Furanocoumarin enantiomers in fig fruits and methods and uses thereof

Abstract

The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L Ficus carica The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried fruit of fig (L The present application relates to a kind of furan coumarins enantiomers in fig fruit and its preparation method and purposes, the method with the dried

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to two new furanocoumarins enantiomers and their use in the preparation of anti-vitiligo drugs. BACKGROUND

[0002] Vitiligo is a chronic acquired skin pigmentation disorder that affects 1-4% of the global population, including adults and children. It is characterized by selective destruction of melanocytes in the skin and hair, mainly manifested as the expansion of skin pigmentation lesions. Although the exact pathogenesis is not clear, the underlying causes include genetics, immunity, oxidative stress, production of inflammatory mediators, etc. According to the regulatory network of melanin formation, the activity of tyrosine (TYR), tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) genes is up-regulated, which ultimately increases melanin formation.

[0003] Furanocoumarins are a class of secondary metabolites in higher plants, which are a general term for compounds and their derivatives formed by condensation of isopentenyl group at 6 or 8 position with 7 position of coumarin nucleus to form furan ring. It is reported that furanocoumarins have significant melanogenesis-promoting effect. Psoralen is the most commonly used photosensitive drug in clinical practice, which is used in combination with long-wave ultraviolet light for early treatment of vitiligo. Ficus carica L. is a plant of Moraceae Ficus, which is an important source of food and health products. It is reported that Ficus carica L. is rich in coumarins. The present application aims to study the anti-vitiligo effect of coumarins in Ficus carica L. fruit, so as to obtain drug lead compounds or even drugs, and to provide a reference for the research and development of anti-vitiligo drugs. Two new furanocoumarin enantiomers were found in the fruit of Ficus carica L., and they were found to have anti-vitiligo activity and could be used for the preparation of anti-vitiligo drugs.

[0004] The present application relates to the field of medicine, in particular to two new furanocoumarins enantiomers and their use in the preparation of anti-vitiligo drugs. SUMMARY

[0005] The application aims to provide a preparation method and use of furanocoumarin enantiomers, which are obtained from fruits of Ficus carica L. by thin layer chromatography and analytical high performance liquid chromatography detection and analysis, extraction with an organic solvent, and then separation by solvent extraction, silica gel column chromatography, MCI column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography and chiral stationary phase method to obtain two furanocoumarin monomer compounds, which are determined to be new furanocoumarin compounds by high resolution mass spectrometry, nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. In addition, the two compounds are subjected to in vitro anti-vitiligo activity detection by using mouse melanoma B16 cells, and the results show that the compounds have different degrees of melanin production promotion and tyrosinase activation activity, and can be used for preparing anti-vitiligo drugs.

[0006] The furanocoumarin enantiomer in the fruits of Ficus carica L. has the structural formula:

[0007]

[0008] Among them:

[0009] The compound formula (I) is named as (S)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin.

[0010] The compound formula (II) is named as (R)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin.

[0011] The preparation method of the furanocoumarin enantiomer is carried out according to the following steps:

[0012] a. The dried fruits of Ficus carica L. are taken as raw materials, crushed, and then subjected to cold soak extraction for 3-5 times at room temperature by using 2-5 times amount of volume concentration of 50-100% ethanol or methanol aqueous solution, and the extraction liquid is combined and concentrated to obtain a crude extract of Ficus carica L.;

[0013] b. The crude extract obtained in step a is dispersed with water, and then 1:2-1:5 volume ratio of petroleum ether and ethyl acetate is added respectively for 3-5 times of extraction, and the petroleum ether extract is concentrated to obtain a petroleum ether extract extract;

[0014] c. The petroleum ether extract extract obtained in step b is subjected to silica gel column chromatography, MCI column chromatography, Sephadex LH-20 column chromatography, semi-preparative high performance liquid chromatography and chiral stationary phase method for separation to obtain the compound formula (I) named as (S)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin and the compound formula (II) named as (R)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin.

