Alkaloids of the skeleton type in nigerica seed of nigerica and preparation method and use thereof

Abstract

This invention relates to a skeleton-type alkaloid from *Nigeria rubra* seeds, its preparation method, and its uses. The chemical name of the alkaloid is 3,3,6,6,11,11-hexamethyl-10,12-(4-hydro)-4H-piperidin-4-one (4a,4b) quinolonesine-8-one. The method involves extraction from *Nigeria rubra* seeds using an organic solvent, followed by acid-soluble and alkali-precipitated total alkaloids. Further purification was then performed using normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography to obtain high-purity skeleton-type alkaloids. Its structure was confirmed by spectroscopic and mass spectrometric data analysis. Bioactivity tests were conducted on this skeleton-type alkaloid, particularly evaluating its ability to promote melanin production in B16 cells. The results showed that the alkaloid's ability to promote melanin production was comparable to the positive control, demonstrating its potential in improving vitiligo symptoms. Therefore, this skeleton-type alkaloid, as a novel lead compound, provides new possibilities for the development of anti-vitiligo drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to alkaloids of the skeleton type in black cumin seeds, their preparation methods, and uses. Background Technology

[0002] Vitiligo is a common acquired skin depigmentation disorder characterized by the selective destruction of melanocytes in the skin and hair, or a decreased ability to synthesize melanin, resulting in white patches on the skin. Research indicates that the direct cause of vitiligo is a decrease or disappearance of tyrosinase activity within melanocytes, thereby hindering melanin synthesis. Therefore, promoting melanin synthesis in the skin is considered an effective approach to treating vitiligo. Developing new drugs for treating vitiligo by promoting melanin synthesis in melanoma cells (specifically, B16) has become a new research direction.

[0003] *Nigella glandulifera* Freyn is an annual herb belonging to the genus *Nigella* in the family Ranunculaceae. This genus contains approximately 20 species, three of which are primarily used medicinally: *Nigella sative* (also known as domestic nigella) and *Nigella demascena* L., and *Nigella glandulifera* Freyn (also known as glandular-haired nigella). As a traditional folk remedy, the seeds of *Nigella glandulifera* are generally used to treat various ailments, including relieving pain and wind, tonifying the kidneys and brain, promoting diuresis and sweating, stimulating digestion, strengthening the stomach, fighting worms, and treating asthma. Modern pharmacological studies have also shown that *Nigella glandulifera* seeds possess multiple effects, including regulating menstruation, anti-oxidation, anti-inflammation, anti-tumor, protecting internal organs, protecting the nervous system, lowering blood sugar, and improving respiratory diseases. Although existing studies have shown that plants of the genus *Nigella* have anti-vitiligo activity, research reports on the improvement of vitiligo by *Nigella nobilis* seed monomers are rare both domestically and internationally. Summary of the Invention

[0004] The present invention aims to provide a skeleton-type alkaloid from *Nigella glandulifera* seeds, its preparation method, and its uses. This alkaloid is obtained from *Nigella glandulifera* seeds through organic solvent extraction, followed by separation using normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography to finally obtain the skeleton-type alkaloid. Its skeleton type and structure are determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Activity testing results indicate that the skeleton-type alkaloid described in this invention can be used in drugs for improving vitiligo symptoms.

[0005] The present invention discloses a skeleton-type alkaloid from *Nigeria nigra* seeds, the structural formula (I) of which is:

[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) quinoloxetine-8-one.

[0008] The preparation method of the skeleton-type alkaloids in the seeds of *Nigeria nigra* is carried out according to the following steps:

[0009] a. Soak crushed black cumin seeds in petroleum ether overnight to degrease, filter, concentrate the filtrate, recover the petroleum ether, and repeat the degreasing process with petroleum ether 6-10 times. Collect the filter residue and dry it. Soak the dried filter residue in a mixture of ammonia and acetone at a volume ratio of 1 / 2 to 1:1 overnight, and then air dry to obtain the dried product.

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

[0011] c. Dissolve the chloroform extract obtained in step b in 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 no alkaloid reaction occurs. Collect the sulfuric acid extracts and combine them. Extract the sulfuric acid extracts with chloroform 2-4 times, recover the sulfuric acid aqueous layer, add NaHCO3 aqueous solution to adjust the pH to 10 to obtain an alkalized solution. Further extract the alkalized solution with chloroform, concentrate the extract, then add NaOH aqueous solution to the alkalized solution to adjust the pH to 12, and extract again until no alkaloid reaction occurs. Collect the extract, concentrate it, and dry it to obtain a dark brown semi-paste-like total alkaloids.

