Sage Journals: Discover world-class research

Abstract

It was reported that 8-hydroxygenistein (8-OHG) was synthesized by methylation, bromination, methoxylation, and demethylation using cheap and readily available biochanin A as raw material. All synthesized products were structurally confirmed by ultra-high-performance liquid chromatography (UHPLC), infrared spectroscopy, mass spectrometry, ¹H-nuclear magnetic resonance (NMR), and ¹³C-NMR. In addition, we examined the antioxidant capacity of 8-OHG using 6 different methods such as 1,1-diphenyl-2-picrylhydrazyl radical scavenging, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS) scavenging, nitric oxide radical (NO) scavenging, superoxide radical (O₂ ^−•) scavenging, reducing power assay, and total antioxidant activity using ascorbic acid (V_C) as a positive control. Compared with V_C, 8-OHG exhibited higher total antioxidant activity and stronger scavenging activity on ABTS, NO, and O₂ ^−•. These results indicate that 8-OHG is an excellent antioxidant agent and may be effective in preventing damage induced by free radical.

Isoflavones are a large class of natural products that almost exclusively exist in Leguminosae plants. Isoflavone compounds are of particular interest due to their broad spectrum of biological activities, including antibacterial, antioxidation, anticancer, anti-inflammatory, and cardiovascular activities, and play an important role in the prevention and treatment of common diseases.¹

Biosynthetically, as the simplest isoflavone of the Leguminosae,² genistein displays a wide range of biological activities, including antidiabetic effect,³ neuroprotective effect,⁴ antioxidant,⁵ anticancer,⁶ and anti-microbial activities.⁷ However, shortcomings still exist, such as low fat solubility and low water solubility,⁸ low bioavailability,⁹ and multiple targets, which obviously confine its clinical and therapeutic application. To acquire drugs that are more pharmacologically active but with less adverse reactions for clinical use, many studies have focused on the synthesis of genistein derivatives.

The genistein derivatives are mainly modified by glycosylation, alkylation, esterification, and hydroxylation.^10,11 Among them, hydroxylation is a common modification of isoflavones occurring in nature. This modification can produce more complex isoflavones which sometimes possess higher bioactivity than their precursors. In structure–activity relationships, the number and positions of hydroxyl groups in the chemical structures significantly affect the functions of the isoflavones, and increasing the number of phenolic hydroxyl groups can increase bioactivity. For example, 3-hydroxygenistein was identified as a potent melanogenesis inhibitor from the biotransformation of genistein by recombinant Pichia pastoris. ^¹²

The hydroxyl group in the molecular structure of isoflavones is the main active group, and increasing the number of hydroxyl groups can increase the antioxidant activity. Due to the fact that polyhydroxy isoflavones are not abundant in nature, it is hard to get enough quantity for biological analysis from natural products. Therefore, it is necessary to prepare biologically active polyhydroxy isoflavones by chemical synthesis.

As shown in Figure 1, 8-hydroxygenistein (8-OHG, 4′,5,7,8-tetrahydoxyisoflavone) is a novel isoflavone derivative with a hydroxyl group at C-8 position of ring A in genistein. Chang et al isolated and purified 8-OHG from soy germ koji by high-performance liquid chromatography (HPLC) method and found that 8-OHG could inhibit both monophenolase and diphenolase activities of tyrosinase.¹³ However, the practical and economical synthetic pathway has not yet been defined.

Figure 1

Structure of 8-hydroxygenistein (8-OHG).

