Skeleton type alkaloid in nigella glandulifera seeds as well as preparation method and application of skeleton type alkaloid

Abstract

The invention relates to a skeleton type alkaloid in nigella glandulifera seeds as well as a preparation method and application of the skeleton type alkaloid. The chemical name of the alkaloid is 3, 3, 6, 6, 11, 11-hexamethyl-10, 12-(4 hydrogen)-4H-piperidine-4-ketone (4a, 4b) quinolizedine-8-ketone. According to the method, an organic solvent is used for extracting from nigella glandulifera seeds, and then the total alkaloids are obtained through an acid dissolution and alkali precipitation method. And then, further purifying by adopting normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, gel column chromatography and a semi-preparative high performance liquid chromatograph to obtain the high-purity skeleton type alkaloid. The structure is confirmed through spectrum and mass spectrum data analysis. The alkaloid of the skeleton type is subjected to a biological activity test, and particularly, the melanogenesis promotion capability of B16 cells is evaluated. A test result shows that the melanogenesis promoting capability of the alkaloid is equivalent to that of a positive control, and the alkaloid shows the potential in the aspect of improving the vitiligo disease. Therefore, as a novel lead compound, the alkaloid of the skeleton type provides a new possibility for developing the anti-vitiligo medicine.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to skeleton-type alkaloids in tuberous black cumin seeds and a preparation method and application thereof. Background Art

[0002] Vitiligo is a common acquired skin depigmentation disease, which is mainly characterized by the selective destruction of melanocytes in the skin and hair or a decrease in their ability to synthesize melanin, resulting in the appearance of white spots on the skin. Studies have shown that the direct cause of vitiligo is the decrease or disappearance of tyrosinase activity in melanocytes, which in turn hinders the synthesis of melanin. Therefore, promoting the synthesis of skin melanin is considered an effective way to treat vitiligo. By promoting the synthesis of melanin in melanoma cells B16, finding new drugs to treat vitiligo has become a new research direction.

[0003] Nigella glandulifera Freyn is an annual herb belonging to the genus Nigella in the family Ranunculaceae. There are about 20 species in this genus, of which three main medicinal plants include: Nigella sative, also known as house black seed, Nigella demascena L. and Nigella glandulifera Freyn, also known as glandular black seed. As a traditional folk herbal medicine, Nigella glandulifera Freyn seeds are generally used as medicine to treat a variety of diseases, including dispelling wind and relieving pain, tonifying the kidney and strengthening the brain, promoting diuresis and sweating, stimulating digestion, strengthening the stomach, resisting worms and asthma. Modern pharmacological studies have also shown that Nigella seeds have a variety of effects such as regulating menstruation, anti-oxidation, anti-inflammatory, anti-tumor, protecting internal organs, protecting nerves, lowering blood sugar and improving respiratory diseases. Although studies have shown that Nigella cumin plants have anti-vitiligo activity, research reports on the improvement of vitiligo by Nigella cumin seeds are relatively rare both at home and abroad. Summary of the invention

[0004] The present invention aims to provide an alkaloid of skeleton type in Nigella glandulifera seeds and a preparation method and use thereof. The alkaloid is extracted from the seeds of Nigella glandulifera by organic solvent extraction, and then separated by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography and semi-preparative high performance liquid chromatography to finally obtain the skeleton type alkaloid. The skeleton type is determined by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy and the structure is identified. Activity test results show that the skeleton type alkaloid of the present invention can be used for the purpose of the medicine for improving the symptoms of vitiligo.

[0005] The skeleton-type alkaloids in the tuberous black cumin seeds of the present invention have a structural formula (I) of:

[0006]

[0007] The chemical name of formula (Ⅰ) is: 3,3,6,6,11,11-hexamethyl-10,12-(4-hydrogen)-4H-piperidin-4-one (4a,4b)-quinolizidine-8-one.

