CN104208141B - Purposes of the palchouli oil in preparation treatment prostate cancer brain metastes tumour medicine - Google Patents

Abstract

The purposes of use on the way that the invention discloses palchouli oils in the health food, food, drug that preparation prevented, treated or assisted in the treatment of prostate cancer；The volatile oil that the palchouli oil is extracted from Pogostemon cablin Pogostemon cablin (Blanco) Benth. or wrinkled giant hyssop Agastache rugosa (Fisch.Et Mey.) O.Ktze..Palchouli oil of the present invention has antineoplastic action, especially significant to the inhibiting effect of prostate cancer, can be effectively used for treatment prostate cancer and its metastatic lesion, and safety is substantially better than chemotherapeutic drug Paclitaxel, has good potential applicability in clinical practice.

Description

Purposes of the palchouli oil in preparation treatment prostate cancer brain metastes tumour medicine

Technical field

The present invention relates to the new application of palchouli oil, in particular to palchouli oil prepare anti-prostate cancer health food, food, Purposes in drug.

Background technique

Prostate cancer is common one of the malignant tumour of elderly men.Worldwide, prostate-cancer incidence is in male Second is occupied in all malignant tumours of property.In the U.S., prostate-cancer incidence occupies first in all male malignancies, extremely The rate of dying occupies second.In China, its disease incidence has also leapt to the third position of genito-urinary system malignant tumour in recent years.Prostate cancer Morbidity clinic early symptom it is few, most of patient reaches an advanced stage when making a definite diagnosis, and loses surgical radical treatment opportunity.The radical cure of row prostate cancer The patient of art has 27%~53% local recurrence or DISTANT METASTASES IN in 10 years after surgery.Endocrine therapy is current advanced stage forefront The primary treatments of gland cancer, but after median time 14~30 months, nearly all patients with prostate cancer finally switchs to Androgen independent prostate cancer (androgen-independent prostate cancer, AIPC), and then it is sharp for developing Plain refractory prostate cancer (hormone-refractory prostate cancer, HRPC).Such prostate cancer is referred to as Castration-resistant prostate cancer (castrate-resistant prostate cancer, CRPC).Castration-resistant prostate Cancer life in patients is poor, and median survival interval 12~20 months.With prostate-cancer incidence, the rising of the death rate, how to have Effect treatment castration-resistant prostate cancer suffers from the hot spot for having become modern medicine study.

The treatment means of castration-resistant prostate cancer are mainly with Docetaxel, mitoxantrone, prednisone etc. at present Medication combined chemotherapy, side effect is obvious, and there is no therapeutic regimen.New type antineoplastic medicine, which is currently in, to be continually developed Conceptual phase.Therefore, the research for finding a kind of efficient, safety, few side effects anti-tumor drug is imperative.

The natural resources of Chinese medicinal materials in China is extremely abundant, and applicating history is long, and many data and warp are had accumulated in long-term practice It tests.Chinese herb prevention tumour has the advantages such as significant in efficacy, Small side effects, expense are few, is continued to use for a long time by vast clinical workers. Some Chinese medicines can block or inhibit the synthesis of DNA of tumor cell, RNA and protein, so that it is suppressed the proliferation of cancer cell, And it interferes tumour cell energetic supersession or there is direct cancer inhibitting and killing cytosis.Such as Rabdosia rubescens, Paris polyphylla, Radix Notoginseng, subprostrate sophora, Tian Nan Star, lucid asparagus, lily, curcuma zedoary, Snakegourd Fruit, semen coicis, black nightshade, Radix Curcumae, fruit of glossy privet etc..Some Chinese medicines can by influence oncogene and The expression of tumor suppressor gene induces and body cell is promoted to generate the apoptosis that cell factor etc. promotes tumour cell, as Radix Astragali, when Return, fructus lycii etc. can induce LAK Apoptosis or increasing by cell factors such as induction interleukin 2 (IL-2), interferon Add the ability that TNF is apoptosis-induced.In addition, gingko exocarp polysaccharide, resveratrol, matrine, trichosanthin, ganoderma lucidum alcohol extracting Object, ginsenoside, Radix Angelicae Sinensis acetone extract play the role of modulating apoptosis Genes bcl-2 and suppression apoptogene Bax expression, from And realize the adjusting to apoptosis of tumor cells, reach function of tumor inhibition.Radix Astragali, ginseng, Radix Codonopsis, Radix Ophiopogonis, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae, female Loyal son, fructus lycii, Radix Rehmanniae, Radix Angelicae Sinensis etc. can improve or restore the T cell immune functional state of tumor patient disorder, increase CD3⁺、 CD4⁺And CD4⁺/CD8⁺Ratio.There are also many Chinese medicines, and physical stress ability can be improved, and mitigates chemotherapy, Esophageal carcinoma, Such as rhizoma polygonati, Radix Astragali, Caulis Spatholobi, pheretima, Schisandra chinensis, the fleece-flower root, semen coicis, thizoma curculiginis, Herba Epimedii, asparagus fern, Radix Ophiopogonis, radices trichosanthis, stone Dry measure used in former times, pyrrosia lingua etc..Therefore Chinese medicine is antitumor potential advantages and wide prospect.

