Furocoumarin enantiomer in fig fruit as well as preparation method and application of furocoumarin enantiomer

Abstract

The invention relates to furocoumarin enantiomers in fig fruits and a preparation method and application of the furocoumarin enantiomers in the fig fruits, according to the method, dried fig fruits are used as raw materials, organic solvents are used for extraction, and then the furocoumarin enantiomers in the fig fruits are obtained through a solvent extraction method, silica gel column chromatography, MCI column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography and a chiral stationary phase method. A pair of enantiomers of furocoumarin are separated from dried fig fruits, the structures of the enantiomers are identified through methods such as high-resolution mass spectrum, nuclear magnetic resonance spectrum and circular dichroism spectrum, and anti-vitiligo activity detection is carried out. Results show that the compounds (I) and (II) have different degrees of melanogenesis promotion and tyrosinase activation activity, and can be used for development of vitiligo treatment drugs.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to two new furanocoumarin enantiomers and uses thereof in preparing anti-vitiligo drugs. Background Art

[0002] Vitiligo is an acquired, chronic skin pigmentation disorder that affects 1-4% of the global population, including adults and children. It is characterized by the selective destruction of melanocytes in the skin and hair, mainly manifested by the expansion of lesions of skin pigmentation. Although the exact pathogenesis is still unclear, potential causes include genetics, immunity, oxidative stress, and the production of inflammatory mediators. According to the regulatory network of melanogenesis, the activity of microsome-associated transcription factor (MITF) tyrosine (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) genes is upregulated, ultimately increasing melanogenesis.

[0003] Furanocoumarins are a class of secondary metabolites present in higher plants. They are a class of compounds and their derivatives formed by the condensation of the 7-position of the coumarin parent nucleus with the 6-position or 8-position substituted isopentenyl group to form a furan ring. It is reported that furanocoumarins have a significant effect on promoting melanin production. For example, psoralen compounds are currently the most commonly used photosensitizers in clinical practice, and are used in combination with long-wave ultraviolet rays for the early treatment of vitiligo. Fig (Ficus carica L.) is a plant of the genus Ficus in the Moraceae family and is an important source of food and health products. It is reported that figs are rich in coumarin compounds. The present invention aims to study the anti-vitiligo effect of coumarin compounds in fig fruits, thereby obtaining drug lead compounds or even the drugs themselves, and further providing a reference for the research and development of anti-vitiligo drugs. The present invention finds two new enantiomers of furanocoumarins from fig fruits, and finds that they have anti-vitiligo activity and can be used to prepare anti-vitiligo drugs.

[0004] Currently, the invention patents related to furanocoumarin compounds for vitiligo treatment are CN201610983128.3, CN201610983060.9, and CN201710995086.X. These three patents are the previous research results of our research group, and psoralen derivatives are obtained by synthetic methods. Different from them, the present invention uses column chromatography and chiral stationary phase separation to separate furanocoumarin enantiomers from fig fruit. Summary of the invention

[0005] The present invention aims to provide a method for preparing and using furanocoumarin enantiomers. The furanocoumarins are extracted from the fruit of fig (Ficus carica L.) by thin layer chromatography and analytical high performance liquid chromatography, extracted with an organic solvent, and then separated 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 enantiomeric furanocoumarin monomer compounds. The two monomers are determined to be new furanocoumarin compounds by high resolution mass spectrometry, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and other methods. In addition, the two compounds are tested for in vitro anti-vitiligo activity using mouse melanoma B16 cells, and the results show that the compounds have different degrees of melanin-promoting and tyrosinase-activating activities, and can be used to prepare anti-vitiligo drugs.

[0006] The furanocoumarin enantiomers in the fig fruit of the present invention have the structural formula:

[0007]

[0008] in:

[0009] The compound of formula (I) is named: (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin;

[0010] The compound of formula (II) is named: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0011] The method for preparing the furanocoumarin enantiomers is carried out according to the following steps:

[0012] a. Take dried fig fruits as raw materials, crush them, and use 2-5 times the volume of 50-100% ethanol or methanol aqueous solution for percolation and cold soaking extraction at room temperature for 3-5 times, combine the extracts, and concentrate to obtain a crude extract of figs;

[0013] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:2-1:5, respectively, extracting for 3-5 times, concentrating the petroleum ether extract, and obtaining a petroleum ether extract concentrate;

[0014] c. Separate the petroleum ether extract obtained in step b by silica gel column chromatography, MCI column chromatography, Sephadex LH-20 column chromatography, semi-preparative high performance liquid chromatography and chiral stationary phase method to obtain compound formula (I) named: (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; compound formula (II) named: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin;

