Preparation method and application of hydroquinone in camphor tree seeds

Abstract

The application discloses a preparation method and application of hydroquinone in camphor tree seeds, and the camphor tree seed kernels are crushed, and then ultrasonic extraction is carried out by adding an ethanol aqueous solution; the obtained extraction liquid is concentrated under reduced pressure by rotary evaporation to obtain camphor tree seed ethanol crude extract; after being suspended by adding an appropriate amount of water, the camphor tree seed ethanol crude extract is extracted by ethyl acetate, and then concentrated under reduced pressure to obtain an ethyl acetate extract; the ethyl acetate extract is eluted by an MCI column chromatography by using a methanol aqueous solution as an eluent, and then the obtained product is eluted by a silica gel column chromatography, and after subsequent treatment, hydroquinone is prepared. The application also provides application of the hydroquinone as a medicine for adjusting protein expression amounts of COX-2, iNOS, NLRP3, P-ERK, P-P38 and P-JNK, and the hydroquinone is helpful to maximize utilization of camphor tree seeds.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction and separation technology, and relates to a method for preparing hydroquinone from camphor tree seeds and its application. Background Technology

[0002] Camphor trees are widely distributed in provinces south of the Yangtze River, possessing excellent landscaping value and significant economic and medicinal value. While the roots, stems, branches, and leaves of camphor trees have long been effectively utilized, camphor seeds, aside from small quantities used for seed production and oil reserves, have remained largely untapped. Their abundant fall to the ground contributes to resource waste and environmental pollution. Further research is urgently needed to investigate the chemical composition of camphor seed oil, hoping to uncover functional molecules and lay the foundation for the in-depth utilization of camphor seed resources, maximizing the use of plant resources. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing hydroquinone from camphor tree seeds and its application. Hydroquinone was extracted from camphor tree seeds, and the application value of hydroquinone was studied.

[0004] This invention discloses a method for preparing hydroquinone from camphor seeds. Camphor seed kernels are pulverized, and extracted with an ethanol-water solution using ultrasound. The resulting extract is then subjected to rotary evaporation and concentrated under reduced pressure to obtain a crude ethanol extract of camphor seeds. This crude extract is then suspended in an appropriate amount of water and extracted with ethyl acetate. The ethyl acetate extract is then concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract is eluted by MCI column chromatography using a 10% (v / v) methanol-water solution as the eluent. The resulting product is then subjected to silica gel column chromatography using dichloromethane-methanol solvents at volume ratios of 10:1, 5:1, and 3:1, resulting in three fractions, Fr.1.1 to Fr.1.2. Fr.1.2 is then processed by preparative high-performance liquid chromatography to obtain hydroquinone.

[0005] Furthermore, the ethyl acetate extract was subjected to MCI column chromatography with gradient elution of 10%, 20%, 30%, 50%, 70%, and 90% methanol aqueous solutions (v / v). The fractions were then divided and combined by thin-layer chromatography to obtain six fractions Fr.1–Fr.6. Fr.1 was then subjected to silica gel column chromatography.

[0006] Furthermore, the volume concentration of the ethanol-water solution is 80%.

[0007] Another aspect of the present invention is a method for preparing hydroquinone from camphor seeds. Camphor seed kernels are pulverized, and an ethanol solution is added for ultrasonic extraction. The resulting extract is then subjected to rotary evaporation and vacuum concentration to obtain a crude ethanol extract of camphor seeds. After suspension with an appropriate amount of water, the extract is extracted with ethyl acetate and concentrated under vacuum to obtain an ethyl acetate extract. The ethyl acetate extract is decolorized, dissolved in an ethanol-water solution, filtered, and then subjected to isocratic elution by MCI column chromatography using a 5% (v / v) methanol-water solution as the eluent. Fifty bottles of elution products are collected, and the products from bottles 20-35 are combined by TLC spotting. Hydroquinone is then obtained by rotary evaporation and vacuum concentration.

[0008] The present invention also provides the application of hydroquinone as a drug for inhibiting NO release from RAW264.7 cells.

[0009] The present invention also provides the use of hydroquinone as a drug for inhibiting the release of TNF-α, IL-6 or IL-1β.