Pogostemon cablin is Labiatae (Labiatae) perennial thorn stamen herbaceous plant Pogostemon cablin (Pogostemon cablin (Blanco) Benth.) also known as branch perfume, the Tropical Asias such as Philippine are originated in, China, India, Japan, India are now widely distributed in Nicaea, Malaysia, Madagascar, Brazil, Paraguay and Russia etc..At present wide flower bud perfume (or spice) the Guangzhou in Guangdong, Zhaoqing, Zhanjiang and Hainan, Guangxi, Sichuan etc., which save (area), cultivation, is one of " ten great Nan medicines ", and medicinal material commodity are different by the place of production It is divided into board fragrant (Guangzhou production), branch fragrant (Zhaoqing production), 4 kinds of Zhan perfume (Zhanjiang production) He Nanxiang (Hainan production).Its acrid flavour, slightly warm in nature are returned Spleen, stomach, lung channel have with all herbal medicine and eliminate dampness with aromatics, the preventing or arresting vomiting that whets the appetite, deliver the effect of relieving summer-heat.It is usually used in turbi damp obstructing in middle-JIAO, gastral cavity Ruffian vomiting, summer-heat and damp burnout, not easypro, cold-dampness uncomfortable in chest close heat, abdominal pain vomiting and diarrhoea, nasosinusitis headache, affection of exogenous wind-cold.The volatile oil of Pogostemon cablin has There are anti-inflammatory, analgesia and antibacterial activity.

Wrinkled giant hyssop is on the dry ground of Lamiaceae plant wrinkled giant hyssop Agastache rugosa (Fisch.Et Mey.) O.Ktze. Part, containing volatile oil, the same Pogostemon cablin of effect.Modern pharmacology shows that Pogostemon cablin has resisting pathogenic microbes, anti-inflammatory, antipyretic, town The effects of pain, is the treatment such as HuoXiangZhengQiShui, antiviral oral liquor influenza, catches a cold and commonly use the main component of Chinese patent drug, but its medicine Effect material base is still not clear and there are no its internal resisiting influenza virus effect report.The volatile oil of wrinkled giant hyssop has antibacterial action.

Summary of the invention

The purpose of the present invention is to provide the new applications of palchouli oil, and using palchouli oil as the health food of active constituent, Food, drug.

The present invention tests discovery, and patchouli oil has good inhibition/apoptosis-induced effect to PC3 and DU145 cell, In, PC3 is androgen-independent prostate cancer cell lines in vitro, DU145 is separated from prostate cancer brain metastes tumour, For the prostate gland cancer cell of androgen independent, there is powerful metastatic potential.It can be seen that patchouli oil can effectively antagonize Prostate gland cancer cell, also, have to Androgen Independent Prostate Cancer and prostate cancer brain metastes class tumour cell good Inhibitory activity.

The present invention provides palchouli oils to prepare the health food, food, medicine for preventing, treating or assisting in the treatment of prostate cancer The purposes of use on the way in product；The palchouli oil from Pogostemon cablin Pogostemon cablin (Blanco) Benth. or The volatile oil that wrinkled giant hyssop Agastache rugosa (Fisch.Et Mey.) O.Ktze. is extracted.

Further, the prostate cancer is Androgen Independent Prostate Cancer or castration-resistant prostate cancer.

Further, the health food, food, drug are to prevent, treat or assist in the treatment of prostate cancer to be transferred to Bone, brain, lung or the health food of liver, food, drug.

Preferably, the health care product, food or drug are the health care product, food or drug of cancer cell specific induction of apoptosis.

Further, the palchouli oil is derived from the volatile oil of Pogostemon cablin Pogostemon cablin (Blanco) Benth., Patchouli alcohol content is not less than 26%w/w.

Further, the palchouli oil is derived from the volatilization of Pogostemon cablin Pogostemon cablin (Blanco) Benth. Oil, patchouli alcohol content are 26~40%w/w.

In the specific embodiment of the present invention, in the Herba Pogostemonis Volatile oil that uses, Bai Qiuli alcohol content is about 40%/ w/w。

Further, the palchouli oil is to be prepared as follows: taking Pogostemon cablin Pogostemon cablin (Blanco) herb of Benth. or wrinkled giant hyssop Agastache rugosa (Fisch.Et Mey.) O.Ktze., is ground into coarse powder, Add water, impregnate, using extraction by steam distillation to get.

In a specific embodiment of the invention, the volatile oil that is prepared using Pogostemon cablin herb.

Wherein, the health care product, food or drug are using palchouli oil as active constituent, in addition health food, food, drug The preparation that upper acceptable auxiliary material or complementary ingredient are prepared.

