CN109364073A - The purposes in drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma - Google Patents
The purposes in drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma Download PDFInfo
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- CN109364073A CN109364073A CN201811284680.9A CN201811284680A CN109364073A CN 109364073 A CN109364073 A CN 109364073A CN 201811284680 A CN201811284680 A CN 201811284680A CN 109364073 A CN109364073 A CN 109364073A
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- Prior art keywords
- piperlongumine
- alkaloid
- drug
- melanin production
- leucoderma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
- A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
- C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
- C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
- C07D317/60—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
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- Medicinal Chemistry (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
The purposes in drug for promoting melanin production in preparation treatment leucoderma the present invention relates to a kind of piperlongumine alkaloid, the amide alkaloid class compound is chavicine, funing, vacation piperlongumine A and false piperlongumine C, and the measurement of melanin production and tyrosinase activity in Murine melanoma B16 cells is carried out to it, the result shows that: the piperlongumine alkaloid can promote melanin production and tyrosinase activity in various degree, can be used for preparing the drug for the treatment of leucoderma.Benign species basis is provided to treat the drug development of leucoderma.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to a kind of piperlongumine alkaloid promotees in preparation treatment leucoderma
Purposes into the drug of melanin production.
Background technique
Fructus piperis longi (Piper longum Linn.) is the perennial herbaceous stem of Piperaceae (Piperaceae) Piper (Piper)
Liana is grown on height above sea level 200 to the river valley of 1000m, the damp-heat area of pelvic floor hernia, in Yunnan Province of China, Vietnam, Ma Laixi
The ground such as Asia, Philippine, India, Indonesia, Nepal, Bhutan, Sri Lanka are distributed, in China Guangdong, Guangxi, Yunnan
Etc. ground have cultivation, resource is very rich.The fruit ear of fructus piperis longi harvests when by green blackening, removes impurity, dries resulting drying
Nearly mature or mature fruit cluster is the medicinal herbs most in use in Chinese medicine, anaesthetic, Tibetan medicine, dimension medicine and India's Ayurveda medicine, has anti-blood
Platelet cohesion, antidepression, anti-diabetic, anti-epileptic, anti-asthma, anti-inflammatory, immunological regulation, lipidemia, anti-infective, anti-Parkinson
A variety of physiological activity such as disease, antiatherosclerosis, antibacterial and radiation protection.Amide alkaloid is the feature in pepper platymiscium
Property secondary metabolite is increasingly becoming the research hotspot of field of medicinal chemistry due to its significant anti-tumor activity in recent years.
Leucoderma is a kind of common depigmentation skin disease, and clinical manifestation forms white for depigmentation at skin lesion
Spot, mainly since the melanocyte of skin and hair is reduced or defunctionalization causes caused by depigmentation.Tyrosinase
It is widely present in microorganism, animals and plants and human body, is the key enzyme of B16 cell, junket in the melanocyte of skin and hair follicle
Propylhomoserin enzymatic activity lowers or disappears, and the progressive that will lead to melanosome generation is reduced or disappeared, and causes limitation or general hair property support
Pigmentosa lesion, so as to cause leucoderma.The mechanism of leucoderma is still unclear at present, and treatment method and means are also a variety of more
Sample.By promoting tyrosinase activity to increase melanin content, so that inducing secondary color is treatment leucoderma medicament research and development one important
Approach.
Summary of the invention
The purpose of the present invention is to provide a kind of piperlongumine alkaloids to promote melanin raw in preparation treatment leucoderma
At drug in purposes.The amide alkaloid class compound is chavicine, funing, vacation piperlongumine A and vacation Bi
Roots of grass amide C, and the measurement of melanin production and tyrosinase activity in Murine melanoma B16 cells has been carried out to it, as a result
Show: the amide alkaloid class compound can promote melanin production and tyrosinase activity in various degree, can be used for
The drug of preparation treatment leucoderma.Benign species basis is provided to treat the drug development of leucoderma.
A kind of piperlongumine alkaloid of the present invention promotes the drug of melanin production in preparation treatment leucoderma
In purposes.
The piperlongumine Alkaloid refers to the list of chavicine, funing, vacation piperlongumine A and false piperlongumine C
Body.
A kind of piperlongumine alkaloid of the present invention promotes the drug of melanin production in preparation treatment leucoderma
In purposes, wherein the piperlongumine Alkaloid is chavicine (isopiperine), funing
(piperlonguminine), vacation piperlongumine A (retrofractamide A) and false piperlongumine C
(retrofractamide C) is the amide alkaloid that applicant separates identification for 2017 from fructus piperis longi, and structure is as follows, arrives
So far, the promotion melanin production of this Alkaloid and tyrosinase activity and the application as drug have not been reported.
