WO2017185899A1 - 组合物、藏红花色素类活性部位及其用途 - Google Patents

组合物、藏红花色素类活性部位及其用途 Download PDF

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WO2017185899A1
WO2017185899A1 PCT/CN2017/076910 CN2017076910W WO2017185899A1 WO 2017185899 A1 WO2017185899 A1 WO 2017185899A1 CN 2017076910 W CN2017076910 W CN 2017076910W WO 2017185899 A1 WO2017185899 A1 WO 2017185899A1
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saffron
gentiobioside
active site
acid
ethanol
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PCT/CN2017/076910
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English (en)
French (fr)
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姚新生
张丹
于洋
鲍秀琦
倪阳
臧彩霞
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暨南大学
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Priority to EP17788552.2A priority Critical patent/EP3449924B1/en
Priority to US16/097,247 priority patent/US10851129B2/en
Priority to JP2018556347A priority patent/JP6782790B2/ja
Priority to AU2017255362A priority patent/AU2017255362B2/en
Publication of WO2017185899A1 publication Critical patent/WO2017185899A1/zh

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/74—Rubiaceae (Madder family)
    • A61K36/744—Gardenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the present invention relates to a composition, and to a traditional Chinese medicine extract, in particular to an active part of saffron pigment extracted from gardenia and use thereof for preventing and treating diseases such as Alzheimer's disease More specifically, it relates to an application of a saffron saffron pigment active site and an active ingredient thereof for the preparation of a medicament or a health care product for preventing and treating a disease associated with Alzheimer's disease such as Alzheimer's disease.
  • Saffron pigment is a kind of water-soluble carotenoid with unique structure, including saffronic acid and its sugar ester combined with different glycosyl groups. It is a common pigment component in saffron and hazelnut. Due to its good water solubility, saffron pigment is widely used as a coloring agent for alcohol, dishes and pastries. A number of studies have shown that saffron crude extract, gardenia yellow pigment and monomer components crocin, saffronic acid and other protection in the central nervous system [1-4] , cardiovascular and cerebrovascular system protection [5-6] , antagonizing malignant tumors [7-9] and other aspects showed high pharmacological activity of low toxicity.
  • Saffron is native to southern Europe, the Mediterranean and Iran. Among them, Iranian saffron production accounts for 95% of the world's total. China's Zhejiang, Jiangsu, Shandong, Beijing and other places have a small amount of cultivation. Saffron is used as a stigma, and its yield is extremely low (less than 1 kg per mu). It is expensive ($2,000/kg) and is known as “plant gold”. With the increasing demand for medicinal and edible saffron pigments, it is of great significance to find and discover other plants rich in saffron pigments.
  • Scorpion also known as hawthorn, scorpion, etc.
  • the scorpion is contained in the "Shen Nong's Herbal Classic", which has been recorded in Chinese Pharmacopoeia and Materia Medica. It is the first batch of medicine and food resources promulgated by the Ministry of Health.
  • the scorpion has the effect of purging fire and removing trouble, clearing heat and diuresis, cooling blood and detoxifying. External use can treat sprains and contusions; industrial is a good raw material for extracting natural pigments.
  • hazelnuts contain chemical components such as iridoid glycosides, saffron pigments, triterpenoids, flavonoids, and quinic acid, among which iridoids and saffron pigments are representative components [10- 11] ;
  • the pharmacological effects of medlar are mainly manifested as anti-inflammatory analgesic, gallbladder, liver, antioxidant and anti-tumor [10-11] .
  • Chinese wolfberry has a wide distribution and high yield (the dried fruit of dried hazelnut can reach 200kg per mu, and the annual output of China can reach 5,000 tons).
  • the price is suitable (price is 15 yuan/kg), and it contains saffron.
  • the pigment content is relatively high in content and rich in type, so the scorpion is expected to assist saffron as an ideal plant for extracting saffron pigment.
  • AD Alzheimer's Disease
  • cognitive impairment characterized by loss of memory, cognitive impairment, and personality changes.
  • AD is the most common type of Alzheimer's disease.
  • the initial symptoms of AD patients are forgetfulness, which leads to the decline of orientation, comprehension, judgment and memory.
  • the patient enters a general recession in the late stage, and the intelligence is completely lost.
  • the movement and language barriers tend to Obviously, staying in bed all the time, life can not take care of themselves, and ultimately die from secondary infection and failure [11] .
  • AD Alzheimer first described the disease in 1906
  • AD has been an irreversible disease for more than 100 years. It is internationally recognized that there is no cure for this disease. It can be seen that in the absence of ideal therapeutic drugs, the screening and research and development of anti-Alzheimer's drugs have a very broad market prospect and far-reaching social significance.
  • the patent document CN104491075A reports a method for extracting and enriching crocin in the scorpion by using a macroporous resin column and a dextran gel column, and verifying the effect of depression treatment by stress depression test. .
  • this patent document is concerned with the 50% ethanol site, and the process of combining the macroporous resin column and the dextran gel column is complicated, and the composition and content of the enriched crocin component of the patent document are unknown.
  • the effective dose in the examples is also high (100 to 400 mg), which is most likely due to the low purity of the enriched crocin portion.
  • Another object of the present invention is to provide a saffron pigment active site which is extracted from scorpion and has a clear composition and content, and a medicament, food or the like for preparing a dementia-related disease for preventing and treating Alzheimer's disease and the like Application in food additives.
  • Still another object of the present invention is to provide a saffron saffron pigment comprising a scorpion A composition of a sexual site and a drug having central nervous system protection, a traditional Chinese medicine, a natural product, and the use of the composition for the preparation of a medicament or a health care product for preventing and treating a disease associated with Alzheimer's disease such as Alzheimer's disease.
  • the present invention adopts the following technical solutions.
  • a composition comprising neocrocin B (5) and saffron bis- ⁇ -D-gentiobioside (1).
  • composition according to [1] which further comprises saffronic acid mono- ⁇ -D-gentiobioside (6).
  • composition according to [1] which further comprises 13Z-saffron bis- ⁇ -D-gentiobioside (4).
  • composition according to [1] which further comprises saffron acid- ⁇ -D-glucopyranosyl- ⁇ -D-gentiobioside (2).
  • composition according to [1] which further comprises saffron acid- ⁇ -D-glucopyranosyl- ⁇ -D-gentiobioside (2), saffron bis- ⁇ - D-glucopyranoside (3), 13Z-saffron bis- ⁇ -D-gentiobioside (4), saffronic acid mono- ⁇ -D-gentiobioside (6), 13Z-saffron acid-8 -O- ⁇ -D- gentiobioside (7), 13Z-saffron acid-8'-O- ⁇ -D- gentiobioside (8), saffronic acid mono- ⁇ -D-glucopyranoside ( 9).
  • a saffron pigment active site comprising the composition according to any one of claims 1 to 6.
  • the retention time of saffronic acid mono- ⁇ -D-gentiobioside is set to 1, and the relative retention time of each chromatographic peak is determined.
  • the retention time of ⁇ -D-gentiobioside is 0.38 ⁇ 0.02, and the retention time of crocin- ⁇ -D-glucopyranosyl- ⁇ -D-gentiobioside is 0.48 ⁇ 0.02, saffronic acid double- ⁇
  • the retention time of -D-glucopyranoside is 0.60 ⁇ 0.02
  • the retention time of 13Z-saffronic acid bis- ⁇ -D-gentiobioside is 0.78 ⁇ 0.02
  • the retention time of neocrocin B is 0.89 ⁇ 0.02
  • saffronic acid single The retention time of ⁇ -D- gentian diglucoside is 1.00
  • the retention time of 13Z-saffron acid-8-O- ⁇ -D-gentiobioside is 1.13 ⁇ 0.02, 13Z-saffron acid-8′-O-
  • the retention time of ⁇ -D-gentiobioside was 1.14 ⁇ 0.02, and the retention time of saffronic acid mono- ⁇ -D-glucopyran
  • the octadecylsilane bond and silica gel were stationary phases, and the gradient was eluted with a mobile phase containing 0.1% formic acid in acetonitrile-water solution.
  • the flow rate was 0.6 mL/min
  • the detection wavelength was 440 nm
  • the column temperature was 35 °C. .
  • a saffron pigment active site characterized in that the saffron pigment active site is prepared by the following method:
  • step (1) The method according to [12], wherein in step (1), four times the amount of 60% ethanol is heated and refluxed for 3 times for 2 hours each time;
  • step (2) elution sequentially with water, 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, eluting 4 bed volumes per gradient, and concentrating the 70% ethanol eluate under reduced pressure to obtain The saffron pigment active site.
  • a pharmaceutical composition comprising the saffron pigment active site according to any one of [7] to [11], one or more other drugs having central nervous system protection, and Proper pharmaceutical excipients.
  • composition of the present invention is composed of several novel structures of saffron pigment compounds
  • the active component of the saffron pigment of the present invention further comprises two novel structures of saffron pigment compounds
  • the preparation process of the present invention is relatively simple, and the present invention adopts an internationally recognized AD pharmacological evaluation model, and proves that the active component of the crocin of the present invention has an excellent effect of treating AD at a low dose.
  • Figure 1 shows the active part of saffron saffron pigment determined by UPLC analysis Characteristic map.
  • Fig. 2 is a chromatographic peak designation map of Compound 1 isolated from the active part of saffron saffron pigment in the same UPLC condition.
  • Fig. 3 is a chromatographic peak designation map of Compound 2 isolated from the active part of saffron saffron pigments under the same UPLC conditions.
  • Fig. 4 is a chromatographic peak designation map of Compound 3 isolated from the active part of saffron saffron pigment in the same UPLC condition.
  • Fig. 5 is a chromatographic peak designation map of Compound 4 isolated from the active portion of saffron saffron pigments under the same UPLC conditions.
  • Fig. 6 is a chromatographic peak designation map of Compound 5 isolated from the active portion of saffron saffron pigments under the same UPLC conditions.