[0015] wherein the silica gel column chromatography is normal pressure column chromatography at room temperature, the mobile phase is petroleum ether-ethyl acetate mixed solvent, and gradient elution is used; the MCI column chromatography is normal pressure column chromatography at room temperature, the mobile phase is methanol-water mixed solvent, and gradient elution is used; the Sephadex LH-20 gel column chromatography is normal pressure column chromatography at room temperature, the mobile phase is methanol-water mixed solvent, and isocratic elution is used; the semi-preparative high performance liquid chromatography is pressurized column chromatography, the mobile phase is methanol-water solution with a volume concentration of 20-99% or acetonitrile-water solution with a volume concentration of 30-99%, and isocratic elution or gradient elution is used; and the chiral stationary phase method is liquid chromatography equipped with a chiral column, the mobile phase is n-hexane-ethanol solution with a volume ratio of 99:1-50:50 or n-hexane-isopropanol solution with a volume ratio of 99:1-50:50, and isocratic or gradient elution is used.

[0016] The separation method in step c is as follows:

[0017] The petroleum ether extract obtained in step b is subjected to normal pressure silica gel column chromatography, gradient elution is performed using petroleum ether-ethyl acetate with a volume ratio of 100:0-0:100 as the mobile phase, and the eluate collected when the volume ratio of petroleum ether to ethyl acetate is 4:1 is concentrated to obtain components (I) (S)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin and (II) (R)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin (Fr. 6); the components Fr. 6 are subjected to MCI column chromatography, gradient elution is performed using methanol-water solution with a volume concentration of 10-90% as the mobile phase, and the eluate collected is concentrated to obtain five components Fr. 6A-Fr. 6E; the components Fr. 6C are subjected to gel column chromatography, isocratic elution is performed using 90% methanol-water or pure methanol, and the eluate collected is concentrated to obtain five components Fr. 6B1-Fr. 6B5; the components Fr. 6B3 are subjected to semi-preparative high performance liquid chromatography, gradient elution is performed using acetonitrile-water solution with a concentration of 10-99% or methanol-water solution with a concentration of 20-99%, and racemates Fr. 6B3A of formula (I) and formula (II) are obtained; and Fr. 6B3A is subjected to high performance liquid chromatography equipped with a chiral column, elution is performed using n-hexane-ethanol solution with a volume ratio of 99:1-50:50 or n-hexane-isopropanol solution with a volume ratio of 99:1-50:50, and compounds of formula (I) (S)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin and formula (II) (R)-8-hydroxy-2', 3', 3'-trimethyl furanocoumarin are obtained.

[0018] The use of the furanocoumarin enantiomers of formula (I) and formula (II) in the fruit of Ficus carica in the preparation of an anti-vitiligo drug.

[0019] The furanocoumarin enantiomers in figs (I) and (II) in the fruits of ficus carica and a preparation method and use thereof are obtained, and in vitro melanogenesis and tyrosinase activity detection of the two new furanocoumarin enantiomers (I) and (II) compounds are carried out, and the results show that the two compounds can significantly promote the generation of melanin in mouse melanoma B16 cells and activate tyrosinase activity.

[0020] The furanocoumarin enantiomers in figs (I) and (II) in the fruits of ficus carica and a preparation method and use thereof are obtained, and in vitro melanogenesis and tyrosinase activity detection of the two new furanocoumarin enantiomers (I) and (II) compounds are carried out, and the results show that the two compounds can significantly promote the generation of melanin in mouse melanoma B16 cells and activate tyrosinase activity.