A kind of piperlongumine alkaloid of the present invention promotes the drug of melanin production in preparation treatment leucoderma
In purposes, wherein the piperlongumine Alkaloid separate identification from the fruit ear of fructus piperis longi, concrete operations are by following step
It is rapid to carry out:
A, dry 4.5 kilograms of fructus piperis longi fruit ear are crushed, ethyl alcohol cold soaking extraction 3 times at room temperature for being 95% with concentration, often
It is secondary to use 20L ethyl alcohol, extracting solution is used into Rotary Evaporators evaporated under reduced pressure under the conditions of temperature 45 C, obtains 450 grams of extract；
B, step a is extract obtained water-dispersible, petroleum ether, ethyl acetate, n-butanol is added and successively extracts 3-5 times,
By extract liquor evaporated under reduced pressure, petroleum ether, ethyl acetate and n-butyl alcohol extract are respectively obtained；
C, by the positive silica gel column chromatography separation of the resulting acetic acid ethyl ester extract of step b, with volume ratio 100:0-1:1
Chloroform-methanol carry out gradient elution, obtain 30 flow point Fr1-Fr30, flow point is analyzed through silica gel thin-layer chromatography (TLC), will
Flow point Fr1-Fr8 merges, and obtains merging flow point F1；
D, the positive silica gel column chromatography separation of flow point F1 will be merged, with petroleum ether-acetic acid second of volume ratio 100:0-1:1
Ester carries out gradient elution, obtains 99 flow point F1-1-F1-99；
E, it will be crystallized in flow point F1-47 and F1-56, and purify to obtain the different pepper of monomeric compound using the method for recrystallization
Alkali (isopiperine) and funing (piperlonguminine)；
F, it is analyzed through silica gel thin-layer chromatography (TLC), flow point F1-81-F1-88 is merged, obtain merging flow point F2；
G, flow point F2 will be merged to be separated with reverse phase C-18 spherical silica gel column chromatography, with volume ratio 40:60-100:0's
Methanol-water carries out gradient elution, obtains 220 flow point F2-1-F2-220；
H, it is analyzed through silica gel thin-layer chromatography (TLC), flow point F2-30-F2-51 is merged, using partly preparing high-efficient liquid phase color
Spectrum is isolated and purified, and is carried out gradient elution with the methanol-water of volume ratio 58:42-67:33, is obtained compound vacation piperlongumine
A (retrofractamide A) and vacation piperlongumine C (retrofractamide C).
Detailed description of the invention
Fig. 1 is the shadow of chavicine of the present invention (isopiperine) to melanin genesis in Murine melanoma B16 cells
It rings, P < 0.01 * P < 0.05, * *；
Fig. 2 is funing of the present invention (piperlonguminine) to melanin genesis in Murine melanoma B16 cells
It influences, P < 0.05 *；
Fig. 3 is present invention vacation piperlongumine A (retrofractamide A) to melanocyte in Murine melanoma B16 cells
The influence of synthesis, P < 0.01 * *；
Fig. 4 is vacation piperlongumine C (retrofractamide C) of the present invention to melanocyte in Murine melanoma B16 cells
The influence of synthesis, P < 0.01 * P < 0.05, * *；
Fig. 5 is chavicine of the present invention (isopiperine) to tyrosinase activity in Murine melanoma B16 cells
It influences, P < 0.01 * *；
Fig. 6 is funing of the present invention (piperlonguminine) to tyrosine enzyme activity in Murine melanoma B16 cells
The influence of property, P < 0.05 *；
Fig. 7 is vacation piperlongumine A (retrofractamide A) of the present invention to junket ammonia in Murine melanoma B16 cells
The influence of phytase activity, P < 0.05 *；
Fig. 8 is present invention vacation piperlongumine C (retrofractamide C) to junket ammonia in Murine melanoma B16 cells
The influence of phytase activity, P < 0.01 * *.
Specific embodiment
Embodiment
Material and reagent: dimethyl sulfoxide (DMSO), L-3,4- Dihydroxyphenylalanine (L-DOPA), mushroom tyrosine
Enzyme and positive control drug 8-methoxyposoralen (8-methoxypsoralen, 8-MOP) are purchased from Sigma Co., USA.DMEM training
Feeding base, trypsase, mycillin are dual anti-and trypan blue dye liquor is purchased from U.S. Gibco company.Fetal calf serum is purchased from Israel
Biological Industries company；
Cell strain: Murine melanoma B16 cells are purchased from Shanghai Cell Bank of the Chinese Academy of Sciences；
Cell culture: recovery mouse black-in tumor cell B16 (passage freezes), complete medium culture to cell confluency degree reach
When to 80% or more, culture medium is abandoned, 1 × phosphate buffer (PBS) washs 1 time, and 0.5-2 milli is added according to culture area size
Pancreatin digestion is risen, microscopic observation cell edges start (about 1 minute) when shrinkage is rounded, and gently piping and druming makes after culture medium termination digestion
Cell takes off wall, and cell counter carries out cell count, adjusts cell density to 2.0 × 105 cells/mls, is inoculated in diameter
In 150 millimeters of Tissue Culture Dish, 37 DEG C of temperature are set, 5% CO2Culture in incubator；
Experimental method:
Melanin content measurement: intracellular melanin content is measured according to sodium hydroxide dissolution method, will be in logarithmic growth