[0008] The method for preparing the skeleton-type alkaloids in the tuberous black cumin seeds is carried out according to the following steps:

[0009] a. The crushed tuber black seed was soaked in petroleum ether overnight for defatting, filtered, the filtrate was concentrated, the petroleum ether was recovered, the filter residue was degreased 6-10 times with petroleum ether, the filter residue was collected and dried, and the dried filter residue was soaked with 1 / 2-1 times the volume ratio of 1:1 ammonia and acetone overnight, and then dried in the shade to obtain a dry product;

[0010] b. Add 2-3 times the volume of chloroform to the dried product obtained in step a, soak overnight and extract 6-8 times, filter, collect the filtrate, and then concentrate to obtain a chloroform extract;

[0011] c. Dissolve the chloroform extract obtained in step b with 1-2 times the volume of chloroform, then add 1-3 times the volume of 0.2%-0.4% sulfuric acid solution for extraction until there is no alkaloid reaction, collect the sulfuric acid extracts and combine them, then extract the sulfuric acid extracts with chloroform for 2-4 times, recover the sulfuric acid water layer, add NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkaline solution, the alkaline solution is further extracted with chloroform, the extract is concentrated, and then, a NaOH aqueous solution is added to the alkaline solution to adjust the pH to 12, and the extraction is repeated until no alkaloid reaction occurs, and the extract is collected, concentrated, and dried to obtain total alkaloids in a dark brown semi-extract form;

[0012] d. The dark brown semi-extract total alkaloids obtained in step c are subjected to normal phase silica gel column chromatography, and the eluent is a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1, and gradient or isocratic elution is performed to collect the 60:1-30:1 fraction E, and the fraction E is subjected to reverse phase silica gel column chromatography, and gradient elution is performed using a 5%-99% methanol aqueous solution or an acetonitrile aqueous solution, and the fraction E or E 20-60 eluted with a 5%-60% methanol aqueous solution or an acetonitrile aqueous solution is collected, and E or E The 20-60 fractions are subjected to gel column chromatography, and the alkaloid color fraction is obtained by TLC analysis; then semi-preparative high performance liquid chromatography is used, and 25% acetonitrile aqueous solution, 5%-99% acetonitrile aqueous solution or 30% methanol aqueous solution is used for isocratic elution to obtain the skeleton type alkaloid separated from the seeds of Nigella cuminata of formula (Ⅰ), and its chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4 hydrogen)-4H-piperidin-4-one (4a,4b) quinolizidine-8-one.

[0013] A use of skeleton-type alkaloids in tuber black cumin seeds in the preparation of a medicine for improving vitiligo symptoms.

[0014] The invention discloses a skeleton-type alkaloid in tuberous black cumin seeds and a preparation method and use thereof. The skeleton-type alkaloid is extracted from tuberous black cumin seeds. The alkaloid can be obtained by separation and purification from tuberous black cumin seeds, or can be synthesized by a chemical modification method.

[0015] The present invention discloses a skeleton-type alkaloid in tuber black cumin seeds, a preparation method and a use thereof. The obtained alkaloid is determined by using modern spectral techniques such as high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and the structure identification process is as follows:

[0016] Structural Identification of Alkaloids:

[0017] The structure of the alkaloids was comprehensively determined by a variety of modern spectral techniques such as nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-ESI-MS), electron circular dichroism (ECD), infrared (IR), etc. The structural identification process is as follows:

[0018] The optical activity of alkaloids is: [α] 25 D -4.0 (c 0.01, MeOH); the maximum absorption wavelengths (logε) of the ultraviolet spectrum (UV) in methanol are 203.0 and 242.0 nm; the main absorption peaks of the infrared spectrum (IR) are 2989, 1676, 1586, 1535, 1371 and 1294 cm -1 ;