Pharmaceutically acceptable auxiliary material of the present invention refers in addition to the active ingredient (s include substance in dosage form, packet Include but be not limited only to filler (diluent), lubricant (glidant or antitack agent), dispersing agent, wetting agent, adhesive, adjusting Agent, solubilizer, antioxidant, bacteriostatic agent, emulsifier, disintegrating agent etc..Adhesive include syrup, Arabic gum, gelatin, sorbierite, Tragacanth, cellulose and its derivates (such as microcrystalline cellulose, sodium carboxymethylcellulose, ethyl cellulose or hydroxypropyl methylcellulose Element etc.), gelatine size, syrup, starch slurry or polyvinylpyrrolidone etc.；Filler include lactose, Icing Sugar, dextrin, starch and its Derivative, cellulose and its derivates, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium monohydrogen phosphate, precipitated calcium carbonate), sorb Alcohol or glycine etc.；Lubricant includes superfine silica gel powder, magnesium stearate, talcum powder, aluminium hydroxide, boric acid, hydrogenated vegetable oil, poly- second Glycol etc.；Disintegrating agent includes starch and its derivative (such as sodium carboxymethyl starch, Explotab, pregelatinized starch, improvement shallow lake Powder, hydroxypropul starch, cornstarch etc.), polyvinylpyrrolidone or microcrystalline cellulose etc.；Wetting agent includes dodecyl sulphate Sodium, water or alcohol etc.；Antioxidant packages are containing sodium sulfite, sodium hydrogensulfite, sodium pyrosulfite, dibutyl benzoic acid etc.；Bacteriostatic agent includes 0.5% phenol, 0.3% cresols, 0.5% anesin etc.；Regulator includes hydrochloric acid, citric acid, potassium hydroxide (sodium), Chinese holly Rafter acid sodium and buffer (including sodium dihydrogen phosphate and disodium hydrogen phosphate) etc.；Emulsifier includes Tween-80, fatty acid mountain Pears are smooth, pluronic gram F-68, lecithin, Fabaceous Lecithin etc.；Solubilizer includes Tween-80, bile, glycerol etc..

The pharmaceutically acceptable complementary ingredient, it has certain physiological activity, but the addition of the ingredient will not change Become the leading position of above compound or derivative in the course of disease treatment, and only plays auxiliary effect, these auxiliary function Effect is only the utilization to the ingredient known activity, is the usual adjuvant treatment modality of field of medicaments.If by it is above-mentioned it is complementary at Divide and be used cooperatively with the compounds of this invention, still should belong to the scope of protection of the invention.

Further, the preparation is liquid preparation, gaseous formulation, solid pharmaceutical preparation, semisolid preparation.

Further, the content of palchouli oil is 0.1%~100% (w/w) in the preparation.

Patchouli oil has antineoplastic action, and to inhibiting prostate cancer growth effect clear, it is non-to can treat androgen Dependence prostate cancer and its each organ metastasis lesion, and activity is better than Bai Qiuli alcohol monomer compound, cost is less expensive, market Application prospect is good.

In addition, inventor is the study found that patchouli oil is living to the inhibition of androgen-independent prostate cancer cell lines in vitro (PC3) Property, which is substantially better than, is applied alone Bai Qiuli alcohol；Bai Qiuli determining alcohol is significantly lower than in the case where being applied alone Bai Qiuli alcohol in patchouli oil (only the 43.7%~67.2% of monomer concentration), patchouli oil, which can reach, is applied alone Bai Qiuli alcohol swollen to prostate cancer brain metastes Patchouli oil can be used directly when specifically used in the inhibiting effect of tumor (DU145), without using purity higher white Autumn Lee's alcohol monomer, hence it is evident that save drug cost.

Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.

The specific embodiment of form by the following examples remakes further specifically above content of the invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention The technology realized all belongs to the scope of the present invention.

Detailed description of the invention

Fig. 1 apoptosis morphology changes (transmission electron microscope, × 5000)

Influence of Fig. 2 various concentration patchouli oil to the PC3 cell cycle；A: control group；B~F: patchouli oil concentration is 40、 60、80、100、120μg/ml

Influence of Fig. 3 various concentration patchouli oil to the DU145 cell cycle；A: control group；B~F: patchouli oil concentration is 40、 60、80、100、120μg/ml

Fig. 4 tests each group nude mice weight variation tendency

Fig. 5 tests each group nude mouse tumor volume change trend

Fig. 6 tests each group nude mouse tumor appreciation rate T/C (%) variation tendency

Fig. 7 tests each group tumor weight

Specific embodiment

The patchouli oil that the present invention uses can purchase commercial product, can also pass through extraction by steam distillation, wherein the wide leaves of pulse plants The quality of sesame oil has to comply with the relevant regulations in " Chinese Pharmacopoeia " one, and patchouli alcohol content is not less than 26%w/w.

The preparation of the palchouli oil of the present invention of embodiment 1

Pogostemon cablin Pogostemon cablin (Blanco) Benth. herb is taken, crushed 20-40 mesh, 10-14 is added The distilled water of times weight, after impregnating 1-5 hours, using extraction by steam distillation 2-6 hours to get palchouli oil of the present invention, again Claim patchouli oil.

Through detecting, in the patchouli oil of the method for the present invention preparation, (the C containing patchouli alcohol₁₅H₂₆O) about 40%w/w.