[0010] The present invention also provides the application of hydroquinone as a drug for regulating the protein expression levels of COX-2, iNOS, NLRP3, P-ERK, P-P38, and P-JNK.

[0011] This invention also provides the application of hydroquinone as a drug for inhibiting NF-κB p65 nuclear transport.

[0012] This invention utilizes hydroquinone extracted from camphor tree seeds. Hydroquinone has effects on NO release, TNF-α, IL-6 or IL-1β release, protein expression levels of COX-2, iNOS, NLRP3, P-ERK, P-P38, and P-JNK, and NF-κB p65 nuclear transport, and can be used to prepare related drugs. Attached Figure Description

[0013] Figure 1 The effects of the compound on NO release and cell survival rate;

[0014] Figure 2 The effects of hydroquinone on TNF-α, IL-1β, and IL-6;

[0015] Figure 3 The effects of hydroquinone on the expression of COX2, iNOS, and NLRP3 proteins;

[0016] Figure 4 The effects of hydroquinone on the expression of iNOS, COX-2, NLRP3, IL-1β, IL-6, and TNF mRNA;

[0017] Figure 5 The effects of hydroquinone on the LPS-induced MAPK signaling pathway in RAW264.7 cells at 1, 2, and 6 h were investigated.

[0018] Figure 6 The effect of hydroquinone on NF-κB p65 nuclear transport. Detailed Implementation

[0019] The present invention will be further explained in detail below with reference to the embodiments.

[0020] 1. Experimental Methods

[0021] 1.1 Separation, Extraction and Identification

[0022] Example 1: Take 2.0 kg of thoroughly dried camphor seed kernels, crush them, add 80% ethanol aqueous solution and ultrasonically extract twice (400W, 30 min). The resulting extract is then concentrated under reduced pressure by rotary evaporation to obtain crude camphor seed ethanol extract. After adding an appropriate amount of water to suspend it, it is extracted with ethyl acetate and dichloromethane in sequence. After concentration under reduced pressure, ethyl acetate extract and dichloromethane extract are obtained respectively.

[0023] The ethyl acetate extract (80 g) was subjected to MCI column chromatography with a gradient elution using methanol-water solutions (10%, 20%, 30%, 50%, 70%, and 90% V / v) as eluents. Thin-layer chromatography (TLC) fragmentation and fractionation yielded six fractions, Fr.1–Fr.6. Fr.1 (500 mg) was subjected to silica gel column chromatography with a dichloromethane-methanol solvent system (10:1, 5:1, and 3:1 V / v) to obtain three fractions, Fr.1.1–Fr.1.3. Fr.1.2 (150 mg) was further fractionated by preparative high-performance liquid chromatography (methanol-water solution = 8:92) to obtain compound 1 (90 mg, t). R =45min).

[0024] Compound 1: 1 H-NMR (400MHz, CD3OD)δ C :6.62(12H,s,H-2,3,5,6), 13 C-NMR (125MHz, CDCl3)δ H :151.3 (C-1, 4), 116.8 (C-2, 3, 5, 6). Compound 1 was identified as hydroquinone.

[0025] Example 2: Take 2.0 kg of thoroughly dried camphor seed kernels, crush them, add 80% ethanol (v / v) and extract twice by ultrasonic extraction (400W, 30 min). The resulting extract is then concentrated by rotary evaporation and vacuum to obtain crude camphor seed ethanol extract. After adding an appropriate amount of water to suspend it, it is extracted with ethyl acetate and dichloromethane in sequence. After vacuum concentration, ethyl acetate extract and dichloromethane extract are obtained respectively.

[0026] The ethyl acetate extract (80g) was decolorized, dissolved and filtered in 80% ethanol aqueous solution, and then subjected to isocratic elution by MCI column chromatography with 5% methanol aqueous solution as eluent. The extracts were collected in 150mL Erlenmeyer flasks, and a total of 50 flasks were collected. The samples were spotted by TLC, and 20-35 flasks were combined and concentrated under reduced pressure by rotary evaporation to obtain hydroquinone (500mg).