The objective of this study was to synthesize (Figure 2) and evaluate the antioxidant activity of 8-OHG. It was found that biochanin A was selectively methylated using different methylation reagents. We could get 4′,5,7-trimethoxyflavone in high yield (97%) using dimethyl sulfate as methylation reagent, but it was hard to get 4′,5,7-trimethoxyflavone in high yield using excessive iodomethane (CH₃I) as methylation agent and prolonging the reaction time. Compound 1a was confirmed by ¹H-nuclear magnetic resonance (NMR) (supplemental Figure S1, signals for 5- and 7-OCH₃ protons in the ¹H-NMR spectrum appeared at δ 3.93 and 3.88 ppm). Brominated isoflavone was the key intermediate in the synthetic route. N-bromosuccinimide (NBS) was chosen to cleave the C-8 position of 4′,5,7-trimethoxyflavone in 82% yield. Compound 1b was confirmed by ¹H-NMR spectroscopy (supplemental Figure S4, loss of H-6 and H-8 signals, unchanged signals for the spin–spin coupling system of the B ring). The methanolysis of 8-bromo-4′,5,7-trimethoxyflavone promoted by the system MeO^–/copper(I) bromide (CuBr) gave the 4′,5,7,8-tetramethoxyflavone in 75% yield. Compound 1 c was confirmed by ¹H-NMR spectroscopy (supplemental Figure S7, signal for 8-OCH₃ proton in the ¹H-NMR spectrum appeared at 3.83 ppm). 8-OHG was obtained by the demethylation reaction of 1 c with boron tribromide (BBr₃) in anhydrous dichloromethane (CH₂Cl₂) in 80% yield (supplemental Figure S10, loss of OCH₃ proton signals, while signals for OH protons appeared in the range δ 12.81-8.70 ppm).

Figure 2

Synthetic route for 8-hydroxygenistein (8-OHG).

8-OHG was obtained with the total yields of 48% (calculated as biochanin A). The purity of 8-OHG determined by ultra-HPLC (UHPLC) (Figure 3) exceeded 95%. The full process provided a pathway for convergent synthesis of 8-OHG starting from the inexpensive biochanin A.

Figure 3

Ultra-high-performance liquid chromatography spectrum of 8-hydroxygenistein.

In this study, the antioxidant activities of 8-OHG were evaluated using 6 different assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), nitric oxide (NO) and superoxide (O₂ ⁻•) radical scavenging, reducing power capability, and total antioxidant capacity (TAC) assay.

DPPH radical scavenging assay has been widely used to measure the antioxidant activity of natural compounds based on their abilities to reduce radicals. The inhibitory effects of the 8-OHG and V_C on DPPH radical were shown in Figure 4(a) and Table 1. 8-OHG and V_C exhibited dose-dependent scavenging activity on DPPH radical with half-maximal inhibitory concentration (IC₅₀) values of 0.29 mmol/mL (r ²: 0.9930) and 0.27 mmol/mL (r ² : 0.9939), respectively. The 8-OHG exhibited a similar DPPH radical scavenging effect with V_C.

Figure 4

Determination of antioxidant activity of 8-hydroxygenistein (8-OHG). (a) 1,1-Diphenyl-2-picrylhydrazyl (DPPH), radical scavenging assay; (b) 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging assay; (c) nitric oxide (NO) radical scavenging assay; and (d) superoxide radical scavenging assay (mean ± SD, n = 3).

Table 1

Determination of Radical Scavenging Effects of 8-OHG.

Compound	DPPH % inhibition		ABTS % inhibition		NO % inhibition		O₂ ⁻ ^•% inhibition
Compound	IC₅₀	r ²	IC₅₀	r ²	IC₅₀	r ²	IC₅₀	r ²
8-OHG	0.29 ± 0.01	0.9930	0.12 ± 0.01	0.9937	0.14 ± 0.01	0.9874	0.24 ± 0.03	0.9854
V_C	0.27 ± 0.01	0.9939	0.78 ± 0.02	0.9904	1.14 ± 0.01	0.9388	0.69 ± 0.01	0.9905

ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate); DPPH, 1,1-diphenyl-2-picrylhydrazyl; NO, nitric oxide; IC₅₀, half-maximal inhibitory concentration; 8-OHG, 8-hydroxygenistein.

The value of IC₅₀ in the table is represented as mean ± SD (n = 3)

ABTS radical scavenging reﬂects the electron or hydrogen donating ability of antioxidants, and it is broadly used to evaluate the antioxidant activity of natural compounds. The inhibitory effects of 8-OHG and V_C on ABTS radical were shown in Figure 4(b) and Table 1. Both 8-OHG and V_C possessed inhibitory effects on ABTS radical in a dose-dependent manner. The IC₅₀ values of 8-OHG and V_C were 0.12 (r ²: 0.9937) and 0.78 (r ²: 0.9904) mmol/mL, respectively. At the concentration of 0.5 mmol/mL, the scavenging rates of 8-OHG and V_C reached to 81.98% and 42.66%, respectively. 8-OHG showed better inhibitory activity against ABTS radical than V_C.