The preparation of the palchouli oil of the present invention of embodiment 2

Wrinkled giant hyssop Agastache rugosa (Fisch.Et Mey.) O.Ktze. herb is taken, crushed 20-40 mesh, is added The distilled water of 10-14 times of weight, after impregnating 1-5 hours, using extraction by steam distillation 2-6 hours to get wrinkled giant hyssop of the present invention Oil.

Palchouli oil of the present invention can also be extracted according to pharmacopeia (version in 2005) annex XD determination of volatile oil method, also can be used organic Solvent extraction, supercritical CO₂The prior arts such as extraction extract, or are obtained by purchase commercial product.

The food of the present invention of embodiment 3

Palchouli oil and red bayberry prepared by Example 1 or embodiment 2, adds appropriate orange peel powder, cinnamomi cortex pulveratus, granulated sugar, flos caryophylli Powder, licorice powder, fennel powder, salt and alum, prepare glazed waxberry.

The red bayberry of one layer of about 20cm thickness is first put after taking red bayberry to be ready for, then puts the salt of one layer of mixing, alum, patchouli alcohol, It compresses at once.It is later same it is alternate put red bayberry and salt alum patchouli alcohol mixture, compressed it is marinated after red bayberry base.It takes After red bayberry base need to add granulated sugar, fragrance powder, orange peel powder, cinnamomi cortex pulveratus, flos caryophylli powder, licorice powder, fennel powder, then through immersion, sunning, Spice, packaging are to get glazed waxberry.

The health food of the present invention of embodiment 4

Palchouli oil prepared by Example 1 or embodiment 2, adds appropriate sodium carboxymethylcellulose, and withers in right amount through withering trough It withers, the oriental wormwood after the water-removing of double pots of green-keeping machines, rolling machines are rubbed, fructus amomi, fructus lycii, fingered citron, folium isatidis fresh tea, chrysanthemum, cassia seed, gold Honeysuckle flower, Rhizoma Atractylodis Macrocephalae, pulp of dogwood fruit, Radix Paeoniae Alba, mulberry leaf, Schisandra chinensis, fennel seeds, Chinese yam are uniformly mixed, and prepare liver-nourishing tea.

The drug of the present invention of embodiment 5

Palchouli oil prepared by Example 1 or embodiment 2, adds appropriate sodium carboxymethylcellulose, and withers in right amount through withering trough It withers, the oriental wormwood after the water-removing of double pots of green-keeping machines, rolling machines are rubbed, fructus amomi, fructus lycii, fingered citron, folium isatidis fresh tea, chrysanthemum, cassia seed, gold Honeysuckle flower, Rhizoma Atractylodis Macrocephalae, pulp of dogwood fruit, Radix Paeoniae Alba, mulberry leaf, Schisandra chinensis, fennel seeds, Chinese yam are uniformly mixed, and prepare liver-nourishing tea.

Beneficial effects of the present invention are proved below by way of pharmacological testing:

The palchouli oil anticancer experiment in vitro of the present invention of experimental example 1

1, material and instrument

1.1 cell strain

PC3 cell (people's androgen dependent/non-dependent prostate gland cancer cell), DU145 cell (people's androgen dependent/non-dependent forefront Adenocarcinoma cell), it is purchased from Shanghai Chinese Academy of Sciences cell bank.

1.2 drugs and control drug

Trial drug: invention drug: patchouli oil prepared by embodiment 1.

Positive drug: paclitaxel injection: specification 6mg/ml, 5ml/ branch.The limited public affairs of Taiji Group Sichuan Tai Ji pharmacy Department's production.Authentication code: national drug standard H19994040, product batch number: 12060014.

1.3 culture mediums, reagent and consumptive material

F-12 culture medium (Gibco^TMCompany)；Fetal calf serum (Lanzhou people's marine growth Engineering Co., Ltd, lot number 20120510)；0.25%EDTA- pancreatin (Kai Ji biotech firm)；Methyl thiazoly tetrazolium assay (MTT) is Amerseo product；It is green Mycin G sodium, streptomysin (North China pharmacy)；Steril cell culture bottle and 96 porocyte culture plates (Corning company, the U.S.).200μ L, the disposable pipette tips of 1ml (Jiangsu Haimen biology consumptive material company).Dimethyl sulfoxide (DMSO): it is purchased from Sigma Co., USA.It spits Temperature -80: it is purchased from Chengdu Ke Long chemical reagent factory.Glutaraldehyde: it is purchased from Tianjin Kermel Chemical Reagent Co., Ltd., lot number: 20130321.Osmium tetroxide: Lu Chang Chemical Co., Ltd., lot number: 20130225 are purchased from.Acetone: it is purchased from Chengdu section Long Huagong Chemical reagent work, lot number: 20130719.Acetic acid uranium: Xi'an Lianhu District wind and cloud chemical products sales department, lot number: 20130305 are purchased from. Lead citrate: Shanghai Rong Bai Bioisystech Co., Ltd, lot number: 20130318 are purchased from.DAPI: it is purchased from PARTEC company, Germany.