  • Fig. 7 is a chromatographic peak designation map of Compound 6 isolated from the active part of saffron saffron pigments under the same UPLC conditions.
  • Fig. 8 is a chromatographic peak designation map of Compound 7 isolated from the active portion of saffron saffron pigment under the same UPLC conditions.
  • Fig. 9 is a chromatographic peak designation map of Compound 8 isolated from the active portion of saffron saffron pigment under the same UPLC conditions.
  • Figure 10 is a chromatographic peak designation of Compound 9 isolated from the active part of saffron saffron pigments under the same UPLC conditions.
  • Figure 11 is a graph showing the protective effect of scorpion safflor pigment active site GJ-4 on scopolamine-induced learning and memory impairment in mice.
  • Figure 12 is a graph showing the protective effect (scheduled test) of scorpion safflor pigment active site GJ-4 on learning and memory impairment in mice induced by A ⁇ 25-35 ventricle injection.
  • Figure 13 is a graph showing the protective effect of the scorpion safflor pigment active site GJ-4 on learning and memory impairment in mice induced by A ⁇ 25-35 ventricle injection (Morris water maze).
  • Example 1 Preparation method of scorpion safflor pigment active site
  • the UPLC characteristic map of the saffron saffron pigment active site prepared in Example 1 is shown in Fig. 1. Under the guidance of the characteristic map, the separation method was prepared by ODS column chromatography and RP-HPLC. The identification methods of UV, MS and NMR were used to identify saffronic acid bis- ⁇ -D-gentiobioside and saffronic acid- ⁇ .
  • the isolated compound was identified under the same chromatographic conditions as the UPLC characteristic map of the saffron saffron active site, and the specific identification process is shown in Fig. 2 to Fig. 10.
  • Compound 1 was determined to be safflower bis- ⁇ -D- gentis diglucoside as compared with the literature [11] , and the 13 C-NMR of Compound 1 is shown in Table 1.
  • Compound 2 was determined to be saffron acid- ⁇ -D-glucopyranosyl- ⁇ -D- gentiobioside compared with the literature [11] , and the 13 C-NMR of Compound 2 is shown in Table 1.
  • Compound 3 was determined to be safflower bis- ⁇ -D-glucopyranoside compared to the literature [12] , and the 13 C-NMR of Compound 3 is shown in Table 1.
  • the types are all ⁇ -type.
  • the correlation peaks H-6/C-1', H-1'/C-6 suggest that the two glucose groups are 1 ⁇ 6 linked to form a gentiobiose group.
  • Sugar hydrolysis derivatization experiments show that the absolute configuration of glucose is in the D configuration.
  • the structural fragment of 3 caffeoylquinic acid in the structure was identified by 1 H- 1 H COSY, HSQC and HMBC spectra, and the 4 position of the caffeic acid was inferred by HMBC pattern to be associated with saffron acid [13] .
  • the compound 6 was determined to be safflower mono- ⁇ -D- gentiobiose, and the 13 C-NMR of the compound 6 is shown in Table 1.
  • Compound 7 is a cis- geometric isomer of Compound 6, except that since the structure of Compound 6 is itself asymmetric, there are two cases for its cis- geometric isomer. After 1 H, 13 C-NMR and two-dimensional nuclear magnetic data analysis, Compound 7 was identified as 13Z-saffloric acid-8-O- ⁇ -D-gentiobioside, and 13 C-NMR of Compound 7 is shown in Table 1.
  • Compound 8 is another geometric isomer of Compound 6. After one-dimensional and two-dimensional nuclear magnetic data analysis, compound 8 was identified as 13Z-saffloric acid-8'-O- ⁇ -D- gentis diglucoside. After searching, compound 8 was a new compound not reported, and its 13 C-NMR See Table 1.
  • Compound 9 was determined to be saffronic acid mono- ⁇ -D-glucopyranoside compared to the literature [11] , and the 13 C-NMR of Compound 9 is shown in Table 1.
  • BEH C18 (3.0 mm ⁇ 150 mm, 1.7 ⁇ m); mobile phase: solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid), gradient elution (0 min-20% B, 0.5 min-20%) B, 19 min - 50% B, 20 min - 100% B, 23 min - 100% B, 24 min - 20% B), flow rate: 0.6 mL / min, column temperature: 35 ° C, detection wavelength: 440 nm.
  • Electrospray positive ion mode capillary voltage: 2.0 kV; desolvation gas flow: N 2 , flow rate 600 L / h, desolvation temperature 300 ° C; cone flow: N 2 , flow rate 50 L / h; ion source temperature: 100 ° C; Extractor : 4.00V; collision gas: argon.
  • Table 2 The mass spectrometric analysis of the nine major chromatographic peaks is shown in Table 2.
  • the platform test device is a rectangular reflecting box with a size of 10cm ⁇ 10cm ⁇ 60cm. It is divided into 5 pieces by black plastic plates. The bottom is covered with copper grid. The spacing is 0.5cm. It can be energized. The voltage intensity is controlled by a transformer. A wooden platform with a height and a diameter of 4.5 cm was placed, and the experiment was conducted with 36 V AC. After the electric shock of the mouse, the normal reaction was to jump back to the safety platform to avoid noxious stimulation. On the first day, the mice were not energized. The mice were placed in a reflective box for 5 minutes to be familiar with the environment.
  • the copper grid power supply (36V AC) was turned on, and the time from the shock of each group to the first jump on the safety platform was recorded.
  • the incubation period was calculated at 5 min.
  • mice male, 160, divided into 8 groups, 20 in each group, each group was control group, model group, donepezil (5 mg/kg) group, memantine (5 mg/kg) group, GJ-4 (12.5) The mg/kg) group, the GJ-4 (25 mg/kg) group, the GJ-4 (50 mg/kg) group, and the GJ-4 (100 mg/kg) group.
  • the mice were continuously administered for 7 days in advance, and the mice were trained on the 5th and 6th days of the platform. After 1 hour of administration on the 7th day, the model group and each of the administration groups were intraperitoneally injected with scopolamine (2 mg/kg), and after 30 minutes, the platform was skipped.
  • the behavioral test was performed to record the time (latency) of the mouse's first jump and the number of jumps (the number of errors) within 5 minutes. The data is shown in Figure 11.
  • saffron saffron pigment GJ-4 showed a good effect on the dementia induced by scopolamine.
  • GJ-4 can significantly prolong the latency of the jumping platform and reduce the number of jumping errors, including 25mg/kg, 50mg/
  • the kg and 100 mg/kg dose groups showed a certain dose-effect relationship.
  • the efficacy of the medium and high dose groups was comparable to the positive control drug donepezil, and the results were reproducible. No dose was observed in all dose groups in the experiment. Administration related toxicity.
  • a ⁇ 25-35 was prepared into 5 ⁇ g/ ⁇ L with sterile trihydrated water, placed in a 37 ° C incubator for 7 days, and stored in a refrigerator at -20 ° C.
  • 4% chloral hydrate (10 mg/kg) was anesthetized by intraperitoneal injection, then fixed on a stereotaxic instrument, and the skin of the mouse's head was cut along the midline with a surgical scissors to expose the front and the person. The word is sewn, and the skull is drilled in the left lateral ventricle with an electric drill, so that the meninges are not injured.
  • the relative coordinates are 2 mm after the anterior iliac crest, 2 mm to the left of the midline, and 1.7 mm under the subdural.
  • the sham-operated mice were injected with 2 ⁇ L of sterile tri-steamed water into the left lateral ventricle of the mouse 2 mm after the anterior iliac crest, 2 mm to the left of the midline, and 1.7 mm under the dura mater.
  • mice injected into the lateral ventricle were randomly divided into model group, GJ-4 (25 mg/kg) group, GJ-4 (50 mg/kg) group, GJ-4 (100 mg/kg) group and donepezil. (5 mg/kg) group, 15 in each group. After the operation, the mice were rested for 3 days. Each group was intragastrically administered with the corresponding dose of the drug. The sham operation group and the model group were given the same dose of physiological saline once a day for 12 consecutive days.
  • the platform test device is a rectangular reflecting box with a size of 10cm ⁇ 10cm ⁇ 60cm. It is divided into 5 pieces by black plastic plates. The bottom is covered with copper grid. The spacing is 0.5cm. It can be energized. The voltage intensity is controlled by a transformer. A wooden platform with a height and a diameter of 4.5 cm was placed, and the experiment was conducted with 36 V AC. After the electric shock of the mouse, the normal reaction was to jump back to the safety platform to avoid noxious stimulation. On the 5th day after administration, the mice were not energized. The mice were placed in a reflective box for 5 minutes, and they were familiar with the environment.
  • the Morris water maze test testing the learning and memory ability of each group of mice for spatial position and sense of direction.
  • the Morris water maze experimental device is a circular pool with a diameter of 120cm and a water depth of 40cm. The inner surface is covered with a layer of black tape. The water temperature is 23-25 °C, and the indoor temperature is controlled at 26-28 °C. The water tank was randomly divided into four quadrants. The position of the platform was fixed during the experiment, and it was placed in the center of the second quadrant, which was 1-2 cm below the water surface. Mark the walls around the room so that the mouse can identify the direction based on the mark.
  • the active part of saffron saffron pigment GJ-4 can significantly prolong the latency of mouse jumping and reduce the number of jumping errors.
  • GJ-4 significantly shortens the latency of finding the platform and increases the crossing platform. The number of times and the length of swimming in the quadrant of the platform.
  • the results showed that GJ-4 showed a good effect on improving learning and memory impairment in mice.
  • Each dose group showed a certain dose-response relationship. Among them, the efficacy of the high-dose group was comparable to or better than the positive control drug donepezil. For the positive drug, no toxicity associated with administration was observed in all dose groups in the experiment.
  • Example 5 Neuroprotective effect of safflor pigment monomer in scorpion in L-glutamate-induced SH-SY5Y cell injury model
  • the SH-SY5Y nerve cells were cultured in DMEM medium (containing a volume fraction of 5% fetal bovine serum), cultured at 37 ° C in an incubator containing 5% CO 2 , and passaged every 3 to 4 days. Experiments were performed on logarithmic growth phase cells.
  • SH-SY5Y cells were seeded in 96-well plates at a concentration of 5 ⁇ 10 3 , cultured for 24 h, and 100 ⁇ L of L-glutamic acid-containing medicinal medium was added to a 96-well plate to make L-glutamic acid
  • the concentration was 160 mM
  • the final concentration of the drug was 10 ⁇ M, 1 ⁇ M, and 0.1 ⁇ M
  • three wells in parallel were set for each concentration, and the culture was continued for 24 hours. After 24 hours, the supernatant was aspirated, and 100 ⁇ L of MTT (0.5 mg/mL) was added to each well. Incubation was continued for 4 hours.