As an abundant reactive radical, NO acts as an important oxidative biological signaling molecule in many diverse physiological processes, such as neurotransmission, blood pressure regulation, defense mechanisms, smooth muscle relaxation, and immune regulation. Nevertheless, excess NO can lead to inﬂammation and destruction of proteins, lipids, and deoxyribonucleic acid.¹⁴ The results of NO radical scavenging effect of 8-OHG and V_C were given in Figure 4(c) and Table 1. Both 8-OHG and V_C showed dose-dependent NO scavenging activities ranging from 10.29% to 81.44% for 8-OHG and from 18.42% to 51.01% for V_C at concentrations of 0.0625-1 mmol/mL. When the concentration further increased, the NO scavenging rate changed slowly. The calculated IC₅₀ values of 8-OHG and V_C were 0.14 mmol/mL (r ²: 0.9874) and 1.14 mmol/mL (r ²: 0.9388), respectively. 8-OHG exhibited significantly higher scavenging activity on NO than V_C.

The superoxide (O₂ ⁻•), generated by mitochondrial electron transport systems, is the most common free radical in the human body.¹⁵ It is a weak free radical but can create other strong free radicals, such as hydroxyl radical (·OH) and hydrogen peroxide that may result in various diseases.¹⁶ The results of the O₂ ⁻• radical scavenging effect of 8-OHG and V_C at various concentration levels were illustrated in Figure 4(d) and Table 1. The superoxide radical scavenging ability of 8-OHG increased with the increasing concentration. The scavenging rates of 8-OHG and V_C were 87.57% and 67.32%, respectively, at 1.0 mmol/mL. The calculated IC₅₀ values of 8-OHG and V_C were 0.24 mmol/mL (r ²: 0.9854) and 0.69 mmol/mL (r ²: 0.9905), respectively. The results suggested that 8-OHG owned higher O₂ ⁻• scavenging activity than V_C.

The antioxidant activity of 8-OHG was also evaluated using reducing power assay and TAC assay. The reducing capacity reflects the electron donation ability of the compound. As shown in Figure 5(a), the reducing power of 8-OHG and V_C increased and correlated well with the increasing concentration. However, the reduction power of V_C was relatively more pronounced than 8-OHG. The TAC of 8-OHG was assessed spectrophotometrically by the phosphomolybdenum method. Results in Figure 5(b) indicated that 8-OHG showed higher TAC than V_C.

Figure 5

Determination of reducing activity of 8-hydroxygenistein (8-OHG). (a) Fe³⁺ reducing capacity and (b) total antioxidant capacity (mean ± SD, n = 3).

In conclusion, 8-OHG was synthesized in good yield via a simple and effective method using biochanin A as a starting material. It was also found that 8-OHG possessed stronger scavenging capacities on ABTS, NO, and superoxide radicals than that of V_C. Moreover, 8-OHG also exhibited higher TAC than V_C. Structurally, the strong antioxidant activity of 8-OHG was attributed to the presence of 4 hydroxyl groups, 2 of which in ortho position in molecular structure. The results of the present study indicated that 8-OHG was assumed to be useful as antioxidant agents for the treatment of oxidative-related diseases. Further investigation will be performed to identify the antioxidant activity of 8-OHG in vivo.

Experimental

General

Melting points (uncorrected) were obtained using an X-4B micro melting point apparatus. ¹H and ¹³C-NMR spectra were recorded on a Bruker Avance III HD spectrometer at 400 MHz and 100 MHz, respectively. Infrared spectra were obtained on a Bruker ALPHA FT/IR spectrometer. The absorptions were reported on the wavenumber (cm⁻¹) scale, in the range 400-4000 cm⁻¹. High-resolution mass spectra (HRMS) were recorded on the Apex II by means of the Electrospray Ionization (ESI) technique. Low-resolution mass spectra were recorded on Agilent 6460 triple-quadrupole mass spectrometer with ESI. Data are quoted: m/z value (relative abundance). The purity was analyzed by UHPLC using a Thermo instrument with a thermo Acclain-C₁₈ column (100 × 2.1 mm, 2.2 µm, USA).

Reagents and Materials

Biochanin A was purchased from Ci Yuan Biotechnology Co., Ltd. (Shanxi, China), BBr₃, potassium persulfate, sodium nitroprusside, sulfanilamide, naphthyl ethylenediamine hydrochloride, and ammonium molybdate were obtained from Aladdin Industrial Co. DPPH, ABTS disodium salt, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), and trichloroacetic acid (TCA) were purchased from Sigma Chemical Co.