1.4 key instrument

Superclean bench (SuZhou Antai Air Tech Co., Ltd., Su Jing group, model: SW-CJ-1F)；

Water isolation type constant temperature CO₂Incubator (Thermo scientific company, the U.S.)；

OLYMPUS inverted microscope (model C KX41, Japanese Olympus company)；

Micropipettor (French GILSON company production)；

Vertical electric pressure steam sterilization boiler (SANYO GS company, model: MLS-3780)；

Table-type low-speed centrifuge (Changsha Xiang Yi centrifuge Instrument Ltd., model: L-600)；

Electronic balance (model JA-2603, Shanghai balance equipment factory).

Microplate reader Thermo acientific varioskan flash (U.S.).

Hitachi's H-600IV transmission electron microscope.

Flow cytometer, Beckman Counter company, the U.S., model: COULTER EPICS ELITE-ESP.

2, experimental method

The pre-treatment of 2.1 drugs and positive control

Invention drug patchouli oil 2ml is taken, 0.2ml Tween-80 hydrotropy is added, 37.8ml pure water is being added to be ground to cream repeatedly White liquid preparation, final concentration of 5% (patchouli oil density is 1.012g/ml, concentration 50.6mg/ml)；Tween-80 is dense eventually Degree is 0.5%, spare after filtration sterilization.

It will be used for antitumor positive control medicine paclitaxel injection and do 1/10 dilution (i.e. 600 μ g/ml) with sterile water With spare.

2.2 patchouli oils inhibit the test of prostate gland cancer cell proliferation function

Logarithmic growth phase cell is selected, cell suspension will be made after cell dissociation with 0.25%EDTA- pancreatin, adjust cell Suspension concentration is 6 × 10⁴/ ml is inoculated in 96 well culture plates, every 100 μ l of hole reaction volume.37 DEG C are set, 5%CO₂It is trained in incubator It supports for 24 hours, after cell adherent growth, abandons supernatant, wash cell face 1 time with PBS buffer solution and inhale abandoning.

It removes after culture solution respectively plus with the cell culture fluid of the patchouli oil containing various concentration, patchouli oil is from 50.6mg/ Ml starts, first 10 times dilutions, does continuous gradient dilutions, every hole reaction volume in the range after determining substantially tumor suppression range 100 μ l continue to set 37 DEG C, 5%CO₂24,48,72h are cultivated in incubator, each concentration sets 3 multiple holes, while setting containing cell not Drug containing contains solvent control group, the blank control group without cell and drug of 0.5% Tween-80.For comparative drug effect, sheet It tests while setting various concentration taxol as positive drug control.Using MTT colorimetric method for determining integral optical density (Integral Optical density, IOD) value, cell survival rate is calculated, thus calculates cell proliferation inhibition rate, then calculate drug IC₅₀。

Mtt assay measures cell activity: terminating preceding 96 orifice plate of culture and discards drug containing maintaining liquid, MTT (5mg/ml) is added in every hole 20 μ l of solution continues to cultivate 4h, terminates culture, carefully suck supernatant, cleaned 1 time with PBS, and 150 μ l DMSO are added in every hole, Low-speed oscillation 5min on shaking table is set, dissolves crystal sufficiently, the suction in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm wavelength Light value (IOD value).Cell proliferation inhibition rate is calculated according to the following formula:

Cell survival rate (%)=(medicine group IOD value-blank control group IOD value/solvent control group that is averaged that is averaged is average IOD value-blank control group is averaged IOD value) × 100%

Cell proliferation inhibition rate (%)=100%- cell survival rate (%)

Half-inhibitory concentration IC50 be that be medicine group IOD value reduce than solvent control group IOD value 50% drug concentration.

IC₅₀It is calculated using SPSS19.0 software.Statistical analysis, experimental data are carried out using SPSS19.0 statistics software With " average ± standard deviation" indicate, the comparison between sample average is examined using t, and group difference uses single factor test side Poor analytical control, P < 0.05 are that difference has conspicuousness.

The metamorphosis of 2.3 Electronic Speculum observation PC3, DU145 cell

2.3.1 cell drug is handled

By PC3 cell inoculation in Tissue Culture Flask, cell density is 0.5 × 10⁶/ ml, volume of culture 6ml.Set 37 DEG C, 5%CO₂It is cultivated in incubator for 24 hours, adds the culture solution used and contain 80 μ g/ml patchouli oils after removing culture solution, volume of culture is 6ml, if the not solvent control group of drug containing.

It is fixed 2.3.2 cell is collected

After cell culture 48h, suspension 4ml is made in 0.25% trypsin digestion culture cell, is put in centrifuge tube, 1500rpm is centrifuged 15 minutes, discards supernatant liquid.About 0.5% glutaraldehyde fixer (stoste PBS1:6 is slowly added to along tube wall Dilution) 4ml, 4 DEG C of standing 10min.Centrifuge 10000rpm is set, is centrifuged 10min again, discards supernatant liquid, be slowly added to 3% glutaraldehyde fixer 2ml is fixed.

2.3.3 electron microscope specimen production observation

3% glutaraldehyde is taken to pre-fix sample, 1% osmium tetroxide is fixed, and acetone is dehydrated step by step, Epon812 embedding, half thinly-sliced Ultra-thin section, acetic acid uranium and lead citrate double staining after piece optical alignment are set and observe cell ultrastructure simultaneously under transmission electron microscope Adopt figure.