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Abstract

一种组合物以及一种从栀子中提取的藏红花色素类活性部位，主要包含下述成分：藏红花酸双-β-D-龙胆二糖苷、藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷、藏红花酸双-β-D-吡喃葡萄糖苷、13Z-藏红花酸双-β-D-龙胆二糖苷、neocrocin B、藏红花酸单-β-D-龙胆二糖苷、13Z-藏红花酸-8-O-β-D-龙胆二糖苷、13Z-藏红花酸-8'-O-β-D-龙胆二糖苷、藏红花酸单-β-D-吡喃葡萄糖苷。药理实验结果表明，所述藏红花色素类活性部位可有效改善东莨菪碱和β淀粉样蛋白所致小鼠学习记忆损伤。

Description

组合物、藏红花色素类活性部位及其用途 技术领域
本发明涉及一种组合物，还涉及一种中药提取物，具体而言，涉及一种从栀子中提取的藏红花色素类活性部位及其在预防和治疗阿尔茨海默症等疾病中的用途，更具体而言，涉及一种栀子藏红花色素类活性部位及其所含的活性成分在制备预防和治疗阿尔茨海默症等老年痴呆相关疾病的药物或保健品中的应用。
背景技术
藏红花色素是一类结构独特的水溶性类胡萝卜素，包括藏红花酸及其与不同糖基结合而成的糖酯，是藏红花和栀子中的共有色素成分。由于其良好的水溶性，藏红花色素广泛用于酒类、菜肴、糕点的着色剂。多项研究表明，藏红花粗提物、栀子黄色素以及单体成分藏红花素、藏红花酸等在中枢神经系统保护[1-4]、心脑血管系统的保护[5-6]、拮抗恶性肿瘤[7-9]等方面表现出高效低毒的药理活性。
藏红花原产于南欧、地中海及伊朗等地，其中伊朗藏红花产量占全球的95％，我国的浙江、江苏、山东、北京等地有少量栽培。藏红花以柱头入药，产量极低(亩产不足1千克)，价格昂贵(2000美元/kg)，素有“植物黄金”之称。随着藏红花色素类成分药用和食用需求量的不断加大，寻找和发现其他富含藏红花色素类成分的植物显得意义重大。
栀子，又名山栀子、黄栀子等，为茜草科栀子属植物，广泛分布于我国中部和南部各省。栀子始载于《神农本草经》，中国历代药典和本草均有记载，是卫生部颁布的首批药食两用资源，栀子内服具有泻火除烦，清热利尿、凉血解毒之功效；外用可治疗扭伤、挫伤；工业上是提取天然色素的良好原料。近代化学和药理学研究 发现，栀子中含有环烯醚萜、藏红花色素、三萜、黄酮、奎宁酸等化学成分，其中环烯醚萜类、藏红花色素类为其代表性成分[10-11]；栀子的药理作用主要表现为抗炎镇痛、利胆保肝、抗氧化和抗肿瘤等[10-11]。
栀子作为常用中药材，种植分布广泛，产量较高(栀子干燥果实亩产可达200kg，我国年产量可达5000吨)，价格适宜(售价在15元/kg)，其所含藏红花色素类成分相对含量较高、类型较为丰富，因而栀子有望辅助藏红花成为提取藏红花色素的理想植物。
阿尔茨海默症(Alzheimer’s Disease，AD)，是一种与衰老相关，以记忆缺失、认知障碍、人格改变为特征的渐进性神经退行性疾病。AD是老年痴呆中最常见的类型，AD患者最初的症状是健忘，进而发展为定向力、理解力、判断力和记忆力的下降，患者晚期进入全面衰退状态，智能完全丧失，运动和语言障碍日趋明显，终日卧床，生活不能自理，最终多死于继发性感染和衰竭[11]。
随着世界老龄化进程的加剧，AD的发病率呈逐年迅速上升的趋势，给各个国家尤其是发展中国家的社会和人民带来沉重的经济和家庭负担。自1906年德国医生Alzheimer首次描述该病迄今100多年来，AD仍是一个不可逆性疾病，国际上公认暂无治愈此病的方法和药物。可见，在尚无理想治疗药物的情况下，抗老年痴呆药物的筛选和研发具有十分广阔的市场前景和深远的社会意义。
另外，专利文献CN104491075A报道了一种联合应用大孔树脂柱和葡聚糖凝胶柱从栀子中提取富集藏红花素部位的方法，并经过应激抑郁实验验证了其在抑郁症治疗的效果。然而，该专利文献关注的是50％乙醇部位，而且联合应用大孔树脂柱和葡聚糖凝胶柱的工艺也较为复杂，此外，该专利文献的富集藏红花素部位的组成及含量不明，而且实施例中的有效剂量也较高(100～400mg)，这很可能是由于其中的富集藏红花素部位的纯度较低造成的。
【参考文献】
[1]Karakani A.-M.，Riazi G.，Mahmood G.-S.，et al.Inhibitory effect of  corcin on aggregation of 1N/4R human tau protein in vitro[J].Iranian journal of basic medical sciences.2015，18(5)，485-92.
[2]Papandreou M.-A.，Kanakis C.-D.，Polissiou M.-G.，et al.Inhibitory Activity on Amyloid-βAggregation and Antioxidant Properties of Crocus sativus Stigmas Extract and Its Crocin Constituents[J].Jouranl of Agriculture and Food Chemistry.2006，54(23)，8762-8768.
[3]Akhondzadeh S.，Sabet M.-S.，Harirchian M.-H.，et al.A 22-week，multicenter，randomized，double-blind controlled trial of Crocus sativus in the treatment of mild-to-moderate Alzheimer’s disease[J].Psychopharmacology.2010，207(4)，637-643.
[4]Farokhnia M.，Shafiee S.-M.，Iranpour N.，et al.Comparing the efficacy and safety of Crocus sativus L.With memantine in patients with moderate to severe Alzheimer’s disease：a double-blind randomized clinical trial[J].Human Psychopharmacology.2014，29(4)，351-359.
[5]Zheng Y.-Q.，Liu J.-X.，Wang J.-N.，et al.Effects of crocin on reperfusion-induced oxidative_nitrative injury to cerebral microvessels after global cerebral ischemia[J].Brain Research.2007，1138，86-94.
[6]Higashino S.，Sasaki Y.，Giddings J.-C.，et al.Crocetin，a Carotenoid from Gardenia jasminoides Ellis，Protects against Hypertension and Cerebral Thrombogenesis in Stroke-prone Spontaneously Hypertensive Rats[J].Phytotherapy Research.2014，28(9)，1315-1319.
[7]董盛宇，刘付梅，李祥勇.藏红花素对CNE2细胞的增殖及迁移抑制作用[J].湖北民族学院学报·医学版.2013，30(2)，6-12.
[8]王新星，于正洪，侍述碌等.藏红花素对人肺腺癌SPC-A1细胞的增殖抑制作用及机制研究[J].临床肿瘤学杂志.2013，18(4)， 295-299.
[9]陈福雄，陶佳，黄穗等.儿童EB病毒感染相关IM和EBV-AHS的临床研究和病毒感染特征[A].中华医学会、中华医学会儿科学分会.中华医学会第十七次全国儿科学术大会论文汇编(上册)[C].中华医学会、中华医学会儿科学分会：，2012：1.
[10]孟祥乐，李红伟，李颜等.栀子化学成分及其药理作用研究进展[J].中国新药杂志.2011，20(11)，959-967.
[11]于洋.栀子抗老痴呆活性成分研究[D].沈阳药科大学，2010.
[12]Calsteren M.-R.-V.，Bissonnette M.C.，Cormier F.，et al.Spectroscopic Characterization of Crocetin Derivatives from Crocus sativus and Gardenia jasminoides[J].Jouranl of Agriculture and Food Chemistry.1997，45(4)，1055-1061.
[13]李海波，于洋，王振中等.热毒宁注射液化学成分研究(II)(J).中草药.2015，46(11)，1597-1602.
