC4, has been involved in colon, ovarian, and gastric cancers, however, HDAC7 plays an important role in cardiovascular developments and diseases.^19

-22

In this article, we report the bioactive compounds: a new glycoside, glutacoside (1), 2 known glycosides (2) and (3), and 4 flavonoids (4-7) isolated from the root of G. usitata. The isolation and structural elucidation as well as HDAC inhibitory and antioxidant activities of the isolated compounds are discussed. In order to search for the isoform-selective inhibitors with different HDAC enzymes, molecular docking studies of the most active compound 7 with HDAC2, HDAC8, HDAC4, and HDAC7 isoforms are described. The antioxidant activities of all compounds were also measured in order to explore the synergistic activity.

Experimental

General Experimental Procedures

Column chromatography (CC) was carried out on a silica gel 60 (Merk, Germany). Melting points were measured on a Gallenkamp SANYO melting point apparatus. ¹H-NMR and ¹³C-NMR were recored on a Varian Mercury Plus spectrometer operated at 400 MHz (¹H) or 100 MHz (¹³C). Mass spectrometer was operated using a Micromass Q-TOF 2 hybrid quadrupole time-of-flight. The fluorescence was measured using spectra Max Gemini XPS microplate spectrofluorometer. The UV spectra were obtained using a model Agilent 8453 UV-Vis Spectrometer. Optical rotation was measured on a JASCO DIP-1000 digital polarimeter.

Plant Material

The fresh roots of G. usitata were collected at Ubolratanaphitthayakhom School, Khon Kaen, in November 2014. The plant was independently verified by Dr Boonchuang Boonsuk. A voucher specimen (P. Kumboonma 01) has been deposited in the Herbarium of Khon Kaen University (KKU).

Extraction and Isolation

Air-dried and powdered root of G. usitata (2 kg) were extracted successively with methanol (3 × 10 L, 3 days each time) at room temperature. The combined extracts were concentrated to dryness under vacuum to obtain a methanol extract (105 g). The methanol extract (105 g) was isolated with a normal phase CC using a gradient of dichloromethane-methanol (10:0 to 5:5), to yield 4 fractions (F₁-F₄) based on Thin layer chromatography (TLC) profiles. Fraction F₁ was purified on silica gel column, eluting with an isocratic of dichloromethane-methanol (9:1) to yield compound 3 (105 mg). Fraction F₂ was further subjected to a normal phase silica gel column using a step gradient of dichloromethane-methanol (9:1 to 7:3) to afford compounds 1 (18 mg) and 2 (10 mg). Fraction F₃ was separated on a silica gel column using a gradient of dichloromethane-methanol (9:1 to 7:3) to yield compounds 4 (75 mg) and 5 (50 mg). Fraction F₄ was further chromatographed on a silica gel column, eluting with a gradient of dichloromethane-methanol (9:1 to 0:10) to afford compounds 6 (95 mg) and 7 (85 mg).

Glutacoside (1)

White powder; mp 142°C to 143°C, $[α]_{D}^{24.6}$ [α]_D ^24.6 -238 (c 0.1, MeOH); HRMS-ESI m/z (m/z 515.1555 [M+Na]⁺) (calcd for C₂₄H₂₈O₁₁Na, 515.1529) (supplemental figure S1), UV (MeOH) λ_max 225 (ԑ 65900), 311 (ԑ 70800); IR (neat) υ _max 3356, 1693, 1631, 1602, 1506 cm⁻¹ (supplemental figure S9); ¹H NMR, ¹³C NMR (CD₃OD/CDCl₃), Distortionless enhancement with polarization transfer (DEPT), Correlation spectroscopy, Nuclear overhauser effect spectroscopy, Heteronuclear multiple-quantum correlation spectroscopy, and Heteronuclear multiple-bond correlation spectroscopy (HMBC) (see supplemental figures S2-S8, respectively).