Same treatment DU145 cell.

Influence of the 2.4 flow cytomery patchouli oils to PC3, DU145 cell cycle

2.4.1 cell drug is handled

By PC3 cell inoculation in 6 orifice plates, cell density is 0.5 × 10⁶/ ml, every hole reaction volume are 2ml.Set 37 DEG C, 5%CO₂12h is cultivated in incubator to adherent, after removing culture solution, with 40,60,80,100, the 120 μ g/ml containing patchouli oil Cell culture fluid handle cell, while setting the solvent control group of not drug containing, every hole reaction volume is 2ml, continue to set 37 DEG C, 5%CO₂It is cultivated in incubator.

It is fixed 2.4.2 cell is collected

After cell culture 48h, 0.25% trypsase 1ml digestion, which is added, in every hole makes attached cell fall off, and 2ml culture solution is whole Cell suspension is collected after only digesting to centrifuge tube, 1500rpm is centrifuged 5min and abandons supernatant, collects cell, and cold PBS liquid 10ml washing is thin Born of the same parents 2 times, centrifugation cell abandons supernatant.Cell is resuspended in the cold PBS liquid of 0.5ml, is rapidly added 3.5ml70% pre-cooled ethanol (4 DEG C), blow and beat uniformly fixed 30min.

2.4.3 cell DAPI dyeing observation

It is centrifuged the fixed cell of ethyl alcohol, abandons supernatant, PBS 5ml washs 2 removal residual ethanols of cell.1ml is added The dyeing of DAPI dye liquor, 4 DEG C were protected from light after 30min using the flow cytometer measurement cell cycle.

2.4.4 data processing

Flow cytomery is repeated 2 times, cell cycle data after obtained various concentration patchouli oil effect, with G₀/ G₁、S、G₂Each phase percentage (%) mean of/MIt indicates.

Same treatment DU145 cell.

3. experimental result

3.1 patchouli oils inhibit prostate gland cancer cell proliferation results

3.1.1 patchouli oil inhibits PC3 cel l proliferation

Solvent control group cell form under light microscopic keeps normal, patchouli oil experimental group cell during the test There is different degrees of condensation, core agglutination, cytoplasm blistering, cell detachment in form, and cell growth is suppressed.MTT testing result Further confirm that patchouli oil has growth inhibition effect to PC3 cell, and when action intensity has m- dose dependent.The wide leaves of pulse plants Sesame oil to PC3 for 24 hours, the maximum suppression concentration of 48h, 72h be respectively 170 μ g/ml, 170 μ g/ml and 140 μ g/ml, inhibiting rate Respectively 98.61%, 97.83% and 90.45%.Be calculated patchouli oil to PC3 for 24 hours, 48h, 72h IC₅₀Respectively 89.68 μ g/ml, 79.89 μ g/ml, 66.74 μ g/ml, 78.77 μ g/ml of mean value, group difference have statistical significance (P < 0.05).It the results are shown in Table 1.

Positive control medicine taxol to PC3 for 24 hours, the maximum suppression concentration of 48h, 72h be respectively 30 μ g/ml, 35 μ g/ Ml and 35 μ g/ml, inhibiting rate are respectively 93.52%, 93.79% and 92.88%.Be calculated taxol to PC3 for 24 hours, 48h、 72h IC₅₀Respectively 13.56 μ g/ml, 15.52 μ g/ml, 15.09 μ g/ml, 14.72 μ g/ml of mean value.It the results are shown in Table 2.

1 patchouli oil of table inhibits test result (x ± s, n=3) to PC3 growth of tumour cell

Note: patchouli oil acts on PC3 cell for 24 hours, IC after 48h, 72h₅₀Comparison among groups have statistical difference, * P < 0.05.

2 taxol of table inhibits test result (x ± s, n=3) to PC3 growth of tumour cell

3.1.2 patchouli oil inhibits DU145 cel l proliferation

Solvent control group cell form under light microscopic keeps normal, patchouli oil experimental group cell during the test There is different degrees of condensation, core agglutination, cytoplasm blistering, cell detachment in form, and cell growth is suppressed.MTT testing result Further confirm that patchouli oil has growth inhibition effect to DU145 cell, and when action intensity has m- dose dependent.Extensively Palchouli oil to DU145 for 24 hours, the maximum suppression concentration of 48h, 72h be respectively 170 μ g/ml, 160 μ g/ml and 140 μ g/ml, suppression Rate processed is respectively 97.53%, 96.31% and 91.28%.Be calculated patchouli oil to DU145 for 24 hours, 48h, 72h IC₅₀Point Not Wei 109.64 μ g/ml, 93.70 μ g/ml, 86.88 μ g/ml, 96.74 μ g/ml of mean value, group difference have statistical significance (P < 0.05).It the results are shown in Table 3.

Positive control medicine taxol to DU145 for 24 hours, the maximum suppression concentration of 48h, 72h be respectively 35 μ g/ml, 30 μ G/ml, 35 μ g/ml, inhibiting rate are respectively 91.83%, 96.03% and 97.70%.Taxol is calculated to DU145's 24h、48h、 72h IC₅₀Respectively 11.07 μ g/ml, 12.34 μ g/ml, 12.59 μ g/ml, 12 μ g/ml of mean value.It the results are shown in Table 4.