[14]陈红，肖永庆，李丽等.栀子化学成分研究[J].中国中药杂志.2007，32(11)，1041-1043.
发明内容
鉴于上述现有技术存在的问题，本发明的一个目的在于提供一种组合物，所述组合物包括若干全新结构的藏红花色素类化合物。
本发明的另一个目的在于提供一种从栀子中提取的、组分及含量明确的藏红花色素类活性部位及其在制备预防和治疗阿尔茨海默症等相关痴呆性疾病的药物、食品或食品添加剂中的应用。
本发明的又一个目的在于提供一种包括栀子藏红花色素类活 性部位与具有中枢神经保护作用的药物、传统中药、天然产物的组合物，以及所述组合物在制备预防和治疗阿尔茨海默症等老年痴呆相关疾病的药物或保健品中的应用。
为了实现上述目的，本发明采用如下技术方案。
[1]一种组合物，其特征在于，所述组合物包括neocrocin B(5)和藏红花酸双-β-D-龙胆二糖苷(1)。
[2]根据项[1]所述的组合物，其特征在于，还包括藏红花酸单-β-D-龙胆二糖苷(6)。
[3]根据项[1]所述的组合物，其特征在于，还包括13Z-藏红花酸双-β-D-龙胆二糖苷(4)。
[4]根据项[1]所述的组合物，其特征在于，还包括藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷(2)。
[5]根据项[1]所述的组合物，其特征在于，还包括藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷(2)、藏红花酸双-β-D-吡喃葡萄糖苷(3)、13Z-藏红花酸双-β-D-龙胆二糖苷(4)、藏红花酸单-β-D-龙胆二糖苷(6)、13Z-藏红花酸-8-O-β-D-龙胆二糖苷(7)、13Z-藏红花酸-8′-O-β-D-龙胆二糖苷(8)、藏红花酸单-β-D-吡喃葡萄糖苷(9)。
[6]根据项[5]所述的组合物，其中，所述组合物中各组分的含量分别为：
Figure PCTCN2017076910-appb-000001
其中，上述各化合物的结构式分别如下所示，
Figure PCTCN2017076910-appb-000002
Figure PCTCN2017076910-appb-000003
[7]一种藏红花色素类活性部位，其特征在于，其包括权利要求1～6中任一项所述的组合物。
[8]根据项[7]所述的藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位是从栀子中提取得到的。
[9]根据项[7]所述的藏红花色素类活性部位，其特征在于，所述 藏红花色素类活性部位的UPLC特征图谱主要包含9个色谱峰，将藏红花酸单-β-D-龙胆二糖苷的保留时间设为1，分别求出各色谱峰的相对保留时间，藏红花酸双-β-D-龙胆二糖苷的保留时间为0.38±0.02，藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷的保留时间为0.48±0.02，藏红花酸双-β-D-吡喃葡萄糖苷的保留时间为0.60±0.02，13Z-藏红花酸双-β-D-龙胆二糖苷的保留时间为0.78±0.02，neocrocin B的保留时间为0.89±0.02，藏红花酸单-β-D-龙胆二糖苷的保留时间为1.00，13Z-藏红花酸-8-O-β-D-龙胆二糖苷的保留时间为1.13±0.02，13Z-藏红花酸-8′-O-β-D-龙胆二糖苷的保留时间为1.14±0.02，藏红花酸单-β-D-吡喃葡萄糖苷的保留时间为1.19±0.02。
[10]根据项[9]所述的藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位的UPLC特征图谱是采用反相超高效液相色谱法建立的，色谱条件是：以十八烷基硅烷键和硅胶为固定相，以含有0.1％甲酸的乙腈-水溶液为流动相，进行梯度洗脱，其中，流速为0.6mL/min，检测波长为440nm，色谱柱温度为35℃。
[11]一种藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位通过下述方法进行制备：
(1)将栀子干燥果实适当粉碎后，用乙醇、甲醇或水，采用不同提取次数和时间，通过热提取或者超声提取的方法进行提取，减压浓缩提取液，得到栀子总提取物；
(2)用适量水溶解所述栀子总提取物，离心，上清液通过大孔吸附树脂开放柱色谱，用水和/或30％～95％的乙醇洗脱适量的柱床体积，收集洗脱液，减压浓缩70％乙醇洗脱液，得到所述藏红花色素类活性部位。
[12]一种制备项[7]～[10]中任一项所述的藏红花色素类活性部位的方法，其特征在于，包括以下步骤：
(1)将栀子干燥果实适当粉碎后，用乙醇、甲醇或水，采用不同提取次数和时间，通过热提取或者超声提取的方法进行提取，减 压浓缩提取液，得到栀子总提取物；
(2)用适量水溶解所述栀子总提取物，离心，上清液通过大孔吸附树脂开放柱色谱，用水和/或30％～95％的乙醇洗脱适量的柱床体积，收集洗脱液，减压浓缩，得到所述藏红花色素类活性部位。
[13]根据项[12]所述的方法，其特征在于，在步骤(1)中，用4倍量的60％乙醇，加热回流提取3次，每次2小时；
在步骤(2)中，用水、30％乙醇、50％乙醇、70％乙醇、95％乙醇依次洗脱，每个梯度洗脱4个柱床体积，减压浓缩70％乙醇洗脱液，得到所述藏红花色素类活性部位。
[14]项[7]～[11]中任一项所述的藏红花色素类活性部位在制备改善学习记忆能力的药物中的应用。
[15]项[7]～[11]中任一项所述的藏红花色素类活性部位在制备预防和治疗阿尔茨海默症的药物中的应用。
[16]一种药物组合物，其特征在于，包括项[7]～[11]中任一项所述的藏红花色素类活性部位、一种或多种其他具有中枢神经系统保护作用的药物以及适当的药物辅料。
[17]项[16]所述的药物组合物在制备预防和治疗中枢神经退行性疾病的药物中的应用。
有益效果
(1)本发明的组合物由若干全新结构的藏红花色素类化合物组成；
(2)本发明的藏红花色素类活性部位还包括2个全新结构的藏红花色素类化合物；
(3)本发明的制备工艺较为简单，而且，本发明采用国际公认的AD药理学评价模型，证明了本发明的藏红花素类活性部位在低剂量下具有治疗AD的优异效果。
附图说明
图1是通过UPLC分析液相确定的栀子藏红花色素类活性部位 的特征图谱。
图2是从栀子藏红花色素类活性部位中分离得到的化合物1在相同UPLC条件下的色谱峰指认图谱。
图3是从栀子藏红花色素类活性部位中分离得到的化合物2在相同UPLC条件下的色谱峰指认图谱。
图4是从栀子藏红花色素类活性部位中分离得到的化合物3在相同UPLC条件下的色谱峰指认图谱。
图5是从栀子藏红花色素类活性部位中分离得到的化合物4在相同UPLC条件下的色谱峰指认图谱。
图6是从栀子藏红花色素类活性部位中分离得到的化合物5在相同UPLC条件下的色谱峰指认图谱。
图7是从栀子藏红花色素类活性部位中分离得到的化合物6在相同UPLC条件下的色谱峰指认图谱。
图8是从栀子藏红花色素类活性部位中分离得到的化合物7在相同UPLC条件下的色谱峰指认图谱。
图9是从栀子藏红花色素类活性部位中分离得到的化合物8在相同UPLC条件下的色谱峰指认图谱。
图10是从栀子藏红花色素类活性部位中分离得到的化合物9在相同UPLC条件下的色谱峰指认图谱。
图11是表示栀子藏红花色素活性部位GJ-4对东莨菪碱造成小鼠学习记忆损伤的保护作用的图。
图12是表示栀子藏红花色素活性部位GJ-4对Aβ25-35脑室注射造成小鼠学习记忆损伤的保护作用(跳台试验)的图。
图13是表示栀子藏红花色素活性部位GJ-4对Aβ25-35脑室注射造成小鼠学习记忆损伤的保护作用(Morris水迷宫)的图。
具体实施方式
以下结合实施例进一步阐述本发明的技术方案，但本发明并不限于这些实施例。
实施例1：栀子藏红花色素类活性部位的制备方法
取栀子干燥成熟果实40.0kg，经适当粉碎后，用4倍量的60％乙醇加热回流提取3次，每次2小时。合并提取液，减压蒸去溶剂，得到栀子总提物6.2kg；以适量水溶解提取物，离心，进行大孔树脂开放柱层析(20.0×90cm)，依次用4倍柱床体积的水、30％、50％、70％、95％的乙醇进行梯度洗脱，收集各部分洗脱液，分别减压回收溶剂，得到水洗脱、30％乙醇洗脱组合部分约4.5kg，50％乙醇洗脱部分710.0g，70％乙醇洗脱部分150.0g，95％乙醇洗脱部分112.0g，其中70％乙醇洗脱部分即为栀子藏红花色素活性部位GJ-4。
实施例2：栀子藏红花色素类活性部位中主要成分的分离及鉴定
实施例1中制备的栀子藏红花色素类活性部位的UPLC特征图谱如图1所示。在特征图谱指导下通过ODS柱色谱、RP-HPLC制备液相等分离手段，利用UV、MS、NMR等分析鉴定方法，鉴定了藏红花酸双-β-D-龙胆二糖苷、藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷、藏红花酸双-β-D-吡喃葡萄糖苷、13Z-藏红花酸双-β-D-龙胆二糖苷、neocrocin B、藏红花酸单-β-D-龙胆二糖苷、13Z-藏红花酸-8-O-β-D-龙胆二糖苷、13Z-藏红花酸-8′-O-β-D-龙胆二糖苷、藏红花酸单-β-D-吡喃葡萄糖苷这9个化合物的结构。