[0021] The racemate of the compounds in figs (I) and (II) is colorless amorphous crystal; UV (MeOH) λmax (lgΔε) 216 (+2.7), 327 (+1.4) nm; HRESIMS [M+H] + 247.0965 m / z (calculated value 247.0965 m / z, C 14 H 15 O4 + ); FT-IR (KBr) υ max 3300, 2968, 1703, 1625, 1447, 1355, 1060, 819 cm -1 ; 1 H and 13 C NMR attribution is shown in Table 1;

[0022] Table 1 shows the 1 H and 13 C NMR data of the compounds in figs (I) and (II) 1 H-600MHz, 13 C-150MHz, CD3OD)

[0023]

[0024] Note: s is a single peak; d is a doublet; q is a quartet; J is a coupling constant;

[0025] The optical rotation value of the compounds in figs (I) and (II) is close to 0, the racemate is separated by using a chiral stationary phase method, and the compounds in figs (I) and (II) are obtained, and the separation chromatogram is shown in Figure 3 ;

[0026] The compound in fig (I) is colorless oily solid; 55.1 (c 0.5, MeOH); ECD (MeOH) 211 (0.33), 254 (-0.57), 326 (-0.40);

[0027] Compound formula (II): colorless oily solid; -55.6 (c 0.5, MeOH); ECD (MeOH) 209 (0.94), 252 (1.15), 330 (0.82). BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Chromatogram of racemic compound formula (I) and (II) of the present application; wherein the liquid chromatogram of compound (I) and (II) is 254 nm, a is racemic, b is compound (I), and c is compound (II); 1 H NMR spectrum (600 MHz, CD3OD);

[0029] Figure 2 Chromatogram of racemic compound formula (I) and (II) of the present application; wherein the liquid chromatogram of compound (I) and (II) is 254 nm, a is racemic, b is compound (I), and c is compound (II); 13 C NMR spectrum (125 MHz, CD3OD);

[0030] Figure 3 Chromatogram of racemic compound formula (I) and (II) of the present application; wherein the liquid chromatogram of compound (I) and (II) is 254 nm, a is racemic, b is compound (I), and c is compound (II);

[0031] Figure 4 Chromatogram of racemic compound formula (I) and (II) of the present application; wherein the liquid chromatogram of compound (I) and (II) is 254 nm, a is racemic, b is compound (I), and c is compound (II); DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with the examples and drawings below, but the use and purpose of these example embodiments are only used to list the present application, and do not constitute any form of any limitation on the actual protection scope of the present application, nor limit the protection scope of the present application to this.

[0033] The solvents used in column chromatography are analytical pure (Tianjin Xinputa Chemical Co., Ltd.); the solvents used in high performance liquid chromatography are chromatographically pure (Merck, Germany); 100-200 mesh silica gel (Qingdao Haoyang Chemical Industry); MCI GEL CHP20 / P120 filler (Mitsubishi Chemical Group); Sephadex LH-20 (Sirtopu Company); high performance liquid chromatography (Hitachi) equipped with DAD full wavelength ultraviolet detector; semi-preparative liquid chromatography (Thermo Fisher Scientific); chiral stationary phase chromatographic column (DAICEL, ID, 10 x 250 mm, 5 μm); mass spectra were determined on a quadrupole-time-of-flight hybrid mass spectrometer (Applied Biosystems, USA); nuclear magnetic resonance was determined on a VARIAN VNMRS 600 MHz nuclear magnetic resonance spectrometer; optical rotation values were recorded using an Autopol VI automatic polarimeter; ECD spectra were obtained using a Chirascan spectropolarimeter (UK).

[0034] Ficus carica L. was collected in September 2019 from the city of Atushi, Xinjiang Uygur Autonomous Region, China, and identified by Dr. Lu Chunfang, Xinjiang Technical Institute of Physics & Chemistry, Chinese Academy of Sciences, as the fruit of Ficus carica L.

[0035] Example 1

[0036] a. 10 kg of dried Ficus carica L. fruit was pulverized and cold-extracted with 20 L of 50% ethanol at room temperature for 5 times, and the extract was combined and concentrated to obtain the crude extract of Ficus carica L.

[0037] b. The crude extract obtained in step a was dispersed with water, and 1:2 petroleum ether and ethyl acetate were added in sequence to extract 5 times, respectively, and the petroleum ether extract was concentrated to obtain the petroleum ether extract extract.