[0019] High resolution mass spectrometry (HR-ESI-MS) showed m / z 339.2077 [M+H] + , the calculated value is C 21 H 27 O 2 N 2 , 339.2068, the molecular formula is determined to be C 21 H 26 O 2 N 2 By H NMR spectroscopy ( 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR), proton correlation spectroscopy ( 1 H- 1 The comprehensive analysis of H COSY, gradient field proton single quantum correlation spectroscopy (HMQC), and proton multiple bond correlation (HMBC) confirmed that the alkaloid was a compound with a new skeleton type; the data of H NMR (deuterated chloroform, 600 MHz) and C NMR (deuterated chloroform, 151 MHz) are shown in Table 1;

[0020] Table 1. Alkaloids of formula (I) 1 H and 13 C NMR data [δ(ppm), J(Hz)]

[0021]

[0022]

[0023] Where s: represents a single peak. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention 1 H NMR (600 MHz, CDCl 3 ) spectrum;

[0025] Figure 2 The present invention 13 C NMR (151 MHz, CDCl 3 ) spectrum;

[0026] Figure 3 HSQC diagram of the present invention;

[0027] Figure 4 HMBC diagram of the present invention;

[0028] Figure 5 The present invention 1 H- 1 H COSY diagram;

[0029] Figure 6 The present invention1 H- 1 H NOESY diagram;

[0030] Figure 7 High-resolution mass spectrometry of the present invention.

[0031] The reagents used in the present invention are all of analytical grade, and the acetonitrile used in the high performance liquid chromatography is of HPLC grade, provided by Merck, Germany. The normal phase silica gel (100-200 mesh and 200-300 mesh) used in column chromatography is produced by Qingdao Ocean Chemical Plant; the reverse phase silica gel ODS is produced by Merck, Germany. The high performance liquid chromatography system (manufactured by Dionex, USA) includes the following configurations: P680 HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser and chameleon chromatography workstation. The preparative high performance liquid chromatography system (also manufactured by Dionex, USA) includes: P680 HPLC pump, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser and chameleon chromatography workstation. Mass spectrometry was performed using a quadrupole-time-of-flight hybrid mass spectrometer provided by Applied Biosystems, USA; NMR was performed using a VARIAN VNMRS 600MHz NMR spectrometer; ECD spectra were obtained using a Chirascan spectropolarimeter produced in the UK; and SpectraMAX M5 microplate reader.

[0032] The tuberous black seed was provided by Xinjiang Renyu Chinese Medicine Pieces Co., Ltd., with the production batch number 210443001. This batch of black seed was identified by Minde Researcher of Xinjiang Uygur Autonomous Region Food and Drug Inspection Institute in August 2022 and confirmed to be the seeds of Nigella glandulifera Freyn of the genus Nigella in the Ranunculaceae family. DETAILED DESCRIPTION

[0033] Example 1

[0034] a. Take 50 kg of tuber black seed, coarsely crush, add 3 times the volume of petroleum ether and soak for 6 times overnight to defat, then filter, concentrate the filtrate, recover the petroleum ether, and repeat the degreasing with petroleum ether 6 times (petroleum ether is reused to replenish the part of volatile loss), collect the filter residue and dry it, soak the dried residue with 1 / 2-1 times the volume ratio of 1:1 ammonia and acetone overnight, and then dry in the shade to obtain a dry product;

[0035] b. Add 2 times the volume of chloroform to the dried product obtained in step a, soak overnight and extract 6 times, extract repeatedly (chloroform is reused to replenish the volatile loss), filter, collect the filtrate, and concentrate the filtrate to obtain a chloroform extract;

[0036] c. The chloroform extract obtained in step b was dissolved with 1 volume of chloroform, and then 1 volume of 0.2% sulfuric acid solution was added for extraction until no alkaloid reaction occurred (detected by alkaloid colorimetric reagent), the sulfuric acid extract was collected, the extracts were combined, and the sulfuric acid extract was extracted twice with chloroform, the sulfuric acid aqueous layer was recovered, and NaHCO was added 3 The aqueous solution is adjusted to pH 10 to obtain an alkaline solution, the alkaline solution is further extracted with chloroform, the extract is concentrated, and then a NaOH aqueous solution is added to the alkaline solution to adjust the pH to 12, and the extraction is repeated again, and the extract is collected, concentrated and dried to obtain total alkaloids in a dark brown semi-extract form;