[0012] d. The dark brown semi-paste-like total alkaloids obtained in step c are subjected to normal-phase silica gel column chromatography with a gradient or isocratic elution using a mixture of petroleum ether-acetone and dichloromethane-methanol (volume ratio 100:0-0:1). The fraction E (60:1-30:1) is collected. Fraction E is then subjected to reversed-phase silica gel column chromatography with a gradient elution using 5%-99% methanol-water or acetonitrile-water solutions. The fraction E eluted with 5%-60% methanol-water or acetonitrile-water solutions, or E20-60, is collected. 20-60 fractions were subjected to gel column chromatography, and the alkaloid color fraction was obtained by TLC analysis. Then, semi-preparative high performance liquid chromatography was used to perform isocratic elution with 25% acetonitrile aqueous solution, 5%-99% acetonitrile aqueous solution or 30% methanol aqueous solution to obtain the skeleton type alkaloid isolated from the seeds of *Nigrum nigrum* of formula (I), whose chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4H)-piperidin-4-one (4a,4b) quinoloxetine-8-one.

[0013] The use of a skeleton-type alkaloid from *Nigrum nigrum* seeds in the preparation of a drug to improve the symptoms of vitiligo.

[0014] The present invention relates to a skeleton-type alkaloid from *Nigrum rubrum* seeds, its preparation method, and its uses. This alkaloid is extracted from *Nigrum rubrum* seeds and can be obtained by separation and purification from *Nigrum rubrum* seeds or by synthesis through chemical modification methods.

[0015] This invention relates to a skeleton-type alkaloid from *Nigeria nigra* seeds, its preparation method, and its uses. The obtained alkaloid's compound structure was determined using modern spectroscopic techniques such as high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy. The structural identification process is as follows:

[0016] Structural identification of alkaloids:

[0017] The structure of the alkaloid was determined using a combination of modern spectroscopic techniques, including nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-ESI-MS), electron circular dichroism (ECD), and infrared spectroscopy (IR). 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 (UV) spectrum in methanol are 203.0 and 242.0 nm; the main absorption peaks of the infrared (IR) spectrum are 2989, 1676, 1586, 1535, 1371 and 1294 cm⁻¹. -1 ;

[0019] High-resolution mass spectrometry (HR-ESI-MS) showed m / z 339.2077 [M+H]. + The calculated value is C 21 H 27 O2N2, 339.2068, molecular formula determined to be C 21 H 26 O2N2. Spectroscopy using nuclear magnetic resonance (NMR) hydrogen spectroscopy (… 1 H-NMR), carbon nuclear magnetic resonance (NMR) 13 C-NMR), proton correlation spectrum ( 1 H- 1 The comprehensive analysis of H COSY, gradient field hydrogen nucleus single quantum correlation (HMQC), and hydrogen nucleus multi-bond correlation (HMBC) confirmed that the alkaloid is a compound with a new skeleton type; the data of the proton NMR spectrum (deuterated chloroform, 600MHz) and carbon NMR spectrum (deuterated chloroform, 151MHz) are detailed 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. Attached Figure Description

[0024] Figure 1 The present invention 1 1H NMR (600MHz, CDCl3) spectrum;

[0025] Figure 2 The present invention 13 C NMR (151MHz, CDCl3) spectrum;

[0026] Figure 3 The HSQC diagram of this invention;

[0027] Figure 4 The HMBC diagram of this invention;

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

[0029] Figure 6 The present invention 1 H- 1 H NOESY plot;

[0030] Figure 7The high-resolution mass spectrum of this invention.