[0038] c. The petroleum ether extract extract obtained in step b was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate with a volume ratio of 100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 as the mobile phase, and the eluent of petroleum ether: ethyl acetate 4:1 was collected and concentrated to obtain the components Fr.6 of compound (I) and compound formula (II); the obtained component Fr.6 was subjected to MCI column chromatography, and gradient elution was performed using methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% as the mobile phase, and the eluent of 50% methanol water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography, and isocratic elution was performed using 90% methanol-water to collect the eluent and concentrate to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography, and isocratic elution was performed using acetonitrile-water solution with a concentration of 42% to obtain the racemate Fr.6B3A of (I) and (II); and then Fr.6B3A was eluted using high performance liquid chromatography equipped with a chiral column, using n-hexane-ethanol solution with a concentration of 90:10 (volume ratio) as the mobile phase to obtain the compound of formula (I) as (S)-8-hydroxy-2',3',3'-trimethyl furanocoumarin and the compound of formula (II) as (R)-8-hydroxy-2',3',3'-trimethyl furanocoumarin.

[0039] Example 2

[0040] a, take 10 kg of dried fig fruits, crush them and extract them with 100 L of 90% ethanol at room temperature for 3 times, combine the extract, and concentrate to obtain the crude extract of figs;

[0041] b, disperse the crude extract obtained in step a with water, and sequentially add petroleum ether and ethyl acetate with a volume ratio of 1:3 to extract them for 3 times respectively, concentrate the petroleum ether extract to obtain the petroleum ether extract infusion;

[0042] c, subject the petroleum ether extract infusion obtained in step b to silica gel column chromatography, adopt normal pressure silica gel column chromatography, use petroleum ether-ethyl acetate with a volume ratio of 100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 as the mobile phase to perform gradient elution, collect the eluent of petroleum ether: ethyl acetate 4:1, and concentrate to obtain the components Fr.6 of compounds (I) and (II); subject the obtained component Fr.6 to MCI column chromatography, use methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% as the mobile phase to perform gradient elution, collect the eluent of 50% methanol-water, and concentrate to obtain Fr.6C; subject the component Fr.6C to gel column chromatography, use 90% methanol-water to perform isocratic elution, and collect the eluent to concentrate to obtain 5 components Fr.6C1-Fr.6C5; subject the component Fr.6C3 to semi-preparative high performance liquid chromatography separation, use acetonitrile-water solution with a concentration of 52% to perform isocratic elution to obtain the racemate Fr.6B3A of (I) and (II); and then subject Fr.6B3A to high performance liquid chromatography equipped with a chiral column, use n-hexane-ethanol solution with a volume ratio of 90:10 as the mobile phase to perform elution, to obtain the compound of formula (I) as (S)-8-hydroxy-2',3',3'-trimethyl furanocoumarin and the compound of formula (II) as (R)-8-hydroxy-2',3',3'-trimethyl furanocoumarin.

[0043] Example 3

[0044] a, take 10 kg of dried fig fruits, crush them and extract them with 20 L of 75% ethanol at room temperature for 3 times, combine the extract, and concentrate to obtain the crude extract of figs;

[0045] b, disperse the crude extract obtained in step a with water, and sequentially add petroleum ether and ethyl acetate with a volume ratio of 1:4 to extract them for 5 times respectively, concentrate the petroleum ether extract to obtain the petroleum ether extract infusion;

[0046] c. The petroleum ether extract of step b was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 by volume) as the mobile phase. The eluate of petroleum ether: ethyl acetate 4:1 was collected and concentrated to obtain the components Fr.6 of compounds (I) and (II); the obtained component Fr.6 was subjected to MCI column chromatography, and gradient elution was performed using methanol-water solution (10%, 30%, 50%, 70% and 90% by volume) as the mobile phase. The eluate of 50% methanol-water was collected and concentrated to obtain Fr.6C; the component Fr.6C was subjected to gel column chromatography, and isocratic elution was performed using 90% methanol-water to obtain 5 components Fr.6C1-Fr.6C5; the component Fr.6C3 was subjected to semi-preparative high performance liquid chromatography, and isocratic elution was performed using acetonitrile-water solution (52% by volume) to obtain the racemates Fr.6B3A of (I) and (II); Fr.6B3A was subjected to high performance liquid chromatography equipped with a chiral column, and elution was performed using n-hexane-ethanol solution (90:10 by volume) as the mobile phase to obtain compound (I) as (S)-8-hydroxy-2',3',3'-trimethylfurocoumarin and compound (II) as (R)-8-hydroxy-2',3',3'-trimethylfurocoumarin.