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

[0016] The separation method in step c:

[0017] The petroleum ether extract obtained in step b is subjected to normal pressure silica gel column chromatography, and 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 with a petroleum ether:ethyl acetate ratio of 4:1 is collected and concentrated to obtain the components (Fr.6) of compound (I) (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound (II) (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; component Fr.6 is subjected to MCI column chromatography, and gradient elution is performed using a methanol-water solution with a volume concentration of 10-90% as the mobile phase, and the eluate is collected and concentrated to obtain 5 components Fr.6A-Fr.6E; component Fr.6C is subjected to gel column chromatography, and isocratic elution is performed using 90% methanol-water or pure methanol. Elution, collecting and concentrating the eluate to obtain five components Fr.6B1-Fr.6B5; separating component Fr.6B3 by semi-preparative high performance liquid chromatography, eluting with a gradient of 10-99% acetonitrile-water solution or 20-99% methanol-water solution to obtain racemic Fr.6B3A of formula (I) and formula (II); using high performance liquid chromatography equipped with a chiral chromatographic column, eluting with a n-hexane-ethanol solution of 99:1-50:50 by volume or a n-hexane-isopropanol solution of 99:1-50:50 by volume to obtain compound formula (I): (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (II): (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0018] The furanocoumarin enantiomers of formula (I) and formula (II) in the fig fruit are used in the preparation of anti-vitiligo drugs.

[0019] The invention discloses a furanocoumarin enantiomer in fig fruit, a preparation method and a use thereof. Two new furanocoumarin enantiomers (I) and (II) compounds obtained are subjected to in vitro melanin production and tyrosinase activity tests, and the results show that the two compounds can significantly promote the production of melanin in mouse melanoma B16 cells and activate tyrosinase activity.

[0020] The furanocoumarin enantiomers in fig fruit and the preparation method and use thereof described in the present invention are determined by high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and optical rotation spectroscopy. The structural identification process is as follows:

[0021] Racemic form of compound (I) and (II): colorless amorphous crystals; 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, 819cm -1 ; 1 H and 13 C NMR assignments are shown in Table 1 ;

[0022] Table 1 Compounds of formula (I) and (II) 1 H and 13 C NMR data ( 1 H-600MHz, 13 C-150MHz, CD3OD)

[0023]

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

[0025] The optical rotation values ​​of compounds (I) and (II) are close to 0. The racemates are separated by chiral stationary phase method to obtain compounds (I) and (II). The separation chromatograms are shown in Figure 3 ;

[0026] Compound formula (I): 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 THE DRAWINGS

[0028] Figure 1 The racemic forms of the compounds of formula (I) and (II) of the present invention are 1 H NMR spectrum (600 MHz, CD3OD);

[0029] Figure 2 The racemic forms of the compounds of formula (I) and (II) of the present invention are 13 C NMR spectrum (125 MHz, CD3OD);

[0030] Figure 3 The chiral stationary phase separation chromatogram of the compounds (I) and (II) of the present invention; wherein the liquid phase analysis diagram of the compounds (I) and (II) is 254 nm, a is a racemic body, b is the compound (I), and c is the compound (II);

[0031] Figure 4 The measured and calculated circular dichroism spectra of the compounds of formula (I) and (II) of the present invention are shown, wherein a is the calculated value of 1'R, b is the calculated value of 1'S, c is the measured value of compound (I), and d is the measured value of compound (II). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with examples and drawings, but the uses and purposes of these exemplary embodiments are only used to list the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the protection scope of the present invention to them.

[0033] The solvents used in column chromatography were analytical grade (Tianjin Xinbote Chemical Co., Ltd.); the solvents used in HPLC were chromatographic grade (Merck, Germany); 100-200 mesh silica gel (Qingdao Ocean Chemical); MCI GEL CHP20 / P120 filler (Mitsubishi Chemical Group); Sephadex LH-20 (Situofan); HPLC (Hitachi) equipped with DAD full-wavelength UV detector; semi-preparative HPLC (Thermo Fisher Scientific); chiral stationary phase chromatographic column (DAICEL, ID, 10×250 mm, 5 μm); mass spectra were measured using a quadrupole-time-of-flight hybrid mass spectrometer (Applied Biosystems, USA); nuclear magnetic resonance was measured using a VARIAN VNMRS 600 MHz nuclear magnetic resonance spectrometer; optical rotation values ​​were recorded using an Autopol VI automatic polarimeter; and ECD spectra were obtained using a Chirascan spectropolarimeter (UK).