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

[0032] The seeds of *Nigella glandulifera* Freyn were supplied by Xinjiang Renyu Traditional Chinese Medicine Pieces Co., Ltd., with production batch number 210443001. In August 2022, this batch of seeds was identified by Researcher Min De of the Xinjiang Uygur Autonomous Region Food and Drug Inspection Institute as seeds of *Nigella glandulifera* Freyn, belonging to the genus *Nigella* in the family Ranunculaceae. Detailed Implementation

[0033] Example 1

[0034] a. Take 50 kg of black cumin seeds, coarsely crush them, add 3 times their volume of petroleum ether and soak them 6 times overnight to degrease them, then filter them, concentrate the filtrate, recover the petroleum ether, and degrease the filter residue 6 times with petroleum ether again (the petroleum ether is reused to replenish the part lost through volatilization), collect the filter residue and dry it, soak the dried residue in a 1 / 2-1 times volume of ammonia water and acetone mixture overnight, and then air dry it to obtain the dried product;

[0035] b. Add 2 times the volume of chloroform to the dried material obtained in step a, soak overnight and extract 6 times. Repeat the extraction (reuse chloroform to replenish the part lost through volatilization), filter, collect the filtrate, concentrate the filtrate, and obtain the chloroform extract.

[0036] c. Dissolve the chloroform extract obtained in step b in 1 volume of chloroform, then add 1 volume of 0.2% sulfuric acid solution for extraction until no alkaloid reaction occurs (detected by alkaloid colorimetric reagent). Collect the sulfuric acid extract, combine the extracts, and extract the sulfuric acid extract twice more with chloroform. Recover the sulfuric acid aqueous layer, add NaHCO3 aqueous solution to adjust the pH to 10 to obtain an alkalized solution. Further extract the alkalized solution with chloroform, concentrate the extract, and then add NaOH aqueous solution to the alkalized solution to adjust the pH to 12. Repeat the extraction process, collect the extract, concentrate and dry to obtain a dark brown semi-paste-like total alkaloid.

[0037] d. The total alkaloids in the dark brown semi-paste obtained in step c were subjected to normal-phase silica gel column chromatography with a gradient elution of a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1. The fraction E of 60:1-30:1 was collected. The fraction E was then subjected to reverse-phase silica gel column chromatography with a gradient elution of 5%-99% methanol aqueous solution. The fraction E eluted with 5%-60% methanol aqueous solution was collected. The fraction E was subjected to gel column chromatography, and the alkaloid color fraction was obtained by TLC analysis. Then, semi-preparative high-performance liquid chromatography was performed with isocratic elution using 30% methanol aqueous solution to obtain the skeleton type alkaloids of formula (Ⅰ) from the seeds of *Nigrum nodosum*, with the chemical name 3,3,6,6,11,11-hexamethyl-10,12-(4-hydro)-4H-piperidin-4-one (4a,4b) quinoloxetine-8-one.

[0038] Example 2

[0039] a. Take 50 kg of black cumin seeds, crush them, soak them overnight in 3 times their volume of petroleum ether to degrease them, filter them, concentrate the filtrate, recover the petroleum ether, and repeat the degreasing process 6 times on the filter residue (the petroleum ether is reused to replenish the part lost through volatilization). Collect the filter residue and dry it. Soak the dried filter residue overnight in a mixture of ammonia water and acetone with a volume ratio of 1 / 2 to 1:4, and then air dry it to obtain the dried product.

[0040] b. Add 2 times the volume of chloroform to the dried material obtained in step a, soak overnight and extract 8 times. Repeat the extraction (reuse chloroform to replenish the part lost through volatilization), filter, collect the filtrate, and then concentrate to obtain the chloroform extract.

[0041] c. Dissolve the chloroform extract obtained in step b in 2 times the volume of chloroform, then add 2 times the volume of 0.3% sulfuric acid solution for extraction until no alkaloid reaction occurs (detected by alkaloid colorimetric reagent). Combine the collected sulfuric acid extracts, and extract the sulfuric acid extract three more times with chloroform. Recover the sulfuric acid aqueous layer, add NaHCO3 aqueous solution to adjust the pH to 10 to obtain an alkalized solution. Further extract the alkalized solution with chloroform, concentrate the extract, and then add NaOH aqueous solution to the alkalized solution to adjust the pH to 12. Extract again until no alkaloid reaction occurs. Collect the extract, concentrate, and dry to obtain a dark brown semi-paste-like total alkaloid.

