CN109234016A - A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect - Google Patents

A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect Download PDF

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Publication number
CN109234016A
CN109234016A CN201811218281.2A CN201811218281A CN109234016A CN 109234016 A CN109234016 A CN 109234016A CN 201811218281 A CN201811218281 A CN 201811218281A CN 109234016 A CN109234016 A CN 109234016A
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tea tree
ethereal oil
tree ethereal
tea
cancer cell
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Inventor
吴亮宇
林金科
李永裕
王文婷
杜之正
黄旭建
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Fujian Agriculture and Forestry University
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Fujian Agriculture and Forestry University
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Priority to CN201811218281.2A priority Critical patent/CN109234016A/en
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/02—Recovery or refining of essential oils from raw materials
    • C11B9/025—Recovery by solvent extraction
    • 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)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
    • 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
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • 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

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medical Informatics (AREA)
  • Epidemiology (AREA)
  • Botany (AREA)
  • Wood Science & Technology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention belongs to plants essential oil extractive technique fields, specifically provide a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect.The present invention is by adding petroleum ether extraction for dry tea crushing, and then tea tree ethereal oil is finally made in water-bath, centrifugation, suction filtration.The product prices of raw materials according to the present invention are cheap, by a series of processing technologys, obtain target product.The product not only remains the active skull cap components of tea tree, but also has the effect of inhibiting tumor cell proliferation, and a kind of pair of beneficial essential oil product of human health can be provided for consumer.