[0047] Example 4

[0048] a. 10 kg of dried fig fruits were crushed and subjected to percolation extraction at room temperature using 30 L of 75% methanol aqueous solution for 3 times. The combined extract was concentrated to obtain the crude extract of figs;

[0049] b. The crude extract of step a was dispersed with water, and 4 times of extraction was performed using petroleum ether and ethyl acetate (1:4 by volume) in sequence. The petroleum ether extract was concentrated to obtain the petroleum ether extract extractum;

[0050] c. The petroleum ether extract of step b was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 by volume) as the mobile phase. The eluate of petroleum ether: ethyl acetate 4:1 was collected and concentrated to obtain component Fr.6 of compounds (I) and (II); the obtained component Fr.6 was subjected to MCI column chromatography, and gradient elution was performed using methanol-water solution (10%, 30%, 50%, 70% and 90% by volume) as the mobile phase. The eluate of 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography, and isocratic elution was performed using 90% methanol-water to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was subjected to semi-preparative high performance liquid chromatography, and gradient elution was performed using acetonitrile-water solution (40-58% by volume) to obtain the racemates Fr.6B3A of (I) and (II); Fr.6B3A was subjected to high performance liquid chromatography equipped with a chiral column, and elution was performed using n-hexane-ethanol solution (90:10 by volume) as the mobile phase to obtain compound (I) as (S)-8-hydroxy-2',3',3'-trimethylfurocoumarin and compound (II) as (R)-8-hydroxy-2',3',3'-trimethylfurocoumarin.

[0051] Example 5

[0052] a. 10 kg of dried fig fruits were crushed and subjected to cold extraction at room temperature using 50 L of 90% methanol-water solution by volume for 3 times. The extraction solutions were combined and concentrated to obtain the crude extract of figs;

[0053] b. The crude extract of step a was dispersed with water, and petroleum ether and ethyl acetate were added in sequence at a volume ratio of 1:5 compared with the water phase for 5 times of extraction respectively. The petroleum ether extract was concentrated to obtain the petroleum ether extract extractum;

[0054] c. The petroleum ether extract of step b was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 by volume) as the mobile phase. The eluate of petroleum ether: ethyl acetate 4:1 was collected and concentrated to obtain component Fr.6 of compounds (I) and (II); the obtained component Fr.6 was subjected to MCI column chromatography, and gradient elution was performed using methanol-water solution (10%, 30%, 50%, 70% and 90% by volume) as the mobile phase. The eluate of 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography, and isocratic elution was performed using 90% methanol-water to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was subjected to semi-preparative high performance liquid chromatography, and isocratic elution was performed using methanol-water solution (42% by volume) to obtain the racemates Fr.6B3A of (I) and (II); Fr.6B3A was subjected to high performance liquid chromatography equipped with a chiral column, and elution was performed using n-hexane-ethanol solution (90:10 by volume) as the mobile phase to obtain compound (I) as (S)-8-hydroxy-2',3',3'-trimethyl furanocoumarin and compound (II) as (R)-8-hydroxy-2',3',3'-trimethyl furanocoumarin.