3 patchouli oil of table inhibits test result (x ± s, n=3) to DU145 growth of tumour cell

Note: patchouli oil acts on DU145 cell for 24 hours, IC after 48h, 72h₅₀Comparison among groups have statistical difference, * P < 0.05。

4 taxol of table inhibits test result (x ± s, n=3) to DU145 growth of tumour cell

3.2 patchouli oils act on apoptosis metamorphosis after PC3, DU145 cell

Control group PC3, DU145 cell adherent growth is secured, and cell membrane is complete, and cytoplasm is evenly distributed, dye in nucleus Chromaticness is evenly distributed, the apparent dense dye glomeration phenomenon of nothing, mitochondria in endochylema, rough surfaced endoplasmic reticulum (RER), and ribosomes etc. is clear in structure；Extensively The feature of the processed experimental group cell apoptosis cells of palchouli oil: nucleus, chromatin show that highlighting for fine and close dense dye is glimmering Light, karyopycnosis, electron density increase, and " crescent ", strip even fragment shape is presented；Cytoplasmic densitometric increases.Reticulum dilatation At blister, become cytoplasm bubble with cell membrane fusion (see Fig. 1).

Influence of 3.3 patchouli oils to PC3, DU145 cell cycle

After various concentration patchouli oil handles PC3 cell 48h, flow cytometry analysis showed, compared with the control group, with Drug concentration increases, G₀/G₁The cell proportion of phase improves；G₂The cell proportion of/M phase reduce in 60,100,120 μ g/ml groups (see Table 5, Fig. 2).And after various concentration patchouli oil processing DU145 cell 48h, flow cytometry analysis showed, with control group ratio Compared with, as drug concentration increases, G₀/G₁The cell proportion of phase improves；G₂The cell proportion of/M phase is in 60,100,120 μ g/ml groups It reduces (being shown in Table 6, Fig. 3).

Influence of the 5 flow cytomery various concentration patchouli oil of table to the PC3 cell cycle

Note: compared with the control group, * P < 0.05；Comparison among groups, ▲ P < 0.05.

Influence of the 6 flow cytomery various concentration patchouli oil of table to the DU145 cell cycle

Note: compared with the control group, * P < 0.05；Comparison among groups, ▲ P < 0.05.

4 brief summaries

4.1 carry out Vitro Tumor inhibition assay result it is found that Pogostemon cablin to two kinds of cells of PC3, DU145 by patchouli oil Oil has different degrees of growth inhibition effect to 2 kinds of prostate gland cancer cells.With IC₅₀Mean value and positive drug taxol are to 2 kinds The half cytostatic concentration IC of prostate gland cancer cell₅₀Compare as reference, patchouli oil to the inhibiting effect effect of PC3 born of the same parents most By force, followed by DU145 cell.

After 4.2 experiments are using 80 μ g/ml concentration of patchouli oil processing PC3, DU145 cell 48h, with transmission electron microscope observing It was found that the highlighted fluorescence of the fine and close dense dye of nucleus, chromatin presentation, karyopycnosis, endochylema is interior to there is the typical apoptosis spy such as a large amount of vacuoles Sign, and solvent control group cellular morphology is normal, prompts patchouli oil that can induce PC3, DU145 apoptosis in vitro, from And inhibit tumour growth.

4.3 cell cycles be adjusted be anti-tumor drug inhibit tumour growth common mechanism.This experimental applications stream The detection discovery of formula cell instrument, after various concentration patchouli oil acts on PC3, DU145 cell 48h, as drug concentration increases, G₀/G₁Phase cell proportion promotes (P < 0.05), G₂The cell proportion of/M phase is reduced in 60,100,120 μ g/ml medication groups.Explanation Patchouli oil can be by influencing PC3, DU145 cell cycle, and blocks tumor cells are in G₀/G₁Phase generates the work for inhibiting tumour growth With.

Anti-tumor experiment in the palchouli oil body of the present invention of experimental example 2

1 experimental material

1.1 experimental drugs and its pretreatment

The extraction preparation of patchouli oil and store method are the same.

1.2 control drug

Paclitaxel injection: specification 6mg/ml, Taiji Group Sichuan Taiji Pharmaceutical Co., Ltd.'s production.Authentication code: National drug standard H19994040, product batch number: 12060014.Paclitaxel injection is antineoplastic, be oophoroma and breast cancer and A line of NSCLC and two wires medication are also used for prostate cancer, the treatment of the tumours such as head and neck cancer, the cancer of the esophagus, seminoma.Face Bed human body list pharmaceutical quantities are 135-200mg/m², diluted with physiological saline or 5% glucose saline, intravenous infusion 3 hours.Drug is in 4 It DEG C saves backup, facing the used time with sterile 0.5% Tween-80 solution is diluted to required concentration.