Description

A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect
Technical field
The invention belongs to plants essential oil extractive technique fields, specifically provide a kind of with the tea for inhibiting cancer cell multiplication effect Set essential oil preparation method.
Background technique
Tea tree ethereal oil, be from Theaceae Camellia (Camellia sinensis) plant blade and tender tip be raw material extract A substance, have the effects that bactericidal antiphlogistic, convergence pore, adjust body immunity.And presently commercially available major part is known as " Australia The substance of continent tea tree ethereal oil ", be from narrow leaved tea tree (Melaleuca ahemifolia) a kind of plant in extract, at Point and the tea tree ethereal oil that refines of effect and Theaceae Camellia it is not identical.
Summary of the invention
The object of the present invention is to provide a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect.This Invention utilizes Theaceae Tea plant, that is, fresh leaf and the tender slightly raw material of traditional tree plant cultivation kind, is mentioned It takes, obtains with a kind of essential oil for inhibiting hepatoma cell proliferation effect.The product has good health-care efficacy.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect, comprising the following steps:
(1) Theaceae Tea plant blade and tender tip are acquired, freeze-drying is placed in and concentrates, be freeze-dried with -25 to -50 DEG C, it will Obtained freeze-drying type dry tea sample is crushed with pulverizer, crosses 40-100 mesh, dry tea sample after being crushed.
(2) dry tea sample after crushing is placed in 500 mL conical flasks, crushes dry tea sample according to every 1g and is added 10g's Petroleum ether is added in petroleum ether ratio.
(3) 24-72 h is extracted in 25-40 DEG C of shaking bath.Conical flask is taken out, is sub-packed in centrifuge tube, item is centrifuged Part are as follows: 4-10 DEG C, 8000-10000 rpm, be centrifuged 10-15 min.
(4) filtrate after centrifugation is filtered, is placed in 10 mL round-bottomed flasks, be concentrated in vacuo, condition: 37-40 DEG C of water temperature, Vacuum degree 100-200 mpa.
(5) tea tree ethereal oil solid then is dissolved with dehydrated alcohol, obtains tea tree ethereal oil.
Remarkable advantage of the invention is: recovery rate is higher, and gained tea tree ethereal oil purity is high is extracted mountain to the maximum extent Effective component in Theaceae plant bud-leaf, utilization efficiency is higher, and moisture content is lower, is conducive to save, and without poisonous and harmful solvent Residual, simple process is few using equipment, is suitable for mass production, and extracting resulting product has good biological activity, has wide Market prospects.
Detailed description of the invention
The survival rate of liver cancer cells after 1 essential oil of the embodiment of the present invention is added in Fig. 1；
The film potential variation of liver cancer cells after 1 essential oil of the embodiment of the present invention is added in Fig. 2.
Specific embodiment
Further to disclose rather than the present invention is limited, the present invention is described in further detail below in conjunction with example.
Embodiment 1
A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect, comprising the following steps:
(1) Theaceae Tea plant blade and tender tip are acquired, freeze-drying is placed in and concentrates, be freeze-dried, will be obtained with -20 DEG C Freeze-drying type dry tea sample crushed with pulverizer, sieve with 100 mesh sieve, dry tea sample after being crushed.
(2) dry tea sample after crushing is placed in 500 mL conical flasks, crushes dry tea sample according to every 1g and is added 10g's Petroleum ether is added in petroleum ether ratio.
(3) 72 h are extracted in 40 DEG C of shaking bath.Conical flask is taken out, is sub-packed in centrifuge tube, centrifugal condition are as follows: 5 DEG C, 10000 rpm are centrifuged 15 min.
(4) filtrate after centrifugation is filtered, is placed in 10 mL round-bottomed flasks, be concentrated in vacuo, condition: 40 DEG C of water temperature, very 150 mpa of reciprocal of duty cycle.
(5) tea tree ethereal oil solid then is dissolved with dehydrated alcohol, obtains tea tree ethereal oil.The tea tree ethereal oil obtained by this method mentions Taking rate is 1%.
Embodiment 2
A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect, comprising the following steps:
(1) Theaceae Tea plant blade and tender tip are acquired, freeze-drying is placed in and concentrates, be freeze-dried with -50 DEG C, by what is obtained Freeze-drying type dry tea sample is crushed with pulverizer, crosses 80 meshes, dry tea sample after being crushed.
(2) dry tea sample after crushing is placed in 500 mL conical flasks, crushes dry tea sample according to every 1g and is added 10g's Petroleum ether is added in petroleum ether ratio.
(3) 72 h are extracted in 35 DEG C of shaking bath.Conical flask is taken out, is sub-packed in centrifuge tube, centrifugal condition are as follows: 4 DEG C, 10000 rpm are centrifuged 15 min.
(4) filtrate after centrifugation is filtered, is placed in 10 mL round-bottomed flasks, be concentrated in vacuo, condition: 35 DEG C of water temperature, vacuum Spend 100 mpa.
(5) tea tree ethereal oil solid then is dissolved with dehydrated alcohol, obtains tea tree ethereal oil.The tea tree ethereal oil obtained by this method mentions Take rate in 1-1.2%.
Embodiment 3
A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect, comprising the following steps:
(1) Theaceae Tea plant blade and tender tip are acquired, freeze-drying is placed in and concentrates, be freeze-dried, will be obtained with -35 DEG C Freeze-drying type dry tea sample crushed with pulverizer, cross 60 meshes, dry tea sample after being crushed.
(2) dry tea sample after crushing is placed in 500 mL conical flasks, crushes dry tea sample according to every 1g and is added 10g's Petroleum ether is added in petroleum ether ratio.
(3) 48 h are extracted in 35 DEG C of shaking bath.Conical flask is taken out, is sub-packed in centrifuge tube, centrifugal condition are as follows: 6 DEG C, 10000 rpm are centrifuged 12 min.
(4) filtrate after centrifugation is filtered, is placed in 10 mL round-bottomed flasks, be concentrated in vacuo, condition: 40 DEG C of water temperature, very 200 mpa of reciprocal of duty cycle.
(5) tea tree ethereal oil solid then is dissolved with dehydrated alcohol, obtains tea tree ethereal oil.The tea tree ethereal oil obtained by this method mentions Taking rate is 0.8-0.9%.
Inhibit liver cancer cells (HepG-2) proliferation experiment
Prefabricated tea tree ethereal oil is pressed into 1/25,1/50,1/100,1/200,1/400 isometric dilution proportion.HepG-2 is thin Born of the same parents cultivate to logarithmic growth phase, and pancreatin digestion is inoculated in 96 orifice plates (every 200 μ L of hole), in incubator, 37 DEG C, and 5%CO2Training It supports, after its degrees of fusion reaches 70-80%, absorbs culture solution, the specific concentrations such as difference that every hole adds the corresponding culture medium of 100 μ L to prepare Tea tree ethereal oil solution of the present invention, continue to cultivate 24 h, then every hole continues to incubate after adding the MTT solution (5 mg/mL) of 20 μ L 4 h are educated, culture solution is removed, 150 μ L dimethyl sulfoxides are added in every hole, and measurement sample is inhaled at concussion 5 min, with 490nm wavelength Luminosity sets zeroing hole for corresponding culture medium to prevent sample itself from influencing, and using sample solvent replace sample as pair According to.
As shown in Figure 1, compared with normally culture liver cancer cells, after the essential oil of different dilution ratios is added, liver cancer cells Survival rate is substantially reduced, and survival rate is 60% hereinafter, illustrate that tea tree ethereal oil generates apparent inhibition to hepatoma cell proliferation and makees With.
Cell membrane JC-1 experiment
JC-1 is that one kind is widely used in the glimmering of detection mitochondrial membrane potential (mitochondrial membrane potential) Light probe.It can detecte cell, tissue or the mitochondrial membrane potential of purifying.The decline of mitochondrial membrane potential is Apoptosis early stage A hallmark events.Cell membrane electricity can be reliably detected by transformation of the JC-1 from red fluorescence to green fluorescence The decline of position, while one of the JC-1 transformation from red fluorescence to green fluorescence as Apoptosis early stage can also be used to detect Index.By prefabricated tea tree ethereal oil by 1/25,1/50,1/100,1/200,1/400 volume proportional diluted by cell culture extremely Logarithmic growth phase, pancreatin digestion, is inoculated in 96 orifice plates (every 200 μ L of hole), in CO2Proportional diluted tea tree is added in incubator culture Culture solution is absorbed after essential oil solution 100 μ l, 24 h, and 50 μ l and JC-1 dyeing liquor of cell culture fluid, 50 μ l is added, mixes well. 20min is incubated in cell incubator.After incubation, supernatant is absorbed, is washed 2 times with JC-1 Buffer (1 ×).100 μ are added L cell culture fluid, is detected using fluorescence microplate reader.Exciting light 490nm is set when detecting JC-1 monomer, and 530nm is arranged in transmitting light； When detecting JC-1 condensate, exciting light 525nm is set, 590nm is arranged in transmitting light.
As shown in Figure 2, compared with normally culture liver cancer cells, after the essential oil of different dilution ratios is added, liver cancer cells Cell membrane potential is remarkably decreased, and as essential oil concentration used increases, and negative correlativing relation is presented in film potential, illustrates that essential oil is added Afterwards, the cell membrane of liver cancer cells can be had an impact, is induced cell apoptosis, to inhibit the proliferation of liver cancer cells.