3,4,5-Trimethoxyphenol-1-O-β-d-Glucopyranoside (2)

White amorphous solid; mp 199°C to 202°C, $[α]_{D}^{24.2}$ [α]_D ^24.2 -64 (c = 0.25, MeOH); HRMS-ESI m/z (m/z 369.1155 [M+Na]⁺) (calcd for C₁₅H₂₂O₉Na, 369.1162) (supplemental figure S10), UV (MeOH) λ_max 216 (ԑ 58700), 262 (ԑ 61200); IR (neat) υ _max 3357, 1644, 1505, 1452, 1413, 1225, 1126, 1075, 1027 cm^-1(supplemental figure S13); ¹H NMR and ¹³C NMR (CD₃OD/CDCl₃) (see supplemental figures S11 and S12).

Acid Hydrolysis of 1 and 2

Acid hydrolysis was performed by heating a solution of compounds 1 and 2 (each 2 mg) in 2 M HCl (2 mL) in a sealed tube at 80°C for 3 hours. The reaction mixtures were extracted with EtOAc (5 mL, 3 times) to separate EtOAc soluble fractions and aqueous fractions. The aqueous fractions were further evaporated to obtain β-d-glucose, $[α]_{D}^{24}$ +30.2 (c = 0.2, MeOH). Identification of sugars was further supported by co-TLC with authentic samples.

β-Sitosterol-3-O-β-d-Glucoside (3)

Light-yellow amorphous solid; IR (neat) υ _max 3369, 2957, 2930, 2867, 1461, 1367, 1068, 1020 cm⁻¹ (supplemental figure S16); ¹H-NMR (400 Hz, CD₃OD): δ (ppm) = 5.36 (bs, 1H, H-6), 4.85 (dd, J = 11.7, 2.5 Hz, 1H, H-6ʹ), 4.84 (dd, J = 11.7, 5.2 Hz, 1H, H-6ʹ), 4.40 (t, J = 4.5 Hz, OH, H-5ʹ), 4.20 (d, J = 7.9 Hz, 1H, H-1ʹ), 3.58 (d, J = 4.5 Hz, OH, H-2′), 3.56 (d, J = 4.5 Hz, OH, H-3′), 3.40 (d, J = 4.5 Hz, OH, H-4′), 3.10 (m, 1H, H-3′), 3.06 (m, 1H, H-5′), 3.00 (m, 1H, H-4′), 2.98 (m, 1H, H-3), 2.89 (m, 1H, H-2′), 2.26 to 2.21 (m, 4H, H-2 and H-4), 0.99 (s, 3H, H-19), 0.89 (d, J = 6.3 Hz, 3H, H-21), 0.84 (t, J = 7.6 Hz, 3H, H-29), 0.81 (d, J = 6.9 Hz, 6H, H-26 and 27), 0.68 (s, 3H, H-18) (supplemental figure S14). ¹³C-NMR (100 Hz, CD₃OD): δ (ppm) = 140.9 (C-5), 121.6 (C-6), 101.3 (C-1′), 77.5 (C-3′), 77.2 (C-3), 77.2 (C-5′), 73.9 (C-2′), 70.5 (C-4′), 61.5 (C-6′) 56.7 (C-14), 55.9 (C-17), 50.1 (C-9), 45.7 (C-24), 42.3 (C-13), 38.8 (C-4), 37.3 (C-12), 36.7 (C-1), 36.7 (C-10), 36.0 (C-20), 33.8 (C-22), 31.9 (C-7), 31.9 (C-8), 29.7 (C-2), 29.2 (C-25), 28.3 (C-16), 26.0 (C-23), 24.3 (C-15), 23.1 (C-28), 21.1 (C-11), 20.1 (C-26), 19.5 (C-19), 19.4 (C-27), 19.1 (C-21), 12.2 (C-29), 12.1 (C-18) (supplemental figure S15).