[0037] d. The dark brown semi-extract total alkaloids obtained in step c are subjected to normal phase silica gel column chromatography, the eluent is a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1, and gradient elution is performed, and a fraction E of 60:1-30:1 is collected, and fraction E is subjected to reverse phase silica gel column chromatography, and gradient elution is performed using a 5%-99% methanol aqueous solution, and fraction E eluted with a 5%-60% methanol aqueous solution is collected, and fraction E is subjected to gel column chromatography, and alkaloid color fractions are obtained by TLC analysis, and then semi-preparative high performance liquid chromatography is used, and 30% methanol aqueous solution is used for isocratic elution, thereby obtaining the skeleton type alkaloid in the black cumin seeds of formula (I), whose chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4 hydrogen)-4H-piperidin-4-one (4a,4b)-quinolizidine-8-one.

[0038] Example 2

[0039] a. Take 50 kg of tuber black seed, crush it, soak it in 3 volumes of petroleum ether overnight for defatting, filter, concentrate the filtrate, recover the petroleum ether, repeat the defatting 6 times (petroleum ether is reused to make up for the loss due to evaporation), collect the filter residue and dry it, soak the dried filter residue with 1 / 2-1 times the volume ratio of 1:4 of ammonia and acetone overnight, and then dry it in the shade to obtain a dry product;

[0040] b. Add 2 times the volume of chloroform to the dried product obtained in step a, soak overnight and extract 8 times, extract repeatedly (chloroform is reused to replenish the volatile loss), filter, collect the filtrate, and then concentrate to obtain a chloroform extract;

[0041] c. The chloroform extract obtained in step b was dissolved with 2 volumes of chloroform, and then 2 volumes of 0.3% sulfuric acid solution were added for extraction until no alkaloid reaction occurred (detected by alkaloid colorimetric reagent), the collected sulfuric acid extracts were combined, and the sulfuric acid extracts were extracted 3 times with chloroform, the sulfuric acid aqueous layer was recovered, and NaHCO was added 3 The aqueous solution is adjusted to pH 10 to obtain an alkaline solution, the alkaline solution is further extracted with chloroform, the extract is concentrated, and a NaOH aqueous solution is added to the alkaline solution to adjust the pH to 12, and the solution is extracted again until no alkaloid reaction occurs, the extract is collected, concentrated, and dried to obtain total alkaloids in a dark brown semi-extract form;

[0042] d. The dark brown semi-extract total alkaloids obtained in step c were subjected to normal phase silica gel column chromatography, and the eluent was a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1, and gradient elution was performed, and the 60:1-30:1 fraction was collected as the E fraction, and the E fraction was subjected to reverse phase silica gel column chromatography, and gradient elution was performed using a 5-99% acetonitrile aqueous solution, and the 5%-60% acetonitrile aqueous solution eluted fraction E20-60 , fraction E20-60 is subjected to gel column chromatography, and the alkaloid color fraction is obtained by TLC analysis; semi-preparative high performance liquid chromatography is then used, and 25% acetonitrile aqueous solution is used for isocratic elution to obtain the skeleton type alkaloid in the seeds of Nigella cumin (I), whose chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4hydrogen)-4H-piperidin-4-one (4a,4b) quinolizidine-8-one.

[0043] Example 3

[0044] a. Take 50 kg of tuber black seed, crush it, soak it in 3 volumes of petroleum ether overnight for defatting, filter, concentrate the filtrate, recover the petroleum ether, repeat the defatting 8 times (petroleum ether is reused to make up for the loss due to evaporation), collect the filter residue and dry it, soak the dried filter residue with 1 / 2-1 times the volume ratio of 1:4 of ammonia and acetone overnight, and then dry it in the shade to obtain a dry product;

[0045] b. Add 2 times the volume of chloroform to the dried product obtained in step a, soak overnight for extraction, extract repeatedly (chloroform is reused to replenish the volatile loss), collect the filtrate, and then concentrate to obtain a chloroform extract;