[0027] 1.2 Determination of NO release by the Griess method

[0028] RAW264.7 macrophages were injected at a rate of 5 × 10⁻⁶ cells per ml. 5 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The experiment included a blank control group, an LPS model group, a control group, and a drug group, with six replicates per group. 50 μL of culture medium was added to the blank control and model groups, and 50 μL of drug-containing culture medium was added to the sample groups. After incubation for 2 h, 50 μL of culture medium was added to the blank control group, and 50 μL of LPS-containing culture medium was added to all other groups. Cells were incubated for another 24 h. 100 μL of the culture supernatant was transferred to an ELISA plate, and an equal volume of Griess reagent was added. After reacting at room temperature for 10 min, the absorbance at 540 nm was measured. The concentration of NO in the cell culture supernatant was calculated based on the NaNO2 standard curve. The final concentrations of the drug and LPS used in the above experiments were 20 μg / mL and 100 ng / mL, respectively.

[0029] 1.3 MTT assay for cell viability

[0030] The plate grafting conditions and model settings are the same as in 1.2. After adding LPS and drugs and incubating for 24 h, add 10 μL of MTT culture medium and incubate in the dark for 4 h. Then aspirate the supernatant, add 150 μL of DMSO to each well and shake on a shaker for 10 min to ensure that the reagents are fully dissolved. Measure the absorbance (A) value of each well at a wavelength of 570 nm using an ELISA reader and calculate the cell viability according to the following formula.

[0031]

[0032] 1.4 Determination of the concentrations of IL-6, IL-1β, TNF-α and PEG2

[0033] The specific procedures for cell seeding, grouping, and drug administration are the same as in 1.2. After adding the drug and LPS, the cells were cultured for 24 hours. The cell supernatant was retained, and the release amounts of TNF-α, IL-1β, IL-6, and PEG2 in the cell supernatant were measured according to the specific procedures of the Mosue TNF-α ELISA kit and the Mosue IL-6 ELISA kit.

[0034] 1.5 Western blot experiment

[0035] RAW 264.7 cells were injected at a rate of 1 × 10⁻⁶ cells per mL. 6 Cells were seeded at a density of 3 mL per well in 6-well plates, divided into a blank control group, a model group, and a drug group. 24 h after seeding, different concentrations of hydroquinone (1.25, 2.5, 5, 10 μg / mL) were added to the drug group and cultured for 2 h. The model group was then cultured for another 24 h with LPS (100 ng / mL). Total protein was extracted using RIPA lysis buffer. Protein concentration was quantified using the BCA method, and after adjusting the concentration, denaturation was performed. A one-step PAGE gel preparation kit was used to prepare the lower and upper gels for gel electrophoresis. The gels were then transferred to polyvinylidene chloride (PVDF) membranes. After blocking with blocking buffer for 30 min, washing three times with TBST, and incubating with the corresponding primary antibody overnight at 4°C, the cells were washed three times with TBST and incubated with the corresponding primary antibody for 1 h at room temperature. ECL staining was performed, and the bands were observed and analyzed using AlphaView and ImageLab software. Simultaneously, using GAPDH as an internal control, the net optical density values ​​of the target band and the internal control band, and their ratio, were analyzed. The results were expressed as the relative expression level of the target protein. Relative expression level of the target protein = Integrated optical density value of the target protein (IOD) / Integrated optical density value of the internal control (IOD).

[0036] 1.6 qRT-PCR assay

[0037] The specific procedures for cell seeding, grouping, and drug administration are the same as in 1.2. After culturing for 24 hours, the supernatant was discarded, and the cells were washed twice with PBS at 4°C. Total RNA was extracted using a total RNA kit. The RNA was reverse transcribed into single-stranded cDNA, and then qRT-PCR was performed using the PCR Easy™-SYBR Green I kit. The qRT-PCR results were analyzed, and the relative expression levels of the target gene were calculated using the 2-ΔΔCt method, with GAPDH as the internal reference gene.