在与栀子藏红花色素类活性部位UPLC特征图谱相同的色谱条件下，对分离得到的化合物进行指认，具体指认过程见图2-图10。
2.1分离过程
对得到的栀子藏红花色素类活性部位进行硅胶柱色谱分离，氯仿-甲醇-水8∶2∶0.2洗脱得到化合物6(约7.0g)，氯仿-甲醇-水9∶1∶0.1洗脱得到化合物9(136.5mg)。ODS开放柱色谱，50％甲醇-水洗脱得到化合物1(545.1mg)，55％甲醇-水洗脱得到化合物2(143.7mg)，50％甲醇-水洗脱得到化合物3(315.7mg)。制备型高效液相ODS柱色谱分离，60％甲醇-水洗脱得到化合物4(265.7mg，tR＝16.6min)，68％甲醇-酸水(0.1％CH3COOH)洗脱得到化合物5(520.9mg，tR＝9.5min)，42％乙腈-酸水(0.1％CH3COOH)分离得到化合物7(8.0mg， tR＝17.9min)和8(16.0mg，tR＝21.5min)。
2.2化合物结构解析
2.2.1化合物1
红色无定型粉末。HR-ESI-MS给出m/z999.3680[M+Na]+(计算值999.3685)，确定分子式为C44H64O24，计算不饱和度为13。
1H-NMR(600MHz，in DMSO-d6)显示特征的藏红花酸的烯氢信号[δ7.35(2H，d，J＝10.8Hz)，6.87(2H，dd，J＝7.8，2.4Hz)，6.82(2H，d，J＝15.0Hz)，6.67(2H，dd，J＝15.0，12.6Hz)，6.53(2H，br.d，J＝9.6Hz)，]；4个两两重叠的糖端基信号[δ5.42(2H，d，J＝8.4Hz)，4.17(2H，d，J＝7.8Hz)]以及4个两两重叠的甲基氢信号[δ2.00(6H，s)，1.97(6H，s)]。
经过与文献[11]比较，化合物1确定为藏红花酸双-β-D-龙胆二糖苷，化合物1的13C-NMR参见表1。
2.2.2化合物2
红色无定型粉末。HR-ESI-MS给出m/z837.3166[M+Na]+(计算值837.3157)，确定分子式为C38H54O19，计算不饱和度为12。
1H-NMR(600MHz，in DMSO-d6)显示特征的藏红花酸的烯氢信号[δ7.35(2H，d，J＝11.4Hz)，6.86(2H，dd，J＝8.4，3.0Hz)，6.82(1H，d，J＝14.4Hz)，6.81(1H，d，J＝15.0Hz)，6.66(2H，dd，J＝15.0，12.0Hz)，6.54(2H，br.d，J＝8.4Hz)，]，4个甲基氢信号[δ1.99(6H，s)，1.97(6H，s)]以及3个糖端基质子信号[δ5.42(2H，d，J＝7.8Hz)，4.17(1H，d，J＝7.8Hz)]。
经过与文献[11]比较，化合物2确定为藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷，化合物2的13C-NMR参见表1。
2.2.3化合物3
红色无定型粉末。ESI-MS(positive)给出m/z675[M+Na]+，1327[2M+Na]+，推测其分子量为652。
1H-NMR(600MHz，in DMSO-d6)显示特征的藏红花酸的烯氢信号[δ7.35(2H，d，J＝11.4Hz)，6.86(2H，dd，J＝8.4，3.0Hz)，6.81(2H， d，J＝15.0Hz)，6.67(2H，dd，J＝15.0，11.4Hz)，6.54(2H，br.d，J＝9.6Hz)，]；2个重叠的糖端基信号[δ5.42(2H，d，J＝7.8Hz)]以及4个两两重叠的甲基氢信号[δ2.00(6H，s)，1.97(6H，s)]。
经过与文献[12]比较，化合物3确定为藏红花酸双-β-D-吡喃葡萄糖苷，化合物3的13C-NMR参见表1。
2.2.4化合物4
红色无定型粉末。HR-ESI-MS给出m/z999.3665[M+Na]+(计算值999.3685)，确定分子式为C44H64O24，计算不饱和度为13。
化合物4和化合物1为同分异构体，对比二者的1H-NMR(600MHz，in DMSO-d6)，化合物4烯氢区域产生了较大变化，其余信号与化合物1基本一致。化合物4的13C-NMR(150MHz，in DMSO-d6)中，因为13位双键的构型变化，打破了化合物的高度对称结构，很多重叠的烯碳信号变成了2个信号，且20位的甲基碳信号向低场位移至δ20.0，且与8位碳相连糖的端基氢信号由5.42变为5.44。
经过与文献[11]比较，化合物4确定为13Z-藏红花酸双-β-D-龙胆二糖苷，化合物4的13C-NMR参见表1。
2.2.5化合物5
红色无定型粉末。ESI-MS(positive)给出m/z1011[M+Na]+，提示化合物分子量为988。HR-ESI-MS给出989.3642[M+H]+(计算值为989.3654)，确定化合物分子式为C48H60O22，计算不饱和度为19。
化合物5的1H-NMR(600MHz，in DMSO-d6)图谱，低场区显示一组反式烯氢信号[δ7.44(1H，d，J＝15.6Hz，H-3″′)，6.16(1H，d，J＝16.2Hz，H-2″′)]；一组相互耦合的芳香质子信号[δ7.03(1H，d，J＝1.8Hz，H-5″′)，6.98(1H，dd，J＝8.4，1.8Hz，H-9″′)，6.74(1H，d，J＝7.8Hz，H-8″′)]，结合13C-NMR(150MHz，in DMSO-d6)信号：δ148.5(C-7″′)，145.6(C-6″′)，125.2(C-4″′)，121.6(C-9″′)，115.7(C-8″′)和114.9(C-5″′)，提示结构中存在1，3，4-三取代苯环。烯氢质子信号H-3″′/C-4″′，C-5″′，C-9″′，C-1″′；H-2″′/C-1″′，C-4″′的HMBC远程相关，提示含有一个C6-C3的咖啡酰基片段。
糖端基质子信号[δ5.42(1H，d，J＝7.8Hz，H-1)和4.17(1H，d，J＝7.8Hz，H-1′)]，提示两个葡萄糖残基的构型均为β型。在HMBC谱中，相关峰H-6/C-1′，H-1′/C-6，提示2个葡萄糖基为1→6连接，形成一个龙胆二糖基。糖水解衍生化实验表明，葡萄糖的绝对构型均为D构型。
除去2个葡糖糖残基，1个C6-C3的咖啡酰基片段，与已知文献比对，可以归属结构中特征的藏红花酸信号。
通过1H-1H COSY、HSQC和HMBC谱鉴定结构中谱中存在3咖啡酰奎宁酸的结构片段，经过HMBC图谱推断该咖啡酸的4位与藏红花酸相连接[13]。
经检索，化合物5为未见报道的新化合物，命名neocrocin B。化合物5的13C-NMR参见表1。
2.2.6化合物6
红色无定型粉末。HR-ESI-MS给出675.2625的[M+Na]+(计算值为675.2629)，确定分子式为C32H44O14，计算不饱和度为11。
1H-NMR(600MHz，in DMSO-d6)显示特征的藏红花酸的烯氢信号、2个糖端基信号以及4个甲基氢信号。
经过与文献[14]比较，化合物6确定为藏红花酸单-β-D-龙胆二糖苷，化合物6的13C-NMR参见表1。
2.2.7化合物7
红色无定型粉末。ESI-MS(positive)给出m/z675[M+Na]+，m/z1327[2M+Na]+，提示分子量为652。HR-ESI-MS给出675.2617的[M+Na]+(计算值为675.2629)，确定分子式为C32H44O14，计算不饱和度为11。
化合物7为化合物6的顺式几何异构体，所不同的是，由于化合物6结构本身不对称，因此其顺式几何异构体存在两种情况。经过1H、13C-NMR以及二维核磁数据解析，化合物7确定为13Z-藏红花酸-8-O-β-D-龙胆二糖苷，化合物7的13C-NMR参见表1。
2.2.8化合物8
红色无定型粉末。ESI-MS(positive)给出m/z675[M+Na]+，m/z1327[2M+Na]+，提示分子量为652。HR-ESI-MS给出675.2617的[M+Na+](计算值为675.2629)，确定分子式为C32H44O14，计算不饱和度为11。
化合物8为化合物6的另一几何异构体。经过一维和二维核磁数据解析，化合物8鉴定为13Z-藏红花酸-8′-O-β-D-龙胆二糖苷，经检索，化合物8为未见报道的新化合物，其13C-NMR参见表1。
2.2.9化合物9
红色无定型粉末。HR-ESI-MS给出513.2095[M+Na]+(计算值为513.2101)，1003.4303[2M+Na]+，确定分子式为C26H34O9，计算不饱和度为10。
1H-NMR(600MHz，in DMSO-d6)显示特征的藏红花酸的烯氢信号、1个糖端基信号以及4个甲基氢信号。
经过与文献[11]比较，化合物9确定为藏红花酸单-β-D-吡喃葡萄糖苷，化合物9的13C-NMR参见表1。