Claims (4)

1. a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect, it is characterised in that: the following steps are included:
(1) Theaceae Tea plant blade and tender tip are acquired, freeze-drying is placed in and concentrates, be freeze-dried with -25 to -50 DEG C, it will Obtained freeze-drying type dry tea sample is crushed with pulverizer, crosses 40-100 mesh, dry tea sample after being crushed；
(2) dry tea sample after crushing is placed in 500 mL conical flasks, petroleum ether is added to dry tea sample is crushed；
(3) 24-72 h is extracted in 25-40 DEG C of shaking bath；Conical flask is taken out, is sub-packed in centrifuge tube, is centrifuged；
(4) filtrate after centrifugation is filtered, is placed in 10 mL round-bottomed flasks, be concentrated in vacuo；
(5) tea tree ethereal oil solid then is dissolved with dehydrated alcohol, obtains tea tree ethereal oil.
2. according to claim 1 a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect, feature Be: every 1g crushes the petroleum ether that 10g is added in dry tea sample in step (2).
3. according to claim 1 a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect, feature It is: step (3) centrifugal condition are as follows: 4-10 DEG C, 8000-10000 rpm, be centrifuged 10-15 min.
4. according to claim 1 a kind of with the tea tree ethereal oil preparation method for inhibiting cancer cell multiplication effect, feature It is: step (4) vacuum concentration condition: 37-40 DEG C of water temperature, vacuum degree 100-200 mpa.
CN201811218281.2A 2018-10-19 2018-10-19 A kind of tea tree ethereal oil preparation method with inhibition cancer cell multiplication effect Pending CN109234016A (en)

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Application publication date: 20190118