[0034] The figs were collected from Atushi City, Xinjiang Uygur Autonomous Region in September 2019 and identified as the fruit of fig (Ficus carica L.) by Associate Researcher Lu Chunfang from the Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0035] Example 1

[0036] a. Take 10 kg of dried fig fruit, crush it, and then use 20 L of 50% ethanol by volume to cold-immerse and extract it 5 times at room temperature, combine the extracts, and concentrate to obtain a crude extract of fig;

[0037] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:2, respectively, and extracting for 5 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

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

[0039] Example 2

[0040] a. Take 10 kg of dried fig fruit, crush it, and then use 100 L of 90% ethanol by volume to cold-immerse and extract it three times at room temperature, combine the extracts, and concentrate to obtain a crude extract of fig;

[0041] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:3, respectively, and extracting for 3 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

[0042] c. The petroleum ether extract obtained in step b was subjected to silica gel column chromatography, and normal pressure silica gel column chromatography was performed, and 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 was used as the mobile phase for gradient elution, and the eluate with a petroleum ether:ethyl acetate ratio of 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 a methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% was used as the mobile phase for gradient elution, and the eluate with 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography The product was separated by isocratic analysis and eluted isocratically with 90% methanol-water, and the eluate was collected and concentrated to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography and isocratically eluted with 52% acetonitrile-water solution to obtain racemic Fr.6B3A (Ⅰ) and (Ⅱ); Fr.6B3A was then eluted using high performance liquid chromatography equipped with a chiral chromatographic column and a n-hexane-ethanol solution with a volume ratio of 90:10 as the mobile phase to obtain the compound of formula (Ⅰ) which is (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and the compound of formula (Ⅱ) which is: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0043] Example 3

[0044] a. Take 10 kg of dried fig fruit, crush it, and then use 20 L of 75% ethanol at room temperature for cold soaking extraction three times, combine the extracts, and concentrate to obtain a crude extract of fig;

[0045] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:4, respectively, and extracting for 5 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

[0046] c. The petroleum ether extract obtained in step b was subjected to silica gel column chromatography, and normal pressure silica gel column chromatography was performed, and 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 was used as the mobile phase for gradient elution, and the eluate with a petroleum ether:ethyl acetate ratio of 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 a methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% was used as the mobile phase for gradient elution, and the eluate with 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography The product was separated by isocratic analysis and eluted isocratically with 90% methanol-water, and the eluate was collected and concentrated to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography and isocratically eluted with 52% acetonitrile-water solution to obtain racemic Fr.6B3A (Ⅰ) and (Ⅱ); Fr.6B3A was eluted using high performance liquid chromatography equipped with a chiral chromatographic column and a n-hexane-ethanol solution with a volume ratio of 90:10 as the mobile phase to obtain compound formula (Ⅰ): (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (Ⅱ): (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0047] Example 4

[0048] a. Take 10 kg of dried fig fruit, crush it, and then percolate and extract it three times with 30 L of 75% methanol aqueous solution at room temperature, combine the extracts, and concentrate to obtain a crude extract of fig;

[0049] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:4, respectively, and extracting 4 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

[0050] c. The petroleum ether extract obtained in step b was subjected to silica gel column chromatography, and normal pressure silica gel column chromatography was performed, and 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 was used as the mobile phase for gradient elution, and the eluate with a petroleum ether:ethyl acetate ratio of 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 a methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% was used as the mobile phase for gradient elution, and the eluate with 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography , eluted isocratically with 90% methanol water, collected and concentrated the eluate to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography, and eluted with a gradient of 40-58% acetonitrile-water solution to obtain racemic Fr.6B3A (Ⅰ) and (Ⅱ); Fr.6B3A was eluted using high performance liquid chromatography equipped with a chiral chromatographic column with a n-hexane-ethanol solution with a volume ratio of 90:10 as the mobile phase to obtain compound formula (Ⅰ) is (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (Ⅱ) is: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0051] Example 5

[0052] a. Take 10 kg of dried fig fruit, crush it, and then use 50 L of 90% methanol aqueous solution at room temperature for cold soaking extraction three times, combine the extracts, and concentrate to obtain a crude extract of fig;

[0053] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:5 to water, respectively, and extracting for 5 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