Fisetin (7)

White-yellow powder; IR (neat) υ _max 3358, 3235, 1727, 1600, 1459, 1378, 1252, 1163, 1105, 1012 cm⁻¹ (supplemental figure S19); ¹H-NMR (400 Hz, CD₃OD): δ (ppm) = 7.97 (d, J = 8.8 Hz, 1H, H-5), 7.77 (d, J = 2.0 Hz, 1H, H-8), 7.65 (dd, J = 8.8, 2.0 Hz, 1H, H-6), 6.92 (m, 1H, H-2′), 6.90 (m, 1H, H-6′), 6.86 (m, 1H, H-5′) (supplemental figure S17). ¹³C-NMR (100 Hz, CD₃OD): δ (ppm) = 173.0 (C-4), 162.8 (C-7), 157.1 (C-8a), 147.3 (C-2), 146.1 (C-4′), 144.8 (C-3′), 137.1 (C-3), 126.1 (C-5), 123.0 (C-1′), 120.2 (C-6′), 114.8 (C-5′), 114.6 (C-2′), 114.5 (C-6), 114.1 (C-4a), 101.6 (C-8) (supplemental figure S18).

The structure elucidation of (−)-eriodictyol (4), (−)-dihydroquercetin (5), and (−)-fustin (6) were determined by Infrared spectroscopy (IR), NMR, and electronic circular dichroism analysis as described in the previous report.⁶

Histone Deacetylase Activity Assay

The isolated compounds were evaluated for their ability to inhibit HDAC enzymes. Inhibition of HDAC activity in vitro was measured using the Fluor-de-Lys HDAC activity assay kit. Trichostatin A was used as the positive control. A 5 µL of each sample (in Dimethyl sulfoxide), 1 µL of the Hela nuclear extract, and 19 µL of buffer were added into the 96-well plate and incubated at 37°C for 5 minutes. Next, the substrate (25 µL) was added and incubated again at 37°C for 15 minutes and then 50 µL of the developer was added to generate a fluorophore. Finally, the fluorescence signals of the fluorophores with excitation at 360 nm and emission at 460 nm were detected. A decrease of fluorescence signal indicated an inhibition of HDAC activity.

Antioxidant Assay

The antioxidant activities of all compounds were measured using 2,2-diphenylpicrylhydrazyl (DPPH) free radical. Gallic acid was used as a positive control. Five different concentrations of each antioxidant were tested. The DPPH solution in methanol (1 × 10^–4 M) 3 mL was added to each sample in methanol. The final concentration of DPPH solution was 0.6 × 10^–4 M. Incubation of the reaction mixtures was done in the dark at room temperature for 30 minutes. The absorbances of the solutions were measured at 517 nm. From the absorbance values monitored, the percentage inhibition was calculated and the result is expressed in terms of IC₅₀. The percentage of DPPH inhibition was calculated as

$(%) i n h i b i t i o n = [(A b s_{c o n t r o l} - A b s_{s a m p l e}) ∕ A b s_{c o n t r o l}] \times 100.$

Molecular Docking Studies

The crystal structures of HDACs were obtained from the Protein Data Bank [PDB entry code: 3MAX (HDAC2), 2VQW (HDAC4), 3C0Z (HDAC7), and 1T64 (HDAC8)]. All water and noninteracting ions as well as ligands were removed. Then, all missing hydrogens and sidechain atoms were added using the ADT program. Gasteiger charges were calculated for the system. For ligand setup, the ligands were drawn in Gauss View 03 program. Energy of the molecules was minimized with the AM1 level using Gaussian program. Docking software AutoDock 4.2 Program supplied with AutoGrid 4.0 and AutoDock 4.2 was used to produce grid maps. The spacing between grid points was 0.375 Å. The Lamarckian Genetic Algorithm was chosen to search for the best conformers. Molecular docking studies were operated for 50 runs.

Results and Discussion

Structure Elucidation

Chromatographic separation of the methanol extract of the dried G. usitata root yielded a new glycoside, glutacoside (1), and 6 known compounds (2-7) (Figure 1). These compounds were identified on the basis of spectroscopic methods and comparison of the data with the literature values as 3,4,5-trimethoxyphenol-1-O-β-d-glucopyranoside (2),²³ β-sitosterol-3-O-β-d-glucoside (3),²⁴ (−)-eriodictyol (4),²⁵ (−)-dihydroquercetin (5),²⁶ (−)-fustin (6),²⁷ and fisetin (7).²⁸ Compounds 4 to 6 were previously isolated from the twigs of this plant.⁶

Figure 1

Structures of isolated compounds from the root of Gluta usitata.