4′,5,7-Trimethoxyflavone (1a)

Commercially available biochanin A (2.84 g, 10 mmol) and potassium carbonate (3.04 g, 22 mmol) were dissolved in acetone (100 mL) and then dimethyl sulfate was added (2.08 mL, 22 mmol) to the solution at room temperature. The mixture was heated to 60°C and stirred for 6 hours (course of reaction monitored by TLC). Then the reaction terminated with ammonium hydroxide (5 mL of 10% solution in water), and acetone was removed under vacuum. The residue was poured into water. The resulting solid was collected, dried under vacuum to give compound 2a as white powders.

Yield: 97%.

MP: 166.5°C-167.9°C (161°C-163°C).¹⁷

IR (KBr): 3096, 2968, 1649, 1610, 1571, 1510, 1462, 1290, 1254, 1239, 1212, 1177, 1155, 1078, 824, 809 cm⁻¹.

¹H-NMR (CDCl₃) δ: 7.75 (1H, s, C2-H), 7.49-7.47 (2H, d, J = 8.8 Hz, C2′, C6′-H), 6.94-6.92 (2H, d, J = 8.8 Hz, C3′, C5′-H), 6.43 (1H, d, J = 2.0 Hz, C6-H), 6.36 (1H, d, J = 2.0 Hz, C8-H), 3.93 (3H, s, CH₃O), 3.88 (3H, s, CH₃O), 3.82 (3H, s, CH₃O) (supplemental Figure S1).

¹³C-NMR (CDCl₃) δ: 175.4 (C-4), 163.8 (C-7), 161.4 (C-5), 159.9 (C-4′), 159.4 (C-9), 149.9 (C-2), 130.3 (C-2′, C6′), 125.9 (C-1′), 124.4 (C-3), 113.7 (C-3′, C5′), 109.9 (C-10), 96.1 (C-6), 92.5 (C-8), 56.3 (CH₃O), 55.7 (CH₃O), 56.3 (CH₃O) (supplemental Figure S2).

ESI-MS (m/z) 313.1 [M + H]⁺ (supplemental Figure S3).

8-Bromo-4′,5,7-Trimethoxyflavone (1b)

NBS (2.14 g, 12 mmol) was added dropwise to a solution of compound 1 (3.12 g, 10 mmol) in dimethylformamide (DMF) (30 mL). Then the reaction mixture was stirred at room temperature for 2.5 hours (course of reaction monitored by TLC). After that, the mixture was poured into cold 2 M hydrochloric acid (HCl) (20 mL). The DMF was removed, and the solids were filtered, washed with water, and then recrystallized from methanol to give compound 1b as white powders.

Yield: 82%.

MP: 202.0°C-204.0°C.

IR (KBr): 2942, 2838, 1648, 1512, 1466, 1397, 1242, 1215, 1089, 823, 810 cm⁻¹.

¹H-NMR (CDCl₃) δ: 7.86 (1H, s, C2-H), 7.48-7.47 (2H, d, J = 7.6 Hz, C2′, C6′-H), 6.95-6.92 (2H, d, J = 8.8 Hz, C3′, C5′-H), 6.40 (1H, s,C6-H), 3.97 (3H, s, CH₃O), 3.96 (3H, s, CH₃O), 3.83 (3H, s, CH₃O) (supplemental Figure S4).

¹³C-NMR (CDCl₃) δ: 175.2 (C-4), 160.9 (C-7), 160.0 (C-5),159.6 (C-4′), 155.4 (C-9), 150.2 (C-2), 130.3 (C-2′, C6′), 125.7 (C-1′), 123.8 (C-3), 113.8 (C-3′, C5′), 110.4 (C-10), 92.2 (C-8), 90.4 (C-6), 56.6 (CH₃O), 56.5 (CH₃O), 55.3 (CH₃O) (supplemental Figure S5).

ESI-MS (m/z) 391.0 [M + H]⁺ (supplemental Figure S6).