[0038] 1.7 MAPK signal path detection

[0039] The specific procedures for cell seeding, grouping, and drug administration are the same as in 1.2. After culturing cells for 24 hours, they were pretreated with different concentrations of hydroquinone for 2 hours, followed by LPS treatment for 1, 2, and 6 hours, respectively. Subsequent procedures were the same as Western blot, measuring the phosphorylation levels of ERK, P38, and JNK proteins.

[0040] 1.8 Immunofluorescence detection of NF-κB nuclear transport

[0041] The specific procedures for cell seeding, grouping, and drug administration are the same as in 1.2. After culturing cells for 24 hours, pretreatment with different concentrations of hydroquinone for 2 hours was followed by LPS treatment for 6 hours. NF-κB nuclear transport was performed according to the instructions of the nuclear transport detection kit. After aspirating the culture medium, the cells were fixed with fixative for 10 minutes. After aspirating the fixative and washing with washing buffer, blocking buffer was added and the cells were blocked at room temperature for 1 hour. Then, the blocking buffer was aspirated, and NF-κB p65 antibody was added and incubated overnight at 4°C. The primary antibody was recovered and washed again. The cells were then incubated with Cy3-conjugated secondary antibody at room temperature for 1 hour. Finally, after DAPI staining, the cells were observed under a microscope.

[0042] 2 Experimental Results

[0043] 2.1 Effects of compounds on NO and cell viability

[0044] Figure 1 In the middle, compared with the blank group, #### P < 0.0001, compared with the model group, ** P<0.01, **** P<0.0001; by Figure 1 It can be seen that, compared with the blank group, the NO level in the model group was significantly increased after stimulation with 100 ng / mL LPS for 24 h. After intervening in the cells with 20 μg / mL hydroquinone, the results showed that hydroquinone had a highly significant inhibitory effect on the NO release of RAW264.7 cells. At the same time, compared with the model group, the compounds in the drug group had no obvious cytotoxic effect on the cells.

[0045] 2.2 Effects of hydroquinone on inflammatory factors

[0046] Figure 2 In the middle, compared with the blank group, # P<0.050 0, #### P < 0.0001; compared with the model group, * P<0.0500, ** P<0.010 0, *** P<0.001 0, **** P<0.0001; by Figure 2 It was found that the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the supernatant of the blank group were all low. After stimulation with 100 ng / mL LPS for 24 h, the expression levels of anti-inflammatory factors in the model group were significantly higher than those in the blank group. Figure 2 As shown in section a, when the drug concentration is between 1.25 and 5 μg / mL, hydroquinone has no inhibitory effect on TNF-α; however, when the hydroquinone concentration reaches 10 μg / mL, hydroquinone has a highly significant inhibitory effect on the release of TNF-α. Figure 2As shown in b, the drug group exhibited a highly significant inhibitory effect on IL-6 levels compared to the model group, and this effect was dose-dependent. Figure 2 As shown in Figure c, the IL-1β content in the drug group was significantly lower than that in the model group, indicating that hydroquinone exhibits a highly significant inhibitory effect on IL-1β release.

[0047] 2.3 Effects of hydroquinone on the expression of COX-2, iNOS, and NLRP3 proteins

[0048] Figure 3 Compared with the blank group, # P<0.05, compared with the model group, * P<0.05, by Figure 3 It can be seen that after 24 hours of stimulation with 100 ng / mL LPS, the expression levels of COX-2, iNOS, and NLRP3 proteins in the model group were significantly increased compared with the blank group; Figure 3 As shown in b, different concentrations of hydroquinone significantly inhibited COX-2 protein expression in a dose-dependent manner; Figure 3 As shown in Figure c, when the hydroquinone concentration was 1.25 μg / mL, the expression level of iNOS protein increased significantly, and at concentrations of 2.5–10 μg / mL, it had a significant inhibitory effect on iNOS in a dose-dependent manner; Figure 3 As shown in Figure d, hydroquinone at a concentration of 1.25 μg / mL did not significantly inhibit the protein expression of NLRP3. However, at drug concentrations of 2.5–10 μg / mL, the protein expression of NLRP3 decreased significantly in a dose-dependent manner.