2.3栀子藏红花色素类活性部位的UPLC-Q/TOF-MS分析
2.3.1色谱条件
BEH C18(3.0mm×150mm，1.7μm)；流动相：溶剂A(水，0.1％甲酸)与溶剂B(乙腈，0.1％甲酸)，梯度洗脱(0min-20％B，0.5min-20％B，19min-50％B，20min-100％B，23min-100％B，24min-20％B)，流速：0.6mL/min，柱温：35℃，检测波长：440nm。
2.3.2质谱条件
电喷雾正离子模式，毛细管电压：2.0kV；脱溶剂气流：N2，流速600L/h，脱溶剂温度300℃；锥孔气流：N2，流速50L/h；离子源温度：100℃；Extractor：4.00V；碰撞气体：氩气。9个主要色谱峰的质谱分析见表2。
[表1]
Figure PCTCN2017076910-appb-000004
“a”means signals could be interchangeable with the corresponding position in one compound.
Figure PCTCN2017076910-appb-000005
实施例3：栀子藏红花色素类活性部位GJ-4改善东茛菪碱致小鼠学习记忆损伤(跳台实验)
3.1跳台实验原理
跳台实验装置为一个长方形反射箱，大小为10cm×10cm×60cm，用黑色塑料板分隔成5间，底面铺以铜栅，间距为0.5cm，可以通电，电压强度由一变压器控制，每间右角置一高度和直径均为4.5cm的木制平台，实验时通以36V交流电，小鼠受到电击后其正常反应是跳回安全平台以躲避伤害性刺激。第一天不通电，将小鼠放入反射箱内自由活动5min，熟悉环境；24h后接通铜栅电源(36V交流电)，记录各组小鼠从受到电击到首次跳上安全平台的时间(反应时间)和5min之内从安全跳台跳下的错误次数(基础错误次数)，作为学习测试成绩；次日重复上述过程，记录各组小鼠第一次跳回安全平台的时间(潜伏期)和5min内受到电击的次数(错误次数)，作为记忆测试成绩。实验时，若小鼠停留在安全平台的时间超过5min，其潜伏期以5min计算。
3.2跳台实验方案
ICR小鼠，雄性，160只，分8组，每组20只，各组分别是对照组、模型组、多奈哌齐(5mg/kg)组、美金刚(5mg/kg)组、GJ-4(12.5mg/kg)组、GJ-4(25mg/kg)组、GJ-4(50mg/kg)组和GJ-4(100mg/kg)组。小鼠提前连续给药7天，第5、6天小鼠跳台实验训练，第7天给药1h后模型组和各给药组分别腹腔注射东茛菪碱(2mg/kg)，30min后跳台法进行行为学测试，记录小鼠第一次跳下的时间(潜伏期)和5min内跳下的次数(错误次数)，数据见图11。
实验结果表明，栀子藏红花色素类活性部位GJ-4表现出良好的改善东莨菪碱所致动物痴呆的作用，GJ-4能明显延长小鼠跳台潜伏期并减少跳台错误次数，其中25mg/kg、50mg/kg以及100mg/kg剂量组表现出了一定的剂量效应关系，中、高剂量组的药效与阳性对照药物多奈哌齐相当，且实验结果具有可重现性，实验中所有剂量组均没有观察到与给药相关的毒性反应。
实施例4：栀子藏红花色素类活性部位GJ-4改善侧脑室注射Aβ25-35致小鼠学习记忆损伤(跳台实验和Morris水迷宫实验)
4.1小鼠侧脑室注射手术、分组及给药
Aβ25-35用无菌三蒸水配制成5μg/μL，37℃孵箱中放置7天使其聚集，冻存于-20℃冰箱。ICR小鼠适应性喂养两天后，腹腔注射4％水合氯醛(10mg/kg)麻醉，然后固定于立体定位仪上，用手术剪沿着中线剪开小鼠头部皮肤，暴露前囟和人字缝，用电钻在左侧侧脑室处钻破头骨，以不伤及脑膜为宜，相对坐标为前囟后2mm，中线左侧2mm，硬脑膜下1.7mm，向小鼠左侧侧脑室内注射Aβ25-352μL(10μg)/只，在1min内注射完，停针3min，然后缓慢拔出注射针，然后用手术线将切口缝合，肌注氨苄西林(5mg/kg)，将小鼠放入笼中。假手术组小鼠于坐标为前囟后2mm，中线左侧2mm，硬脑膜下1.7mm，向小鼠左侧侧脑室内注射2μL无菌三蒸水。手术后，将侧脑室注射Aβ25-35小鼠随机分为模型组、GJ-4(25mg/kg)组、GJ-4(50mg/kg)组、GJ-4(100mg/kg)组和多奈哌齐(5mg/kg)组，每组15只。手术后小鼠休息3天，各组灌胃给予相应剂量的药物，假手术组和模型组给予同样剂量的生理盐水，每天一次，连续给药12天。
4.2行为学测试
4.2.1跳台实验
给药第7天通过跳台实验检测小鼠的学习记忆能力。跳台实验装置为一个长方形反射箱，大小为10cm×10cm×60cm，用黑色塑料板分隔成5间，底面铺以铜栅，间距为0.5cm，可以通电，电压强度由一变压器控制，每间右角置一高度和直径均为4.5cm的木制平台，实验时通以36V交流电，小鼠受到电击后其正常反应是跳回安全平台以躲避伤害性刺激。给药第5天不通电，将小鼠放入反射箱内自由活动5min，熟悉环境；24h后接通铜栅电源(36V交流电)，记录各组小鼠从受到电击到首次跳上安全平台的时间(反应时间)和5min之内从安全跳台跳下的错误次数(基础错误次数)，作为学 习测试成绩；给药第7天重复上述过程，记录各组小鼠第一次跳下安全平台的时间(潜伏期)和5min内受到电击的次数(错误次数)，作为记忆测试成绩。实验时，若小鼠停留在安全平台的时间超过5min，其潜伏期以5min计算，结果见图12。
4.2.2 Morris水迷宫实验
跳台实验结束后第二天(即给药第8天)通过Morris水迷宫实验进一步检测小鼠的学习记忆能力(测试各组小鼠对空间位置感和方向感的学习记忆能力)。Morris水迷宫实验装置为一个直径120cm，水深40cm，内表面贴有一层黑色胶带的圆形水池，水温23-25℃，室内温度控制在26-28℃。将水箱随机分为四个象限，实验时平台位置固定不变，置于第二象限中央，低于水面1-2cm。室内四周墙壁上做上明显的标记，以便小鼠能够根据标记辨认方向。实验过程中室内所有物体的摆放位置固定，以免对小鼠产生干扰。实验历时5天，每天进行2次。前4天为定位航行实验，将小鼠面朝池壁先后从两个象限轻轻放入水中，避免应激和将小鼠头部进入水中，同时记录小鼠1min内找到安全平台的潜伏期，并让其在安全平台上停留30s，然后取出放回笼子。若小鼠1min内未找到安全平台，则将其放到安全平台上停留30s，潜伏期记为60s。小鼠每天两次找到安全平台的潜伏期的平均值为小鼠此天游泳的结果，进行统计学分析，结果见图13。第五天进行空间探索实验，撤去安全平台，选择一个象限将小鼠头朝池壁放入水中，记录小鼠1min内穿越平台所在位置的次数以及在平台所在象限的游泳时间，进行统计分析，见图13。
在跳台实验中，栀子藏红花色素类活性部位GJ-4能明显延长小鼠跳台潜伏期并减少跳台错误次数；水迷宫实验中，GJ-4明显缩短小鼠找到平台的潜伏期，同时增加穿越平台的次数及延长在平台所在象限的游泳时间。实验结果表明，GJ-4表现出了良好的改善小鼠学习记忆障碍的作用，各剂量组表现出了一定的剂量效应关系，其中，高剂量组的药效与阳性对照药物多奈哌齐相当甚至优于阳性药，实验中所有剂量组均没有观察到与给药相关的毒性反应。
实施例5：栀子中的藏红花色素单体在L-谷氨酸致SH-SY5Y细胞损伤模型中的神经保护作用
5.1 SH-SY5Y神经细胞培养方法
SH-SY5Y神经细胞培养于DMEM培养基(含体积分数为5％胎牛血清)中，置于37℃，含5％CO2的培养箱中培养，每3～4天传代一次。选取对数生长期细胞进行实验。
5.2 L-谷氨酸损伤模型筛选方法
将SH-SY5Y细胞以5×103的浓度接种于96孔板中，继续培养24h，加入含L-谷氨酸的药液培养基100μL至96孔板中，使L-谷氨酸的终浓度为160mM，药物终浓度为10μM、1μM、0.1μM，每个浓度设定平行的3个孔，继续培养24h。24h后，吸弃上清液，每孔加MTT(0.5mg/mL)100μL，继续孵育4h，吸弃上清液，每孔加入150μL的DMSO，震荡10min，选择570nm波长，在酶标仪上测定吸光度值[13]。(有效率％＝(OD药物-OD模型)/(OD对照-OD模 型)*100)，结果见表3。
[表3]
化合物 药物浓度(moL/L) 加药细胞存活率
neocrocin B(5) 10-5 40.03±3.91**
  10-6 27.63±5.36*
  10-7 9.89±2.93
藏红花酸单-β-D-吡喃葡萄糖苷(9) 10-5 0.00±0.00
  10-6 0.00±0.00
  10-7 0.00±0.00
藏红花酸双-β-D-吡喃葡萄糖苷(3) 10-5 0.00±0.00
  10-6 0.00±0.00
  10-7 0.00±0.00
藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷(2) 10-5 0.00±0.00
  10-6 0.00±0.00
  10-7 0.00±0.00
*P＜0.1，**P＜0.05，***P＜0.01.