4′,5,7,8-Tetramethoxyflavone (1c)

A solution of 25% sodium methoxide in methanol (20 mL, 0.2 moL) was added to the suspension of CuBr (0.7 g, 5 mmol) in DMF (15 mL). The mixture was stirred at room temperature for 1 hour and then added in 1 portion to a solution of 1b (1.49 g, 5 mmol) in DMF (25 mL) at 120°C. After stirring for 40 minutes (course of reaction monitored by TLC), the reaction mixture was poured into ice-water and adjusted to pH 6 using 20% HCl (aq), and extracted by ethyl acetate (20 mL × 3), dried over anhydrous sodium sulfate. The solvent was removed, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate, v/v, 3:1) to give 1 c as white crystals.

Yield: 75%.

MP: 142.7°C-143.5°C.

IR (KBr): 2943, 2838, 1651, 1608, 1575, 1504, 1467, 1313, 1249, 1209, 1176, 1086, 1063, 831, 813 cm⁻¹.

¹H-NMR (CDCl₃) δ: 7.85 (1H, s, C2-H), 7.49-7.47 (2H, d, J = 8.8 Hz, C2′, C6′-H), 6.95-6.93 (2H, d, J = 8.8 Hz, C3′, C5′-H), 6.44 (1H, s, C6-H), 3.99 (3H, s, CH₃O), 3.96 (3H, s, CH₃O), 3.91 (3H, s, CH₃O), 3.83 (3H, s, CH₃O) (supplemental Figure S7).

¹³C-NMR (CDCl₃) δ: 175.5 (C-4), 159.5 (C-4′), 156.9 (C-5), 156.2 (C-7), 152.1 (C-2), 150.1 (C-9), 130.4 (C-2′, C6′), 130.3 (C-8), 125.5 (C-1′), 124.2 (C-3), 113.7 (C-3′, C5′), 109.7 (C-10), 92.7 (C-8), 61.6 (CH₃O), 56.7 (CH₃O), 56.3 (CH₃O), 55.3 (CH₃O) (supplemental Figure S8).

HRMS-ESI: m/z [M + H]⁺ calcd for C₁₉H₁₈O₆: 343.1176; found: 343.1178 (supplemental Figure S9).

8-Hydroxygenistein

1M BBr₃(2 mL) in CH₂Cl₂ was added dropwise to a stirred solution of 1 c (328 mg, 1 mmol) in anhydrous CH₂Cl₂(10 mL) at −15°C. The reaction mixture was stirred at room temperature for another 12 hours (course of reaction monitored by TLC). Then the mixture was quenched with cold water. CH₂Cl₂ was removed under vacuum. The residue was poured into water. The obtained solid was collected, dried under vacuum, and recrystallized from aqueous methanol (2:1) to give compound 1 as yellow crystals.

Yield: 80%.

MP >320°C (330°C).¹⁸

IR (KBr): 3356, 3222, 1669, 1628, 1571, 1537, 1516, 1446, 1379, 1287, 1239, 1015, 823 cm⁻¹.

¹H-NMR (DMSO-d6) δ: 12.39 (1H, s, C5-OH), 10.55 (1H, s, C4′-OH), 9.58 (1H, s, C7-OH), 8.75 (1H, s, C8-OH), 8.37 (1H, s, C2-H), 7.40-7.38 (2H, d, J = 8.6 Hz, C2′, C6′-H), 6.84-6.82 (2H, d, J = 8.0 Hz, C3', C5'-H), 6.30 (1H, s, C6-H) (supplemental Figure S10).

¹³C-NMR (DMSO-d ₆) δ: 181.0 (C-4), 157.8 (C-4′), 154.3 (C-2), 153.9 (C-5), 153.7 (C-7), 146.3 (C-9), 130.7 (C-2′, C6′), 125.3 (C-8), 122.3 (C-1′), 121.9 (C-3), 115.5 (C-3′, C5′), 104.5 (C-10), 99.2 (C-6) (supplemental Figure S11).

ESI-MS (m/z) 287.1 [M + H]⁺ (supplemental Figure S12).

Purity Test

The purity of 8-OHG detected using UHPLC with a thermo Acclain-C18 column (100 × 2.1 mm, 2.2 µm, USA). Isocratic elution was performed using water (H₂O) (A) and methanol (B) with the following gradient combination: 40% B (0-10 min). The column temperature was 25°C. The flow rate was 0.2 mL/min, and 10 µL of the sample was injected. The detection wavelength was 254 nm.