The B16 cell dissociation of phase, is inoculated in 6 orifice plates, concentration is 2 × 105 cells/mls, and every hole adds 2 milliliters of cell suspending liquids,
It is placed in the CO of 37 DEG C of temperature, 5%2It is cultivated 12 hours in incubator；The medication after cell is completely adherent, and continue culture 48
Hour；Supernatant is abandoned, after cleaning 2 times with phosphate buffer (PBS)；100 microlitres of cell pyrolysis liquids are added in every hole, use cytobrush
Cell is taken off into wall, then 12,000rpm is centrifuged 20 minutes；Supernatant is taken to measure protein concentration；150 microlitres of 1M are added in sediment
Sodium hydroxide (NaOH) solution containing 10% dimethyl sulfoxide (DMSO), dissolved 1 hour at 80 DEG C of temperature, and in 405nm
The absorbance of place's measurement dissolution melanin；Final melanin content is normalized with relative protein concentration；Melanocyte is calculated to contain relatively
Amount, melanocyte relative amount=melanocyte OD405/ protein concentration C, melanogenesis activity ratio=experimental group melanocyte relative amount/solvent
Control group melanocyte relative amount × 100%；
Tyrosinase activity measurement: intracellular tyrosinase activity uses L-3,4- Dihydroxyphenylalanine (L-
DOPA) oxidizing process detects: it will be in the B16 cell dissociation of logarithmic growth phase, is inoculated in 12 orifice plates, concentration is 3 × 105/ml,
Every hole adds 1 milliliter of cell suspending liquid, is placed in the CO of 37 DEG C of temperature, 5%2It is cultivated 12 hours in incubator；It is pasted completely to cell
Medication after wall, and continue culture at 24 hours；Liquid is discarded supernatant, phosphate buffer (PBS) washs 2 times, and every hole, which is added, to be contained
100 microlitres of lysate of 1% NaTDC and 1% Triton X-100 (Triton X-100), at 80 DEG C of temperature
Cracking 30 minutes；After being put into 4 DEG C of temperature dissolutions, then it is centrifuged 15 minutes with per minute 12,000 turns of speed；Take supernatant solution
90 microlitres of 96 orifice plates of addition in, every hole is added after 10 microlitres of L-DOPAs (L-DOPA) mix in temperature
37 DEG C are incubated for 60 minutes in the dark, and absorbance is detected at 490nm；Remaining supernatant is used to measure protein concentration；Most
Whole tyrosinase content is normalized with opposite protein concentration；Calculate tyrosinase activity, tyrosinase relative amount=junket
Propylhomoserin enzyme OD490/ protein concentration C, tyrosinase relative activity=experimental group tyrosinase relative amount/solvent control group junket ammonia
Sour enzyme relative amount × 100%；
Experimental result: melanocyte in Murine melanoma B16 cells is closed from amide alkaloid isolated in fructus piperis longi
At and the influence of tyrosinase activity see Tables 1 and 2:
Influence of 1. compound of table to melanin genesis in Murine melanoma B16 cells
Influence of 2. compound of table to tyrosinase activity in Murine melanoma B16 cells
As seen from table, chavicine (isopiperine), funing (piperlonguminine), false piperlongumine A
(retrofractamide A) and vacation piperlongumine C (retrofractamide C) can be remarkably promoted with concentrationdependent manner
Melanin content generation and tyrosinase activity in Murine melanoma B16 cells.
Claims (2)
1. the purposes in the drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma.
2. purposes as described in claim 1, it is characterised in that: the piperlongumine Alkaloid refers to chavicine, fructus piperis longi
Rather, the monomer of vacation piperlongumine A and false piperlongumine C.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811284680.9A CN109364073A (en) | 2018-10-31 | 2018-10-31 | The purposes in drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma |
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| CN201811284680.9A CN109364073A (en) | 2018-10-31 | 2018-10-31 | The purposes in drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109730006A (en) * | 2019-03-19 | 2019-05-10 | 西华大学 | The method for improving Animal melanin content |
| CN112266352A (en) * | 2020-11-20 | 2021-01-26 | 绍兴文理学院 | Piperlongumine derivative and preparation method and application thereof |
| CN114028389A (en) * | 2021-11-25 | 2022-02-11 | 中国科学院新疆理化技术研究所 | Application of three virtually screened compounds based on molecular docking in preparation of anti-vitiligo drugs |
-
2018
- 2018-10-31 CN CN201811284680.9A patent/CN109364073A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109730006A (en) * | 2019-03-19 | 2019-05-10 | 西华大学 | The method for improving Animal melanin content |
| CN112266352A (en) * | 2020-11-20 | 2021-01-26 | 绍兴文理学院 | Piperlongumine derivative and preparation method and application thereof |
| CN114028389A (en) * | 2021-11-25 | 2022-02-11 | 中国科学院新疆理化技术研究所 | Application of three virtually screened compounds based on molecular docking in preparation of anti-vitiligo drugs |
| CN114028389B (en) * | 2021-11-25 | 2024-02-09 | 中国科学院新疆理化技术研究所 | Application of three compounds based on virtual screening of molecular docking in preparation of anti-vitiligo drugs |
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Application publication date: 20190222 |