[0042] d. The dark brown semi-paste-like total alkaloids obtained in step c are subjected to normal-phase silica gel column chromatography with a gradient elution of a mixture of petroleum ether-acetone and dichloromethane-methanol at a volume ratio of 100:0-0:1. The fraction 60:1-30:1 is collected as fraction E. Fraction E is then subjected to reverse-phase silica gel column chromatography with a gradient elution of 5-99% acetonitrile aqueous solution. The fraction E20-60 eluted with 5%-60% acetonitrile aqueous solution is collected. The E20-60 fraction was subjected to gel column chromatography, and the alkaloid color fraction was obtained by TLC analysis. Then, semi-preparative high performance liquid chromatography was used to elute isocratically with 25% acetonitrile aqueous solution to obtain the skeleton type alkaloid of formula (Ⅰ) in black cumin seeds, with the chemical name 3,3,6,6,11,11-hexamethyl-10,12-(4hydro)-4H-piperidin-4-one (4a,4b) quinoloxetine-8-one.

[0043] Example 3

[0044] a. Take 50 kg of black cumin seeds, crush them, soak them overnight in 3 times their volume of petroleum ether to degrease them, filter them, concentrate the filtrate, recover the petroleum ether, and repeat the degreasing process 8 times on the filter residue (the petroleum ether is reused to replenish the part lost through volatilization). Collect the filter residue and dry it. Soak the dried filter residue overnight in a mixture of ammonia water and acetone with a volume ratio of 1 / 2 to 1:4, and then air dry it to obtain the dried product.

[0045] b. Add 2 times the volume of chloroform to the dried material obtained in step a, soak overnight and extract, repeat the extraction (reuse chloroform to replenish the part lost through volatilization), collect the filtrate, and then concentrate it to obtain the chloroform extract;

[0046] c. Dissolve the chloroform extract obtained in step b in 2 times the volume of chloroform, then add 2 times the volume of 0.4% sulfuric acid solution for extraction until no alkaloid reaction occurs (detected by alkaloid color reagent). Collect the sulfuric acid extracts and combine them. Extract the sulfuric acid extracts four times with chloroform to remove the non-alkaloid portion (the chloroform layer is the non-alkaloid portion). Recover the sulfuric acid aqueous layer and adjust the pH to 10 with NaHCO3 aqueous solution to obtain an alkalized solution. Further extract the alkalized solution with chloroform and concentrate the extract. Then, add NaOH aqueous solution to the alkalized solution to adjust the pH to 12 and extract with chloroform again. Collect the extract, concentrate and dry it to obtain a dark brown semi-paste-like total alkaloid.

[0047] d. The dark brown semi-paste-like total alkaloids obtained in step c are subjected to normal-phase silica gel column chromatography with a gradient elution of a mixture of petroleum ether-acetone and dichloromethane-methanol (v / v) at a ratio of 100:0-0:1. The fraction E (60:1-30:1) is collected. Fraction E is then subjected to reverse-phase silica gel column chromatography with a gradient elution of acetonitrile-water solution. The fraction E20-60 eluted with 5-60% acetonitrile-water solution is collected. The fraction was subjected to gel column chromatography, and the alkaloid color fraction was obtained by TLC analysis. Then, semi-preparative high performance liquid chromatography was used to perform isocratic elution with 5%-99% acetonitrile aqueous solution to obtain the skeleton type alkaloid isolated from the seeds of Nori-nodosa (I), whose chemical name is 3,3,6,6,11,11-hexamethyl-10,12-(4-hydrogen)-4H-piperidin-4-one (4a,4b) quinoloxetine-8-one.

[0048] Example 4

[0049] The application of the skeleton-type alkaloid (chemical name 3,3,6,6,11,11-hexamethyl-10,12-(4-hydro)piperidin-4-one (4a,4b) quinoloxetine-8-one) obtained from Nigella oleifera seeds in Examples 1-3 in the preparation of a drug to improve the symptoms of vitiligo, in particular by promoting melanin production, using the melanin content of B16 melanoma cells as an example, verified the potential therapeutic effect of this 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 a temperature of 37°C and a CO2 environment.