DPPH Radical Scavenging Assay

The DPPH radical scavenging activity was determined according to the Zhang method with some modifications.^19,20 For this, 50 µL of various concentrations (0.0625-2.0 mmol/mL) of samples were added to 150 µL of 0.2 mM DPPH radical solution in ethanol and the resulting mixture incubated for 0.5 hours in the dark at room temperature. The absorbance of the mixture was measured at 517 nm using a microplate reader (Spectramax i3, Molecular Devices). The absorbance of the positive control (ascorbic acid, V_C) and control (DPPH radical without sample) was also measured. The percentage of DPPH radical scavenging activity was calculated using the following equation:

Scavenging activity (%) = (1 - A_{1} ∕ A_{0}) \times 100,

(1)

A ₁ and A ₀ were the absorbance of sample and control, respectively.

Antioxidant activity was expressed as IC₅₀ (mmol/mL) values. Here, IC₅₀ values were referred to as a concentration of a sample with a 50% DPPH radical scavenging rate. IC₅₀ was calculated by the linear equation obtained based on the concentration and inhibition percentage. A lower IC₅₀ value corresponds with higher antioxidant activity. All samples were analyzed in triplicate.

ABTS Radical Scavenging Assay

The ABTS radical scavenging activity was measured based on the following method.²¹ Brieﬂy, ABTS-mixture solution was prepared by mixing an equal amount of 7 mM ABTS and 2.45 mM potassium peroxodisulfate solutions for 16 hours rotation in the dark at room temperature. Thereafter, ABTS-working solution was prepared after dilution of the ABTS-mixture solution (1 mL) in methanol (3.9 mL) before use. The reaction was started by the addition of the ABTS-working solution (200 µL) into varying concentrations of test samples (50 µL) and then allowed to proceed at room temperature for 10 minutes in the dark. The absorbance of the resulting solution was measured at 734 nm in a microplate reader (Spectramax i3, Molecular Devices). V_C was used as a reference standard. The percentage of scavenging was calculated according to Eq. (1).

NO Radical Scavenging Assay

The NO radical scavenging assay was measured by the Griess reaction with some modifications.²² In brief, 50 µL of various concentrations (0.0625-2.0 mmol/mL) of samples were added to 50 µL of sodium nitroprusside (20 mmol/L in phosphate buffer, pH 7.4). The reaction mixture was incubated for 150 minutes under light at room temperature. After incubation, 50 µL of 0.33% (w/v) sulfanilamide (in 20% glacial acetic acid) were added and the whole kept standing for 10 minutes. Then 50 µL of 0.1% (w/v) naphthyl ethylenediamine hydrochloride were added, and the resulting solution was further incubated for 30 minutes. The absorbance was measured at 540 nm in a microplate reader (Spectramax i3, Molecular Devices). V_C was used as a reference standard. The NO radical scavenging activity was calculated according to Eq. (1).

Superoxide Radical Scavenging Assay

The superoxide scavenging activity was determined by the PMS–NADH–NBT system with slight modifications.²³ For this, 50 µL of NBT solution (0.2 mM in distilled water), 50 µL of NADH solution (0.5 mmol/L in 0.1M Tris–HCl, pH 8.0), and 100 µL of samples with different concentrations (0.0625-2.0 mmol/mL) were mixed and treated with 50 µL of PMS solution (25 µM PMS in distilled water). The reaction mixture was incubated at room temperature for 10 minutes, and the absorbance at 570 nm in a microplate reader (Spectramax i3, Molecular Devices) was measured. V_C was used as a positive control. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The percentage of scavenging was calculated according to Equation (1).

Reducing Power Assay

The reducing power assay was performed according to the method of Oztaskin.²⁴ For this, 100 µL of various concentrations (0.03125-0.5 mmol/mL) of samples were mixed with 2.5 mL of 0.2 mol/L sodium phosphate buffer (pH = 6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide. The mixture was incubated for 30 minutes at 50°C and then 2.5 mL of 10% TCA was added. Subsequently, the mixture was centrifuged at 3000 rpm for 10 minutes. The upper layer fraction (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride. The absorbance was measured at 700 nm after 10 minutes (Spectramax i3, Molecular Devices). V_C was used as a positive control. A higher absorbance indicates a higher reducing power. All samples were tested in triplicate.