[0055] Example 6

[0056] a. 10 kg of dried fig fruits were crushed and subjected to percolation extraction at room temperature using 20 L of ethanol (100% by volume) for 3 times, and the crude extract of fig was obtained by concentration;

[0057] b. The crude extract of step a was dispersed with water, and petroleum ether and ethyl acetate (1:3 by volume) were added successively for extraction 5 times. The petroleum ether extract was concentrated to obtain the petroleum ether extract of the crude extract;

[0058] c. The petroleum ether extract of step b was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1 and 2:1 by volume) as the mobile phase. The eluate of petroleum ether: ethyl acetate 4:1 was collected and concentrated to obtain the components Fr.6 of compounds (I) and (II); the obtained component Fr.6 was subjected to MCI column chromatography, and gradient elution was performed using methanol-water solution (10%, 30%, 50%, 70% and 90% by volume) as the mobile phase. The eluate of 50% methanol-water was collected and concentrated to obtain Fr.6C; the component Fr.6C was subjected to gel column chromatography, and isocratic elution was performed using 90% methanol-water to obtain 5 components Fr.6C1-Fr.6C5; the component Fr.6C3 was subjected to semi-preparative high performance liquid chromatography, and isocratic elution was performed using 52% methanol-water solution to obtain the racemates Fr.6B3A of compounds (I) and (II); Fr.6B3A was subjected to high performance liquid chromatography equipped with a chiral column, and elution was performed using n-hexane-ethanol solution (99:1-70:30 by volume) as the mobile phase to obtain compound (I) as (S)-8-hydroxy-2',3',3'-trimethylfurancoumarin and compound (II) as (R)-8-hydroxy-2',3',3'-trimethylfurancoumarin.

[0059] Example 7

[0060] Detection of melanin synthesis and tyrosinase activity in melanoma B16 cells by the compounds (I) and (II) obtained from Examples 1-7:

[0061] Cell survival rate detection: cell viability was detected using a cell viability detection kit (CCK-8). Cells were resuscitated and inoculated into a 96-well plate. The cells were cultured until they adhered to the plate. The supernatant was discarded, and culture medium containing the test compound at a concentration of 1 μM, 2 μM, 10 μM, 20 μM, 40 μM, 50 μM, 60 μM, 80 μM or 100 μM was added. After 24 h of continuous culture, 10 μL of CCK-8 solution was added, and the cells were cultured for another 2 h. The absorbance value was measured at 450 nm. Untreated cells were used as the blank control. Each sample was repeated three times. Cell survival rate (%) = (OD 450 sample-OD 450 sample control) / (OD 450 control-OD 450 culture medium) x 100%;

[0062] Melanin content detection: The melanin content in B16 cells was determined using the sodium hydroxide (NaOH) lysis method. B16 cells were digested, seeded in 6-well plates, and cultured overnight at 37℃. After cell attachment, the cells were drug-treated and cultured for another 48 hours. The supernatant was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Cells were scraped, and 100 μL of lysis buffer was added to each well. After complete lysis, the cells were centrifuged at 12000 rpm for 20 min, and the protein concentration in the supernatant was measured. 190 μL of 1M NaOH lysis buffer was added to the precipitate, and the cells were incubated at 80℃ for 1 h. The absorbance was measured at 405 nm. Dimethyl sulfoxide (DMSO) was used as a blank control, and 8-methoxypsoralen (8-MOP) was used as a positive control. Each sample was repeated three times. The relative melanin content was calculated as follows: (OD...) 405 (sample / protein concentration) / (OD) 405 (blank / protein concentration) × 100%;

[0063] Tyrosinase activity assay: using levodopa (LDOPA) L -DOPA oxidation method was used to detect the activity of intracellular tyrosinase in B16 cells; cells were revived, seeded into 6-well plates, and cultured overnight at 37°C; after cell attachment, drugs were administered, and cultured for another 24 h; the supernatant was aspirated, the cells were washed twice with PBS, and 100 μL of lysis buffer containing 1% sodium deoxycholate and 1% Triton X-100 was added to each well; the cells were frozen at -20°C for 30 min, thawed at 4°C, and centrifuged at 12000 rpm for 15 min; the supernatant was added to 96-well plates, and 10 μL of 10 mM sodium hydroxychloroquine was added to each well. L Mix DOPA thoroughly and incubate at 37°C for 30 min until a light brown color appears in each well. Measure the absorbance at 490 nm. Determine the protein concentration using the remaining supernatant. Use DMSO as a blank control and 8-MOP as a positive control. Each sample is repeated three times. Tyrosinase relative activity = (OD200 + DOPA) / (8-MOP) = 0.05% DOPA. 490 (sample / protein concentration) / (OD) 490 (blank / protein concentration) × 100%;

[0064] Experimental results: Compounds (I) and (II) did not show obvious cytotoxicity at the test concentration of 50 μM. The test results of their relative melanin content and relative tyrosinase content are shown in Table 2.