[0049] 2.4 Effects of hydroquinone on the mRNA expression levels of iNOS, COX-2, NLRP3, IL-1β, IL-6, and TNF

[0050] Experimental results are as follows Figure 4 As shown, after stimulation with 100 ng / mL LPS for 24 h, compared with the blank group, the expression levels of TNF-α, IL-6, IL-1β, and iNOS mRNA in the LPS model group were significantly increased. Compared with the model group, hydroquinone had no inhibitory effect on the expression level of iNOS mRNA. Figure 4 In the middle (a), the inhibitory effect on the mRNA expression of COX-2 and IL-1β was only significant at concentrations of 5–10 μg / mL. Figure 3 (b, c) while NLRP3 and TNF showed significant inhibitory effects at concentrations of 5–10 μg / mL. Figure 4 In the middle (d, e), IL-6 has an inhibitory effect at concentrations of 2.5–10 μg / mL. Figure 4 (f), and it is dose-dependent.

[0051] 2.5 Effects of hydroquinone on the MAPK signaling pathway

[0052] like Figure 5 As shown in Figures a, b, and c, after 1 hour of LPS stimulation, compared with the control group, the protein expression levels of P-ERK, P-P38, and P-JNK in the model group were increased, indicating that the MAPK signaling pathway had been activated. Compared with the model group, different concentrations of hydroquinone did not inhibit the MAPK signaling pathway. Figure 5 As shown in Figures c, d, and e, after stimulation with 100 ng / mL LPS for 2 h, compared with the model group, the protein expression of P-ERK was significantly inhibited at a hydroquinone concentration of 10 μg / mL, while the protein expression of P-P38 and P-JNK was inhibited at hydroquinone concentrations ranging from 1.25 to 10 μg / mL. Figure 5 As shown in f, j, and h, after stimulation with 100 ng / mL LPS for 6 h, hydroquinone significantly inhibited the protein expression of P-ERK and P-P38 compared with the model group, while the protein expression of P-JNK was only inhibited when hydroquinone was 2.5 μg / mL.

[0053] 2.6 Effects of hydroquinone on NF-κB nuclear transport

[0054] Depend on Figure 6 It can be seen that, compared with the blank group, after LPS stimulation, the expression level of NF-κB p65 protein in the cell nucleus of the model group was significantly increased and nuclear translocation was increased. Compared with the model group, each dose of hydroquinone had a highly significant inhibitory effect on NF-κB p65 nuclear translocation, and the effect was dose-dependent.

[0055] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing hydroquinone from camphor tree seeds, characterized in that, Camphor seed kernels were pulverized and extracted with an ethanol aqueous solution by ultrasonication. The resulting extract was then concentrated under reduced pressure by rotary evaporation to obtain a crude ethanol extract of camphor seeds. After suspension with an appropriate amount of water, the extract was extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was subjected to MCI column chromatography with gradient elution using 10%, 20%, 30%, 50%, 70%, and 90% methanol aqueous solutions (v / v). Thin-layer chromatography was used to divide and combine the fractions to obtain six fractions Fr.1 to Fr.

6. Fr.1 was subjected to silica gel column chromatography with dichloromethane-methanol solvents at v / v ratios of 10:1 and 5:1 to obtain two fractions Fr.1.1 to Fr.1.

2. Fr.1.2 was then prepared by preparative high-performance liquid chromatography to obtain hydroquinone.

2. The method for preparing hydroquinone from camphor seeds according to claim 1, characterized in that, The volume concentration of the ethanol aqueous solution is 80%.

3. A method for preparing hydroquinone from camphor tree seeds, characterized in that, The camphor seed kernels were crushed, added to an ethanol solution and ultrasonically extracted. The resulting extract was then concentrated under reduced pressure by rotary evaporation to obtain a crude ethanol extract of camphor seeds. After being suspended in an appropriate amount of water, it was extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was decolorized, dissolved and filtered in an ethanol-water solution, and then subjected to isocratic elution by MCI column chromatography with a 5% (v / v) methanol-water solution as the eluent. Fifty bottles of eluent were collected, and the products from bottles 20 to 35 were combined by TLC spotting. Hydroquinone was then concentrated under reduced pressure by rotary evaporation.

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