Claims (17)

  1. 一种组合物，其特征在于，所述组合物包括neocrocin B和藏红花酸双-β-D-龙胆二糖苷，所述neocrocin B的结构式为
    Figure PCTCN2017076910-appb-100001
  2. 根据权利要求1所述的组合物，其特征在于，还包括藏红花酸单-β-D-龙胆二糖苷。
  3. 根据权利要求1所述的组合物，其特征在于，还包括13Z-藏红花酸双-β-D-龙胆二糖苷。
  4. 根据权利要求1所述的组合物，其特征在于，还包括藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷。
  5. 根据权利要求1所述的组合物，其特征在于，还包括藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷、藏红花酸双-β-D-吡喃葡萄糖苷、13Z-藏红花酸双-β-D-龙胆二糖苷、藏红花酸单-β-D-龙胆二糖苷、13Z-藏红花酸-8-O-β-D-龙胆二糖苷、13Z-藏红花酸-8′-O-β-D-龙胆二糖苷、藏红花酸单-β-D-吡喃葡萄糖苷。
  6. 根据权利要求5所述的组合物，其中，所述组合物中各组分的含量分别为：
    Figure PCTCN2017076910-appb-100002
  7. 一种藏红花色素类活性部位，其特征在于，包括权利要求1～6中任一项所述的组合物。
  8. 根据权利要求7所述的藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位是从栀子中提取得到的。
  9. 根据权利要求7所述的藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位的UPLC特征图谱主要包含9个色谱峰，将藏红花酸单-β-D-龙胆二糖苷的保留时间设为1，分别求出各色谱峰的相对保留时间，藏红花酸双-β-D-龙胆二糖苷的保留时间为0.38±0.02，藏红花酸-β-D-吡喃葡萄糖基-β-D-龙胆二糖苷的保留时间为0.48±0.02，藏红花酸双-β-D-吡喃葡萄糖苷的保留时间为0.60±0.02，13Z-藏红花酸双-β-D-龙胆二糖苷的保留时间为0.78±0.02，neocrocin B的保留时间为0.89±0.02，藏红花酸单-β-D-龙胆二糖苷的保留时间为1.00，13Z-藏红花酸-8-O-β-D-龙胆二糖苷的保留时间为1.13±0.02，13Z-藏红花酸-8′-O-β-D-龙胆二糖苷的保留时间为1.14±0.02，藏红花酸单-β-D-吡喃葡萄糖苷的保留时间为1.19±0.02。
  10. 根据权利要求9所述的藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位的UPLC特征图谱是采用反相超高效液相色谱法建立的，色谱条件是：以十八烷基硅烷键和硅胶为固定相，以含有0.1％甲酸的乙腈-水溶液为流动相，进行梯度洗脱，其中，流速为0.6mL/min，检测波长为440nm，色谱柱温度为35℃。
  11. 一种藏红花色素类活性部位，其特征在于，所述藏红花色素类活性部位通过下述方法进行制备：
    (1)将栀子干燥果实适当粉碎后，用乙醇、甲醇或水，采用不同提取次数和时间，通过热提取或者超声提取的方法进行提取，减压浓缩提取液，得到栀子总提取物；
    (2)用适量水溶解所述栀子总提取物，离心，上清液通过大孔吸附树脂开放柱色谱，用水和/或30％～95％的乙醇洗脱适量的柱床体积，收集洗脱液，减压浓缩70％乙醇的洗脱液，得到所述藏红花色 素类活性部位。
  12. 一种制备权利要求7～10中任一项所述的藏红花色素类活性部位的方法，其特征在于，包括以下步骤：
    (1)将栀子干燥果实适当粉碎后，用乙醇、甲醇或水，采用不同提取次数和时间，通过热提取或者超声提取的方法进行提取，减压浓缩提取液，得到栀子总提取物；
    (2)用适量水溶解所述栀子总提取物，离心，上清液通过大孔吸附树脂开放柱色谱，用水和/或30％～95％的乙醇洗脱适量的柱床体积，收集洗脱液，减压浓缩，得到所述藏红花色素类活性部位。
  13. 根据权利要求12所述的方法，其特征在于，在步骤(1)中，用4倍量的60％乙醇，加热回流提取3次，每次2小时；
    在步骤(2)中，用水、30％乙醇、50％乙醇、70％乙醇、95％乙醇依次洗脱，每个梯度洗脱4个柱床体积，减压浓缩70％乙醇洗脱液，得到所述藏红花色素类活性部位。
  14. 权利要求7～11中任一项所述的藏红花色素类活性部位在制备改善学习记忆能力的药物中的应用。
  15. 权利要求7～11中任一项所述的藏红花色素类活性部位在制备预防和治疗阿尔茨海默症的药物中的应用。
  16. 一种药物组合物，其特征在于，包括权利要求7～11中任一项所述的藏红花色素类活性部位、一种或多种其他具有中枢神经系统保护作用的药物以及适当的药物辅料。
  17. 权利要求16所述的药物组合物在制备预防和治疗中枢神经退行性疾病的药物中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913029A (zh) * 2021-11-13 2022-01-11 福建师范大学 一种制备黄栀子有效成分的方法