Glutacoside (1) was isolated as a white powder with melting points of 142°C to 143°C. Its molecular formula was designated as C₂₄H₂₈O₁₁ (m/z 515.1555 [M+Na]⁺) based on High-resolution mass spectrometry-Electrospray ionization (HRMS-ESI) data. The UV spectrum showed absorption maxima due to a conjugated ester at 225 and 311 nm. The IR spectrum showed absorption bands at 3356, 1693, 1631, 1602, and 1506 cm⁻¹, indicating the presence of hydroxy group, conjugated ester carbonyl, double bond, and aromatic ring, respectively. The ¹H NMR spectrum (Table 1) showed the characteristic signals of a p-coumaroyl unit [δ_H 7.58 (d, J = 16.0 Hz, 1H) and 6.21 (d, J = 16.0 Hz, 1H) and δ_H 7.35 (d, J = 8.8 Hz, 2H), and 6.76 (d, J = 8.8 Hz, 2H)], 2 symmetrical aromatic protons of a 1,3,4,5-tetrasubstituted aromatic ring (δ_H 6.41, s, 2H), 3 methoxy groups [δ_H 3.77 (s, 6H) and 3.69 (s, 3H)], and a sugar moiety [δ_H 4.81 (d, J = 8.0 Hz, 1H), 4.58 (dd, J = 12.0, 4.0 Hz, 1H), 4.31 (dd, J = 12.0, 4.0 Hz, 1H), 3.68 (m, 1H), 3.50 (dd, J = 8.0, 3.5 Hz, 2H), and 3.40 (m, 1H)]. The ¹³C NMR and DEPT spectra indicated 19 signals for 24 carbons (3CH₃, 1CH₂, 13CH, and 7C), including an ester moiety (δ_C 168.0), a p-substituted aromatic carbons [δ_C 161.8, 130.0 (2C), 124.6, 116.3 (2C)], 2 olefinic carbons (δ_C 146.1 and 112.5), 2 symmetrical methine carbons, and 3 quaternary carbons of the 1,3,4,5-tetrasubstituted aromatic ring carbons [δ_C 154.3, 153.3 (2C), 132.4, 95.3 (2C)], 3 methoxy carbons [δ_C 60.4 and 55.6 (2C)], and 5 oxymethine carbons and an oxymethylene carbon of the sugar unit carbons [δ_C 101.8, 76.3, 74.4, 73.3, 70.3, and 63.7] (Table 1). The sugar unit was assigned as β-d-glucose by the acid hydrolysis. The HMBC data (Figure 2) showed the anomeric proton of the sugar unit at H-1′ (δ_H 4.81) correlating with C-1 (δ_C 154.3), suggesting that C-1′ of the sugar unit was linked to C-1 of the 3,4,5-trimethoxyphenyl ring. The methoxy groups located at 3, 4, and 5 positions of the phenyl ring were confirmed by comparison with the reported literature values.^29,30 The p-coumaroyl moiety was linked with the sugar unit at C-6′ (δ_C 63.7) according to the HMBC correlation between H-6′ (δ_H 4.58 and 4.31) and C-9′′ (δ_C 168.0). Therefore, glutacoside¹ was identified as 3,4,5-trimethoxybenzene-1-O-β-d-(6′-O-p-coumaroyl) glucoside.

Figure 2

Key correlations observed in the 2D NMR spectra of compound 1.

Table 1

NMR Data of Glutacoside (1) and 3,4,5-Trimethoxyphenol-1-O-β-d-Glucopyranoside (2).