[0046] c. The chloroform extract obtained in step b was dissolved with 2 volumes of chloroform, and then 2 volumes of 0.4% sulfuric acid solution were added for extraction until no alkaloid reaction occurred (detected by alkaloid colorimetric reagent), the sulfuric acid extracts were collected and combined, and then the sulfuric acid extracts were extracted 4 times with chloroform to remove the non-alkaloid part (the chloroform layer is the non-alkaloid part), the sulfuric acid aqueous layer was recovered, and NaHCO3 The aqueous solution is adjusted to pH 10 to obtain an alkaline solution, the alkaline solution is further extracted with chloroform, the extract is concentrated, and then a NaOH aqueous solution is added to the alkaline solution to adjust the pH to 12, and the solution is extracted with chloroform again, and the extract is collected, concentrated and dried to obtain total alkaloids in a dark brown semi-extract form;

[0047] d. The dark brown semi-extract total alkaloids obtained in step c were subjected to normal phase silica gel column chromatography, with the eluent being a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1, and gradient elution was performed to collect the 60:1-30:1 fraction E, and fraction E was subjected to reverse phase silica gel column chromatography, with an acetonitrile aqueous solution for gradient elution, and the fraction E20-60 eluted with 5-60% acetonitrile aqueous solution was collected, and E20-60 was separated. The 0 fraction is subjected to gel column chromatography, and the alkaloid color fraction is obtained by TLC analysis; then semi-preparative high performance liquid chromatography is used, and 5%-99% acetonitrile aqueous solution is used for isocratic elution to obtain the skeleton type alkaloid separated from the seeds of Nigella cuminata of formula (Ⅰ), and its chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4hydrogen)-4H-piperidin-4-one (4a,4b) quinolizidine-8-one.

[0048] Example 4

[0049] The skeleton-type alkaloid obtained from the seeds of Nigella sativa L. in Example 1-3 (chemical name: 3,3,6,6,11,11-hexamethyl-10,12-(4-hydrogen)piperidin-4-one (4a,4b)-quinolizidine-8-one) was used in the preparation of a drug for improving the symptoms of vitiligo, especially by promoting melanin production, taking the melanin content of B16 melanoma cells as an example, to verify the potential therapeutic effect of the alkaloid;

[0050] Cell culture:

[0051] B16 cells (purchased from BeNa Culture Collection, BNCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose medium, purchased from Giboco, USA) containing 10% fetal bovine serum (FBS) (purchased from Zeta Life, USA) at 37°C and 5% CO 2 environment;

[0052] The effects on B16 cell viability and melanin production were tested as follows:

[0053] The skeleton-type alkaloids were first dissolved in dimethyl sulfoxide (DMSO), and the B16 cells in the logarithmic growth phase were selected to determine the intracellular melanin content using the NaOH lysis method. After the B16 cells in the logarithmic growth phase were digested, they were inoculated in a 6-well plate at a cell density of 2×10 5 / well (In the initial drug screening, the cell plating density corresponding to 1μM drug concentration is 1×10 5 / well) and incubated at 37°C and 5% CO 2 After the cells adhered to the wall, the cells were treated with drugs and cultured for 48 hours (the action time of 1 μM drug concentration was 72 hours in the initial drug screening); after removing the supernatant, the cells were washed twice with PBS (phosphate-balanced saline), and the cells were collected with a cell scraper, and 100 μL of cell lysis solution was added to each well; after fully lysing at 4°C for 40 minutes, the cells were centrifuged at 12,000 rpm for 20 minutes; the supernatant was transferred to an EP tube to determine the protein concentration, and 190 μL of 1 mol / L NaOH containing 10% dimethyl sulfoxide (DMSO) lysis solution was added to the precipitate, and the precipitate was fully dissolved in a water bath at 80°C for 1 hour, and then the absorbance was measured at 405 nm, and the melanin content was finally normalized with the relative protein concentration;