Families Citing this family (4)

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CN105935363B (zh) * 2016-04-29 2017-03-08 暨南大学 组合物、藏红花色素类活性部位及其用途
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CN109045047B (zh) * 2018-08-06 2020-04-28 暨南大学 藏红花色素类组合物在制备用于治疗帕金森症的药物中的应用
US20230210803A1 (en) * 2020-04-09 2023-07-06 L.E.A.F. Holdings Group Llc Trans-crocetin compositions and treatment regimens

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1347868A (zh) * 2000-10-08 2002-05-08 程锦雁 藏红花的提取物具有改善脑机能的功能
CN102516325A (zh) * 2011-11-15 2012-06-27 江西中天农业生物工程有限公司 一种以栀子为原料生产纯度大于95%的藏红花素的方法
CN105935363A (zh) * 2016-04-29 2016-09-14 暨南大学 组合物、藏红花色素类活性部位及其用途

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100478348C (zh) * 2007-02-02 2009-04-15 华东理工大学 栀子果实中环烯醚萜苷和藏红花素的分离富集方法
JP2010195779A (ja) * 2009-02-18 2010-09-09 Omnica Gmbh クロシンの加水分解物
CN102432455B (zh) * 2011-12-14 2014-02-19 广西大学 一种制备藏花酸和藏花素的方法
CN102898864B (zh) * 2012-11-09 2013-12-18 苏州衷中医药科技有限公司 一种采用溶剂结晶精制栀子黄色素的方法
US9211298B2 (en) * 2012-11-16 2015-12-15 Song Gao Compositions containing enriched natural crocin and/or crocetin, and their therapeutic or nutraceutical uses
CN103387489B (zh) * 2013-07-11 2015-05-06 苏州大学 一种高纯度藏红花素和栀子苷的制备方法
CN105906672B (zh) * 2016-04-29 2017-12-29 暨南大学 藏红花色素类化合物及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1347868A (zh) * 2000-10-08 2002-05-08 程锦雁 藏红花的提取物具有改善脑机能的功能
CN102516325A (zh) * 2011-11-15 2012-06-27 江西中天农业生物工程有限公司 一种以栀子为原料生产纯度大于95%的藏红花素的方法
CN105935363A (zh) * 2016-04-29 2016-09-14 暨南大学 组合物、藏红花色素类活性部位及其用途

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913029A (zh) * 2021-11-13 2022-01-11 福建师范大学 一种制备黄栀子有效成分的方法
CN113913029B (zh) * 2021-11-13 2023-11-24 福建师范大学 一种制备黄栀子有效成分的方法

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