[0065] Table 2 Results of anti-vitiligo activity tests for compounds (I) and (II)

[0066]

[0067] Experimental results show that both compounds (I) and (II) can promote melanin production in B16 cells and activate tyrosinase activity at the tested concentrations, and can be used for the development of drugs for the treatment of vitiligo.

Claims

1. A furanocoumarin compound from fig fruit, characterized in that... The structural formula of this compound is:

2. A method for preparing enantiomers of furanocoumarins, characterized in that... Follow these steps: a. Take dried fig fruit as raw material, crush it, and then extract it by percolation and cold soaking at room temperature 3-5 times with 2-5 times the volume of 50-100% ethanol or methanol aqueous solution. Combine the extracts, concentrate them, and obtain crude extract of fig. b. Disperse the crude extract obtained in step a with water, and extract it 3-5 times by adding petroleum ether and ethyl acetate in a volume ratio of 1:2-1:

5. Concentrate the petroleum ether extract to obtain petroleum ether extract paste. c. The petroleum ether extract obtained in step b was separated by silica gel column chromatography, MCI column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography, and chiral stationary phase method to obtain compound (Ⅰ) and compound (Ⅱ); the structural formula of the compound is: The silica gel column chromatography method used was room temperature and atmospheric pressure column chromatography, with a petroleum ether-ethyl acetate mixed solvent as the mobile phase and gradient elution. The MCI column chromatography method used was room temperature and atmospheric pressure column chromatography, with a methanol-water mixed solvent as the mobile phase and gradient elution. The Sephadex LH-20 gel column chromatography method used was room temperature and atmospheric pressure column chromatography, with a methanol-water mixed solvent as the mobile phase and isocratic elution. The semi-preparative high-performance liquid chromatography method used was pressurized column chromatography, with a methanol-water solution of 20-99% (v / v) or an acetonitrile-water solution of 30-99% (v / v) and isocratic or gradient elution. The chiral stationary phase method used was liquid chromatography equipped with a chiral column, with a hexane-ethanol solution or a hexane-isopropanol solution of 99:1-50:50 (v / v) as the mobile phase and isocratic or gradient elution.

3. The method for preparing furanocoumarin enantiomers according to claim 2, characterized in that... The separation method in step c: The petroleum ether extract obtained in step b was subjected to atmospheric pressure silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate mobile phase at a volume ratio of 100:0-0:

100. The eluent of petroleum ether:ethyl acetate at a ratio of 4:1 was collected and concentrated to obtain fraction Fr.6 of compounds (I) and (II). Fraction Fr.6 was subjected to MCI column chromatography with a gradient elution using a methanol-water solution at a volume concentration of 10-90%. The eluent was collected and concentrated to obtain five fractions Fr.6A-Fr.6E. Fraction Fr.6C was subjected to Sephadex chromatography. LH-20 gel column chromatography was performed, with 90% methanol-water isocratic elution. The eluent was collected and concentrated to obtain five fractions Fr.6B1-Fr.6B5. Fraction Fr.6B3 was separated by semi-preparative high performance liquid chromatography, with gradient elution of 30-99% acetonitrile-water solution or 20-99% methanol-water solution to obtain the racemic mixture of compound (I) and compound (II) Fr.6B3A. Fr.6B3A was prepared using high performance liquid chromatography with a chiral column and eluted with a hexane-ethanol solution or a hexane-isopropanol solution with a volume ratio of 99:1-50:50 to obtain compounds (Ⅰ) and (Ⅱ).

4. The use of the furanocoumarin compound (Ⅰ) in the fig fruit according to claim 1 in the preparation of an anti-vitiligo drug.

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