Phosphomolybdenum Assay (TAC)

TAC of samples was determined by the phosphomolybdate method according to Albayrak.²⁵ For this, 100 µL of various concentrations (0.0625-1.0 mmol/mL) of samples were mixed with 1.0 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The reaction mixture was incubated for 90 minutes at 95°C in a water bath. Then the resulting solution was rapidly cooled to room temperature. The absorbance of the resulting solution was measured at 695 nm. V_C was used as a positive control. A higher absorbance indicates a higher total antioxidant activity. All samples were tested in triplicate.

Statistical Analyses

The experiments were carried out in triplicate, and the results were expressed as a mean ± standard deviation (SD). The half-maximal effective concentration values were calculated via regression analysis. Student’s t-test was used for comparing two groups. Statistical significance was defined as P < 0.05.

Supplemental Material

Supplementary material - Supplemental material for Synthesis, Characterization, and Antioxidant Activity of 8-Hydroxygenistein

Supplemental material, Supplementary material, for Synthesis, Characterization, and Antioxidant Activity of 8-Hydroxygenistein by Jin Shao, Tong Zhao, Hui-Ping Ma, Zheng-Ping Jia and Lin-Lin Jing in Natural Product Communications

Footnotes

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 study was supported by grants from the National Natural Science Foundation of China (Grant No. 81872796, 81202458, 81571847), Natural Science Foundation of Gansu Province (Grant No. 18JR3RA408, 1308RJYA061, 145RJZA089), China Postdoctoral Science Foundation (Grant No. 2015ZXKF09) and The Logistics Research Program of PLA (Grant No. CWH17J010).

ORCID ID

Lin-Lin Jing

References

Chen

. Isoflavones: anti-inflammatory benefit and possible caveats. Nutrients. 2016;8(6):361.doi:10.3390/nu8060361

Kaufman

PB.

Duke

JA.

Brielmann

Boik

Hoyt

. A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: implications for human nutrition and health. J Altern Complement Med. 1997;3(1):7-12.doi:10.1089/acm.1997.3.7

Gilbert

ER.

Pfeiffer

Zhang

Liu

. Genistein ameliorates hyperglycemia in a mouse model of nongenetic type 2 diabetes. Appl Physiol Nutr Metab. 2012;37(3):480-488.doi:10.1139/h2012-005

Pisani

SL.

Neese

SL.

Doerge

DR.

Helferich

WG.

Schantz

SL.

Korol

. Acute genistein treatment mimics the effects of estradiol by enhancing place learning and impairing response learning in young adult female rats. Horm Behav. 2012;62(4):491-499.doi:10.1016/j.yhbeh.2012.08.006

Record

IR.

Dreosti

IE.

McInerney

. The antioxidant activity of genistein in vitro. J Nutr Biochem. 1995;6(9):481-485.doi:10.1016/0955-2863(95)00076-C

Jiang

Fan

Cheng

Liu

. The anticancer activity of genistein is increased in estrogen receptor beta 1-positive breast cancer cells. Onco Targets Ther. 2018;11:8153-8163.doi:10.2147/OTT.S182239

Park

Kim

YI.

Shin

et al. The dietary ingredient, genistein, stimulates cathelicidin antimicrobial peptide expression through a novel S1P-dependent mechanism. J Nutr Biochem. 2014;25(7):734-740.doi:10.1016/j.jnutbio.2014.03.005

Waldmann

Almukainzi

Bou-Chacra

et al. Provisional biopharmaceutical classification of some common herbs used in Western medicine. Mol Pharm. 2012;9(4):815-822.doi:10.1021/mp200162b

Sepehr

Cooke

Robertson

Gilani

. Bioavailability of soy isoflavones in rats Part I: application of accurate methodology for studying the effects of gender and source of isoflavones. Mol Nutr Food Res. 2007;51(7):799-812.doi:10.1002/mnfr.200700083

10.

Shi

D-H.

Yan

Z-Q.

Zhang

L-N.

Wang

Y-R.

Jiang

C-P.

J-H

. A novel 7-O-modified genistein derivative with acetylcholinesterase inhibitory effect, estrogenic activity and neuroprotective effect. Arch Pharm Res. 2012;35(9):1645-1654.doi:10.1007/s12272-012-0916-y

11.

Jesus

AR.

Dias

Matos

et al. Exploiting the therapeutic potential of 8-β-d-glucopyranosylgenistein: synthesis, antidiabetic activity, and molecular interaction with islet amyloid polypeptide and amyloid β-peptide (1-42). J Med Chem. 2014;57(22):9463-9472.doi:10.1021/jm501069h

12.