[0054] c. The petroleum ether extract obtained in step b was subjected to silica gel column chromatography, and normal pressure silica gel column chromatography was performed, and 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 was used as the mobile phase for gradient elution, and the eluate with a petroleum ether:ethyl acetate ratio of 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 a methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% was used as the mobile phase for gradient elution, and the eluate with 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography The product was separated by isocratic analysis and eluted isocratically with 90% methanol-water, and the eluate was collected and concentrated to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography and isocratically eluted with a methanol-water solution with a concentration of 42% to obtain racemic Fr.6B3A of (Ⅰ) and (Ⅱ); Fr.6B3A was eluted using a high performance liquid chromatography equipped with a chiral chromatographic column and a n-hexane-ethanol solution with a concentration of 90:10 by volume as the mobile phase to obtain compound formula (Ⅰ): (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (Ⅱ): (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0055] Example 6

[0056] a. Take 10 kg of dried fig fruit, crush it, and then use 20 L of 100% ethanol by volume to percolate and extract three times at room temperature, and concentrate to obtain a crude extract of fig;

[0057] b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:3, respectively, and extracting for 5 times, concentrating the petroleum ether extract to obtain a petroleum ether extract concentrate;

[0058] c. The petroleum ether extract obtained in step b was subjected to silica gel column chromatography, and normal pressure silica gel column chromatography was performed, and 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 was used as the mobile phase for gradient elution, and the eluate with a volume ratio of petroleum ether:ethyl acetate of 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 a methanol-water solution with a volume concentration of 10%, 30%, 50%, 70% and 90% was used as the mobile phase for gradient elution, and the eluate with 50% methanol-water was collected and concentrated to obtain Fr.6C; component Fr.6C was subjected to gel column chromatography, and The product was isocratically eluted with 90% methanol-water, and the eluate was collected and concentrated to obtain 5 components Fr.6C1-Fr.6C5; component Fr.6C3 was separated by semi-preparative high performance liquid chromatography, and isocratically eluted with 52% methanol-water solution to obtain racemic compounds Fr.6B3A (Ⅰ) and (Ⅱ); Fr.6B3A was eluted using high performance liquid chromatography equipped with a chiral chromatographic column and a n-hexane-ethanol solution with a volume ratio of 99:1-70:30 as the mobile phase to obtain compound formula (Ⅰ): (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (Ⅱ): (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

[0059] Example 7

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

[0061] Cell viability detection: Cell viability was detected using a cell viability detection kit (CCK-8), and the cells were revived and inoculated into a 96-well plate; the cells were continued to be cultured until they adhered to the wall, the supernatant was discarded, and the culture medium with 1 μM, 2 μM, 10 μM, 20 μM, 40 μM, 50 μM, 60 μM, 80 μM, and 100 μM concentrations of the test compound was added, and the culture was continued for 24 hours, and then 10 μL of CCK-8 solution was added, and the absorbance was measured at 450 nm after continuing to culture for 2 hours. The untreated cells were used as blank controls, and each sample was repeated three times; cell viability (%) = (OD 450 sample-OD 450 sample control) / (OD 450 Control-OD 450 Culture medium) × 100%;

[0062] Melanin content detection: The sodium hydroxide (NaOH) lysis method was used to measure the melanin content in B16 cells. B16 cells were digested and inoculated in a 6-well plate, cultured at 37°C overnight, and the drug was administered after the cells adhered to the wall, and the culture was continued for 48 hours; the supernatant was aspirated, washed twice with phosphate buffered saline (PBS), and the cells were scraped, and 100 μL of lysis solution was added to each well. After sufficient lysis, centrifugation was performed at 12000 rpm for 20 minutes, and the protein concentration was measured in the supernatant; 190 μL of 1M NaOH lysis solution was added to the precipitate, and the absorbance was measured at 405 nm after a water bath at 80°C for 1 hour; dimethyl sulfoxide (DMSO) was used as a blank control, 8-methoxypsoralen (8-MOP) was used as a positive control, and each sample was repeated 3 times; relative melanin content = (OD 405 sample / protein concentration) / (OD 405 Blank / protein concentration)×100%;