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

[0053] Skeletal alkaloids were first dissolved in dimethyl sulfoxide (DMSO). B16 cells in logarithmic growth phase were selected, and intracellular melanin content was determined using the NaOH lysis method. After digestion, the logarithmic growth phase B16 cells were seeded in 6-well plates at a cell density of 2 × 10⁶ cells / well. 5 Cells / well (During initial drug screening, the cell plating density corresponding to a 1 μM drug concentration is 1 × 10⁻⁶ cells / well) 5 (each hole), and at a temperature of 37°C and 5% CO2 2 Cells were cultured overnight under the specified conditions. After cell adhesion, they were treated with the drug and cultured for another 48 hours (for initial drug screening, the treatment time for a 1 μM drug concentration was 72 hours). After removing the supernatant, the cells were washed twice with PBS (phosphate-balanced saline), and the cells were collected using a cell scraper. 100 μL of cell lysis buffer was added to each well. After complete lysis 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. 190 μL of 1 mol / L NaOH containing 10% dimethyl sulfoxide (DMSO) lysis buffer was added to the precipitate, and the cells were incubated in a water bath at 80°C for 1 hour to fully dissolve the precipitate. The absorbance was then measured at 405 nm. The melanin content was finally normalized to the relative protein concentration.

[0054] The formula for calculating melanin-promoting activity is as follows:

[0055] Relative melanin content = (OD 405 (sample / protein concentration) / (OD) 405 (blank / protein concentration) × 100%, each sample was measured 3 times to ensure the accuracy of the results;

[0056] Experimental results: The results of the experiments on the skeleton-type alkaloids extracted from Nigella sativa seeds and the positive control drug 8-methoxypsoralen (8-MOP) are detailed in Table 2.

[0057] Table 2. Alkaloids by Skeletal Type (Mean ± Standard Deviation)

[0058]

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

[0060] Experimental results confirmed that the skeleton-type alkaloids extracted from the seeds of *Nigeria nigra* have a significant effect on promoting melanin production, with an effect comparable to that of the positive control drug. The skeleton-type alkaloids can effectively enhance melanin production, indicating their potential application value in the treatment of vitiligo and other related skin depigmentation diseases.

Claims

1. A skeleton-type alkaloid from *Nigeria nigra* seeds, characterized in that... The structural formula (Ⅰ) of this alkaloid is: （Ⅰ）。 2. A method for preparing a skeleton-type alkaloid from *Nigeria nigra* seeds as described in claim 1, characterized in that, Follow these steps: a. Soak crushed black cumin seeds in petroleum ether overnight to degrease, filter, concentrate the filtrate, recover the petroleum ether, and repeat the degreasing process with petroleum ether 6-10 times. Collect the filter residue and dry it. Soak the dried filter residue in a mixture of ammonia and acetone at a volume ratio of 1 / 2 to 1:1 overnight, and then air dry to obtain the dried product. b. Add 2-3 times the volume of chloroform to the dried material obtained in step a, soak overnight and extract 6-8 times, filter, collect the filtrate, and then concentrate to obtain the chloroform extract; c. Dissolve the chloroform extract obtained in step b in 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 no alkaloid reaction occurs. Collect the sulfuric acid extracts and combine them. Extract the sulfuric acid extracts with chloroform 2-4 times, recover the sulfuric acid aqueous layer, add NaHCO3 aqueous solution to adjust the pH to 10 to obtain an alkalized solution. Further extract the alkalized solution with chloroform, concentrate the extract, then add NaOH aqueous solution to the alkalized solution to adjust the pH to 12, and extract again until no alkaloid reaction occurs. Collect the extract, concentrate it, and dry it to obtain a dark brown semi-paste-like total alkaloids. d. The total alkaloids in the dark brown semi-paste obtained in step c were subjected to normal-phase silica gel column chromatography with a mixture of petroleum ether-acetone and dichloromethane-methanol in a volume ratio of 100:0-0:1 for gradient or isocratic elution. The fraction E of 60:1-30:1 was collected. The fraction E was then subjected to reverse-phase silica gel column chromatography with gradient elution using 5%-99% methanol aqueous solution or acetonitrile aqueous solution. The fraction E or E20-60 eluted with 5%-60% methanol aqueous solution or acetonitrile aqueous solution was collected. The fraction E or E20-60 was subjected to gel column chromatography and the alkaloid colorimetric fraction was obtained by TLC analysis. Then, semi-preparative high-performance liquid chromatography was used with isocratic elution using 25% acetonitrile aqueous solution, 5%-99% acetonitrile aqueous solution or 30% methanol aqueous solution to obtain the skeleton-type alkaloids isolated from the seeds of *Nigrum nodosum* (I).

3. The use of a skeleton-type alkaloid from *Nigeria nigra* seeds in the preparation of a drug for improving vitiligo symptoms, wherein the structural formula (Ⅰ) of the skeleton-type alkaloid from *Nigeria nigra* seeds is: （Ⅰ）。

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