Position	Compound 1		Compound 2
Position	δ _H ^a, mult., J in Hz	δ _c ^b, type	δ _H ^a, mult., J in Hz	δ _c ^b, type
1		154.3, C		154.4, C
2, 6	6.41 (s, 2H)	95.3, CH	6.45 (s, 2H)	94.9, CH
3, 5		153.3, C		153.3, C
4		132.4, C		133.2, C
3-, 5-OCH₃	3.77 (s, 6H)	55.6, CH₃	3.80 (s, 6H)	55.6, CH₃
4-OCH₃	3.69 (s, 3H)	60.4, CH₃	3.70 (s, 3H)	60.4, CH₃
1′	4.81 (d, 8.0, 1H)	101.8, CH	4.80 (d, 7.9, 1H)	101.8, CH
2′	3.50 (dd, 8.0, 3.5, 1H)	76.3, CH	3.90 (m, 1H)	76.8, CH
3′	3.40 (m, 1H)	70.3, CH	3.80 (m, 1H)	70.2, CH
4′	3.50 (dd, 8.0, 3.5, 1H)	73.3, CH	3.45 (m, 1H)	73.4, CH
5′	3.68 (m, 1H)	74.4, CH	3.42 (m, 1H)	76.6, CH
6′	4.58 (dd, 12.0, 4.0, 1H) 4.31 (dd, 12.0, 4.0, 1H)	63.7, CH₂	3.40 (m, 2H)	61.5, CH₂
	4.58 (dd, 12.0, 4.0, 1H) 4.31 (dd, 12.0, 4.0, 1H)	63.7, CH₂
1′′		124.6, C
2′′, 6′′	7.35 (d, 8.8, 2H)	130.0, CH
3′′, 5′′	6.76 (d, 8.8, 2H)	116.3, CH
4′′		161.8, C
7′′	7.58 (d, 16.0, 1H)	146.1, CH
8′′	6.21 (d, 16.0, 1H)	112.5, CH
9′′		168.0, C

^aRecorded at 400 MHz in CD₃OD/CDCl₃.

^bRecorded at 100 MHz in CD₃OD/CDCl₃.

Histone Deacetylase Inhibitory Activity

Histone deacetylase inhibitory activities of all compounds were initially tested at 1 and 100 µM by a fluorimetric assay (Fluor de Lyse) (see supplemental tables S1 and S2). The results are shown in Table 2. All compounds demonstrated weak HDAC inhibitory activities at 1 µM. However, most compounds showed high percentage inhibitions against HDAC at 100 µM. Glycosides (1-3) displayed moderate percentage inhibitions at 100 µM. Whereas, flavonoids (4-6) showed good percentage inhibitions against HDAC at 100 µM according to the previous report.⁶ Fisetin (7), the newly isolated compound from the root of G. usitata, was the most potent HDAC inhibitor among the tested compounds. Therefore, it was further subjected into molecular docking studies.

Table 2

Histone Deacetylase Inhibitory Activity of the Compounds.

Compound	% HDAC inhibitory activity
Compound	At 1 µM	At 100 µM
1	3.7 ± 0.2	64.6 ± 0.6
2	5.1 ± 0.3	59.4 ± 0.6
3	1.7 ± 0.1	63.3 ± 0.8
4 ^a	17.3 ± 0.4	85.3 ± 0.7
5 ^a	13.1 ± 0.8	80.8 ± 0.8
6 ^a	12.3 ± 0.6	74.1 ± 0.6
7	18.2 ± 0.7	88.3 ± 0.8
TSA ^b	-	96.1 ± 1.4

HDAC, histone deacetylase; TSA, trichostatin A.

^aKumboonma et al.⁶

^bPositive control at 25 µM.