[0063] Tyrosinase activity detection: L-dopa ( L -DOPA) oxidation method was used to detect the activity of tyrosinase in B16 cells; the cells were revived and inoculated into 6-well plates and cultured overnight at 37°C; the drugs were administered after the cells attached to the wall and cultured for 24 hours; the supernatant was removed and washed twice with PBS, 100 μL of lysis solution containing 1% sodium deoxycholate and 1% Triton X-100 was added to each well, frozen at -20°C for 30 minutes, thawed at 4°C, and centrifuged at 12000 rpm for 15 minutes; the supernatant was added to a 96-well plate, and 10 μL of 10 mM L -DOPA, mix well, incubate at 37°C for 30 min until each well turns light brown, measure absorbance at 490 nm, and use the remaining supernatant to detect protein concentration; DMSO is used as blank control, 8-MOP is used as positive control, and each sample is repeated 3 times; tyrosinase relative activity = (OD 490 sample / protein concentration) / (OD 490 Blank / protein concentration)×100%;

[0064] Experimental results: Compounds of formula (I) and (II) showed no obvious cytotoxicity at a test concentration of 50 μM, and the test results of their relative melanin content and relative tyrosinase content are shown in Table 2;

[0065] Table 2 Anti-vitiligo activity test results of compounds (I) and (II)

[0066]

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

Claims

1. A furanocoumarin enantiomer in fig fruit, characterized in that The structural formula of the compound is: in: The compound of formula (I) is named: (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; The compound of formula (II) is named: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

2. The method for preparing furanocoumarin enantiomers according to claim 1, characterized in that Follow these steps: a. Take dried fig fruits as raw materials, crush them, and use 2-5 times the volume of 50-100% ethanol or methanol aqueous solution for percolation and cold soaking extraction at room temperature for 3-5 times, combine the extracts, and concentrate to obtain a crude extract of figs; b. Dispersing the crude extract obtained in step a with water, sequentially adding petroleum ether and ethyl acetate in a volume ratio of 1:2-1:5, respectively, extracting for 3-5 times, concentrating the petroleum ether extract, and obtaining a petroleum ether extract concentrate; c. Separate the petroleum ether extract obtained in step b by silica gel column chromatography, MCI column chromatography, Sephadex LH-20 column chromatography, semi-preparative high performance liquid chromatography and chiral stationary phase method to obtain compound formula (I) named: (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; compound formula (II) named: (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; The silica gel column chromatography method used is room temperature and atmospheric pressure column chromatography, the mobile phase is a petroleum ether-ethyl acetate mixed solvent, and gradient elution is adopted; the MCI column chromatography method used is room temperature and atmospheric pressure column chromatography, the mobile phase is a methanol-water mixed solvent, and gradient elution is adopted; the Sephadex LH-20 gel column chromatography method used is room temperature and atmospheric pressure column chromatography, the mobile phase is a methanol-water mixed solvent, and isocratic elution is adopted; the semi-preparative high performance liquid phase used is a pressurized column chromatography, the mobile phase is a methanol-water solution with a volume concentration of 20-99% or an acetonitrile-water solution with a volume concentration of 30-99%, and isocratic elution or gradient elution is adopted; the chiral stationary phase method used is a liquid chromatography equipped with a chiral chromatographic column, the mobile phase is a n-hexane-ethanol solution with a volume ratio of 99:1-50:50 or a n-hexane-isopropanol solution with a volume ratio of 99:1-50:50, and isocratic or gradient elution is adopted.

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 is subjected to normal pressure silica gel column chromatography, and 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 with a petroleum ether:ethyl acetate ratio of 4:1 is collected and concentrated to obtain the components (Fr.6) of compound (I) (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound (II) (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin; component Fr.6 is subjected to MCI column chromatography, and gradient elution is performed using a methanol-water solution with a volume concentration of 10-90% as the mobile phase, and the eluate is collected and concentrated to obtain 5 components Fr.6A-Fr.6E; component Fr.6C is subjected to gel column chromatography, and isocratic elution is performed using 90% methanol-water or pure methanol. Elution, collecting and concentrating the eluate to obtain five components Fr.6B1-Fr.6B5; separating component Fr.6B3 by semi-preparative high performance liquid chromatography, eluting with a gradient of 10-99% acetonitrile-water solution or 20-99% methanol-water solution to obtain racemic Fr.6B3A of formula (I) and formula (II); using high performance liquid chromatography equipped with a chiral chromatographic column, eluting with a n-hexane-ethanol solution of 99:1-50:50 by volume or a n-hexane-isopropanol solution of 99:1-50:50 by volume to obtain compound formula (I): (S)-8-hydroxy-2',3',3'-trimethylfuranocoumarin and compound formula (II): (R)-8-hydroxy-2',3',3'-trimethylfuranocoumarin.

4. Use of the furanocoumarin enantiomers of formula (I) and formula (II) in fig fruit according to claim 1 in the preparation of anti-vitiligo drugs.

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