[0054] The calculation formula of melanin production activity is as follows:

[0055] Melanin relative content = (OD 405 Sample / protein concentration) / (OD 405 Blank / protein concentration) × 100%, each sample was measured three times to ensure the accuracy of the results;

[0056] Experimental results: The experimental results of skeleton-type alkaloids extracted from Nigella sativa seeds and positive control drug 8-methoxypsoralen (8-MOP) are shown in Table 2;

[0057] Table 2. Alkaloids of skeleton type (mean ± SD)

[0058]

[0059] Note: ****Compared with blank control group (NC), P<0.001.

[0060] The experimental results confirmed that the skeleton-type alkaloids extracted from the seeds of the tuber black cumin have a significant effect in promoting melanin production, and the effect is equivalent to that of the positive control drug; the skeleton-type alkaloids can effectively enhance the production of melanin, indicating its potential application value in the treatment of vitiligo and other related skin depigmentation diseases.

Claims

1. A skeleton-type alkaloid in tuber black cumin seeds, characterized in that The structural formula (I) of the alkaloid is: The chemical name of formula (Ⅰ) is: 3,3,6,6,11,11-hexamethyl-10,12-(4-hydrogen)-4H-piperidin-4-one (4a,4b)-quinolizidine-8-one.

2. A method for preparing the skeleton-type alkaloids in the tuberous black cumin seeds as claimed in claim 1, characterized in that: Follow these steps: a. The crushed tuber black seed was soaked in petroleum ether overnight for defatting, filtered, the filtrate was concentrated, the petroleum ether was recovered, the filter residue was degreased 6-10 times with petroleum ether, the filter residue was collected and dried, and the dried filter residue was soaked with 1 / 2-1 times the volume ratio of 1:1 ammonia and acetone overnight, and then dried in the shade to obtain a dry product; b. Add 2-3 times the volume of chloroform to the dried product obtained in step a, soak overnight and extract 6-8 times, filter, collect the filtrate, and then concentrate to obtain a chloroform extract; c. The chloroform extract obtained in step b is dissolved with 1-2 times the volume of chloroform, and then 1-3 times the volume of a 0.2%-0.4% sulfuric acid solution is added for extraction until there is no alkaloid reaction, the sulfuric acid extract is collected and combined, and then the sulfuric acid extract is extracted 2-4 times with chloroform, the sulfuric acid aqueous layer is recovered, and an aqueous NaHCO3 solution is added to adjust the pH to 10 to obtain an alkalized solution, and the alkalized solution is further extracted with chloroform, and the extract is concentrated, and then an aqueous NaOH solution is added to the alkalized solution to adjust the pH to 12, and the extract is extracted again until there is no alkaloid reaction, and the extract is collected, concentrated, and dried to obtain a dark brown semi-extract of total alkaloids; d. The dark brown semi-extract total alkaloids obtained in step c are subjected to normal phase silica gel column chromatography, and the eluent is a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1, and gradient or isocratic elution is performed to collect the 60:1-30:1 fraction E, and the fraction E is subjected to reverse phase silica gel column chromatography, and gradient elution is performed using a 5%-99% methanol aqueous solution or an acetonitrile aqueous solution, and the fraction E or E 20-60 eluted with a 5%-60% methanol aqueous solution or an acetonitrile aqueous solution is collected, and E or E The 20-60 fractions are subjected to gel column chromatography, and the alkaloid color fraction is obtained by TLC analysis; then semi-preparative high performance liquid chromatography is used, and 25% acetonitrile aqueous solution, 5%-99% acetonitrile aqueous solution or 30% methanol aqueous solution is used for isocratic elution to obtain the skeleton type alkaloid separated from the seeds of Nigella cuminata of formula (Ⅰ), and its chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4 hydrogen)-4H-piperidin-4-one (4a,4b) quinolizidine-8-one.

3. Use of a skeleton-type alkaloid in Nigella sativa seeds in the preparation of a drug for improving the symptoms of vitiligo.

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