Molecular Docking Studies

To explore a possibility of being HDAC isoform-selective inhibitor, the most active in vitro compound 7 was docked into the catalytic pockets of representative isoforms of class I (HDAC2 and HDAC8) and class II (HDAC4 and HDAC7). The free energy of binding (∆G) and the calculated inhibition constant (K _i) for each enzyme-inhibitor complex are shown in Table 3. Compound 7 showed moderate HDAC inhibitions against HDAC4 (∆G = −5.74, K _i = 61.87 µM) and HDAC7 (∆G = −6.15, K _i = 30.80 µM). Interestingly, compound 7 showed good HDAC inhibitions against HDAC2 (∆G = −6.76, K _i = 11.02 µM) and HDAC8 (∆G = −8.08, K _i = 1.20 µM). The fisetin 7-HDAC8 complex presented a zinc chelation via the hydroxy group with a distance of 2.48 Å and 3 hydrogen bonds as demonstrated in Figure 3. Two hydrogen bonds occurred between the hydroxy groups of 7 and the carbonyl group of Asp101 (2.21 Å) and the amino group of His180 (2.04 Å). Another hydrogen bond occurred between the carbonyl group of 7 and Phe208 (2.94 Å). The results confirmed that the hydroxy group and carbonyl group played important roles through the hydrogen bond and coordination to the zinc ion. Overexpression of HDAC8 plays a significant role in some of the adult tissue cancers like colon, breast, lung, pancreas, liver, and childhood neuroblastoma.^31,32 Therefore, this compound could be potential lead compound for safe and selective anticancer agents with HDAC inhibitory activity.

Figure 3

The interaction mode of 7 in the active site of histone deacetylase 8.

Table 3

The I n Silico Histone Deacetylase Inhibitory Activity of Compound 7.

Compound	Class I HDACs				Class II HDACs
	HDAC2		HDAC8		HDAC4		HDAC7
	∆G (kcal/mol)	K _i (µM)	∆G (kcal/mol)	K _i (µM)	∆G (kcal/mol)	K _i (µM)	∆G (kcal/mol)	K _i (µM)
7	−6.76	11.02	−8.08	1.20	−5.74	61.87	−6.15	30.80

HDAC, histone deacetylase.

Antioxidant Studies

The antioxidant activities of the isolated compounds were evaluated by DPPH method. The DPPH assay is expressed in terms of IC₅₀ values and the results are demonstrated in Table 4. Glycosides (1-3) showed weak activity against DPPH. Whereas, flavonoids (4-7) demonstrated good activities. Dihydroquercetin (5) displayed the most effective antioxidant activity among the isolated compounds. The results revealed that the introduction of the hydroxy group at the C-3 position increased the oxidation abilities as compared to eriodictyol (4). Fisetin (7) showed better antioxidant property than fustin (6). These results indicated that the presence of the double bond at C-2 and C-3 was essential for the activity.³³ Furthermore, fisetin (7) possessed the antioxidant potentials superior to eriodictyol (4). The results revealed that the introduction of the hydroxy group at the C-3 position have more effective antioxidant than the hydroxyl group at the C-5 position.

Table 4

The I n Vitro Antioxidant Activity of the Compounds.

Compound	IC₅₀ (µM)
1	>100
2	>100
3	>100
4	6.3 ± 0.5
5	0.9 ± 0.1
6	7.6 ± 0.7
7	1.7 ± 0.3
Gallic acid	17.9 ± 1.3

Conclusion

A new glycoside, glutacoside (1), along with 2 glycosides (2), (3) and 4 flavonoids (4-7) was isolated from the root of G. usitata. The HDAC inhibitory and antioxidant activities of the isolated compounds were evaluated. Eriodictyol (4), dihydroquercetin (5), fustin (6), and fisetin (7) acted as good HDAC inhibitors and antioxidants. Molecular docking data showed that the phenolic group and the carbonyl group played major roles in HDAC-inhibitor binding through the zinc ion-coordination and the hydrogen bonds. The results demonstrated that G. usitata can be utilized as the excellent source of bioactive compounds.

Supplemental Material

Supplementary material - Supplemental material for Histone Deacetylase Inhibitors and Antioxidants From the Root of Gluta usitata

Supplemental material, Supplementary material, for Histone Deacetylase Inhibitors and Antioxidants From the Root of Gluta usitata by Pakit Kumboonma, Somprasong Saenglee, Thanaset Senawong and Chanokbhorn Phaosiri in Natural Product Communications

Footnotes

Acknowledgments

This work was supported by Rajamangala University of Technology Isan (RMUTI). We also would like to thank Mr. Kittisak Poopasith for the excellent NMR data.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is funded by Rajamangala University of Technology Isan (RMUTI).

ORCID ID

Pakit Kumboonma

Chanokbhorn Phaosiri

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