Ding

H-Y.

Chiang

C-M.

Tzeng

W-M.

Chang

T-S

. Identification of 3′-hydroxygenistein as a potent melanogenesis inhibitor from biotransformation of genistein by recombinant Pichia pastoris. Process Biochem. 2015;50(10):1614-1617.doi:10.1016/j.procbio.2015.06.007

13.

Chang

T-S

. Two potent suicide substrates of mushroom tyrosinase: 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. J Agric Food Chem. 2007;55(5):2010-2015.doi:10.1021/jf063095i

14.

Valko

Leibfritz

Moncol

Cronin

MT.

Mazur

Telser

. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39(1):44-84.doi:10.1016/j.biocel.2006.07.001

15.

Murphy

. How mitochondria produce reactive oxygen species. Biochem J. 2009;417(1):1-13.doi:10.1042/BJ20081386

16.

Brieger

Schiavone

Miller

FJ.

Krause

. Reactive oxygen species: from health to disease. Swiss Med Wkly. 2012;142:w13659.doi:10.4414/smw.2012.13659

17.

Wang

YL.

Huang

Chen

SQ.

Wang

. Synthesis,structure and tyrosinase inhibition of natural phenols derivatives. J Chin Pharm Sci.. 2011;2011(3):235-244.

18.

Farkas

Varady

. Ring isomerization of isoflavones. I. Synthesis of 5,7,8,4'-tetrahydroxyisoflavone and of 7,4'-di-O-methyltectorigenin. Acta Chim Acad Sci Hung. 1960;24:225-230.

19.

Zhang

Tan

Wang

et al. Synthesis, characterization, and the antioxidant activity of N,N,N-trimethyl chitosan salts. Int J Biol Macromol. 2018;118(Pt A):9-14.doi:10.1016/j.ijbiomac.2018.06.018

20.

Bandeira

PT.

Dalmolin

MC.

de Oliveira

et al. Synthesis, antioxidant activity and cytotoxicity of N-functionalized organotellurides. Bioorg Med Chem. 2019;27(2):410-415.doi:10.1016/j.bmc.2018.12.017

21.

Gallardo

Palma-Valdés

Sarriá

et al. Synthesis and antioxidant activity of alkyl nitroderivatives of hydroxytyrosol. Molecules. 2016;21(5):656.doi:10.3390/molecules21050656

22.

Suluvoy

JK.

Berlin Grace

. Phytochemical profile and free radical nitric oxide (NO) scavenging activity of Averrhoa bilimbi L. fruit extract. 3 Biotech. 2017;7(1):85.doi:10.1007/s13205-017-0678-9

23.

Wang

. Synthesis, characterization and antioxidant activity of selenium modified polysaccharides from Hohenbuehelia serotina. Int J Biol Macromol. 2018;120(Pt B):1362-1368.doi:10.1016/j.ijbiomac.2018.09.139

24.

Öztaşkın

Kaya

Maraş

Şahin

Gülcin

İ.

Göksu

. Synthesis and characterization of novel bromophenols: determination of their anticholinergic, antidiabetic and antioxidant activities. Bioorg Chem. 2019;87:91-102.doi:10.1016/j.bioorg.2019.03.010

25.

Albayrak

Aksoy

Sagdic

Hamzaoglu

. Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from turkey. Food Chem. 2010;119(1):114-122.doi:10.1016/j.foodchem.2009.06.003

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.92 MB

0.00 MB

Synthesis,Characterization,and Antioxidant Activity of 8-Hydroxygenistein

Abstract

Experimental

General

Reagents and Materials

4′,5,7-Trimethoxyflavone (1a)

8-Bromo-4′,5,7-Trimethoxyflavone (1b)

4′,5,7,8-Tetramethoxyflavone (1c)

8-Hydroxygenistein

Purity Test

DPPH Radical Scavenging Assay

ABTS Radical Scavenging Assay

NO Radical Scavenging Assay

Superoxide Radical Scavenging Assay

Reducing Power Assay

Phosphomolybdenum Assay (TAC)

Statistical Analyses

Supplemental Material

Supplementary material - Supplemental material for Synthesis, Characterization, and Antioxidant Activity of 8-Hydroxygenistein

Footnotes

Declaration of Conflicting Interests

Funding

ORCID ID

References

Supplementary Material