1.3 cell strains and culture

PC3 cell (people's androgen dependent/non-dependent prostate gland cancer cell) is purchased from Shanghai Chinese Academy of Sciences cell bank.Cell culture is set In the F-12 culture solution containing 10% inactivated fetal bovine serum, 100U/ml penicillin and 100U/ml streptomysin, 37 DEG C of 5%CO₂And It is cultivated in saturated humidity incubator, every 48h is changed liquid 1 time, 0.25%EDTA- pancreatin had digestive transfer culture.Cell is grown in monolayer adherence, Select logarithmic growth phase cell experiment.

1.4 experimental animals and raising

Balb/c nude mouse: being purchased from Chinese Academy of Sciences Shanghai Experimental Animal Center, male, 4 week old, and totally 40, SPF grades of rings Border is raised, and mouse is raised in SPF grades of environment in this experimentation.The mouse adaptable fed newly bought enters experiment after 1 week.

1.5 reagents and consumptive material

F-12 culture medium: it is purchased from Gibco^TMCompany

Fetal calf serum (FBS): Lanzhou people marine growth Engineering Co., Ltd, lot number 20120510 are purchased from

Novocillin, streptomysin: it is purchased from North China drugmaker

0.25%EDTA- pancreatin: it is purchased from Kai Ji biotech firm

PBS phosphate buffer solution: it is purchased from Thermo Scientific company

Experimental water is produced with Milli-Q ultrapure water system (being purchased from U.S. Millipore company)

Tween-80: it is purchased from Chengdu Ke Long chemical reagent factory

Trypan blue: it is purchased from Sigma Co., USA

50ml, 15ml aseptic plastic centrifuge tube: steril cell culture bottle is purchased from U.S. Corning company

The disposable pipette tips of 200 μ l, 1ml: it is purchased from Jiangsu Haimen biology consumptive material company

75% cotton ball soaked in alcohol, cotton swab, 1ml asepsis injector (No. 7 syringe needles)

1.6 main solutions are prepared

F-12 culture solution: contain 10% inactivated fetal bovine serum in 1000ml F-12 culture solution, Benzylpenicillin sodium salt and streptomysin are each 100U/ml, 200mmol/L glutamine 10ml；

0.5% Tween-80 (for making up a prescription): being added 0.5ml Tween-80 in 95.5ml pure water, filtration sterilization is spare.

1.7 instrument

Superclean bench, SuZhou Antai Air Tech Co., Ltd., Su Jing group, model: SW-CJ-1F

Table-type low-speed centrifuge, Changsha Xiang Yi centrifuge Instrument Ltd., model: L-600

Water isolation type CO₂Constant incubator, Thermo scientific company, the U.S., model: 3111 types

OLYMPUS inverted microscope, Japanese Olympus company, model: CKX41

Micropipettor, French GILSON company

Electronic balance, Shanghai balance equipment factory, model: JA-2603

Blood cell counting plate, vernier caliper, ophthalmic tweezers, eye scissors

2 experimental methods

2.1 prostate gland cancer cell PC3 subcutaneous transplantation Balb/c nude mouses

2.1.1 cell suspension prepares

Well-grown is taken, the PC3 cell number bottle of adherent rate > 50% is in superclean bench, under aseptic condition, absorbs Old culture solution in culture bottle after PBS liquid cleans culture bottle 2 times, after 0.25%EDTA- pancreatin 1ml digestion 3-5min is added, is finished Full F-12 culture solution (containing 10% fetal calf serum) 3ml terminates digestion, and suction pipe gently blows and beats attached cell repeatedly, cell suspension is made It is collected into 15ml centrifuge tube, 1500rpm, centrifugation 5min, the F-12 culture solution suspension cell of serum-free, again 1500rpm, from Heart 5min.Cell is resuspended in the F-12 culture solution of serum-free again.Adjusting cell number is 8 × 10⁶A/ml, trypan blue count living thin Born of the same parents' quantity, is placed in centrifuge tube, send to SPF grades of toilets.

2.1.2 cell subcutaneous inoculation modeling

Animal is under non-narcotization, and the fixed mouse of assistant is sterilized small in prone position, operator with 75% cotton ball soaked in alcohol The left ear rear side of mouse and the nape of the neck skin, gently lift mouse skin of neck, will inhale the 1ml syringe needle for having cell suspension and are pierced into Subcutaneously, injection point is located at outside in the middle part of the forelimb armpit of left side, injects cell suspension 0.2ml, forms skin mound, extracts wine after injection needle Smart cotton balls sterilizes entry point, pays attention to that cell suspension is not allowed to leak out pin hole.

The grouping of 2.2 experimental animals and drug concentration setting

2.2.1 model is identified

The mouse of injection PC3 cell suspension continues raising in SPF environment, observes Subcutaneous Tumor Growth situation every 2d, to Gross tumor volume > 60mm³, mouse ordinary circumstance is good, and knurl judges that subcutaneous tumors are transplanted successfully, into next step without red and swollen ulceration Drug study.2.2.2 animal packet

It is chosen after experiment modeling Balb/c nude mouse totally 40,3 weeks and reaches drug study master pattern totally 30, weight is flat Equal 23.73 ± 1.03g, 67.09 ± 4.58mm of mean tumour volume³.It is randomly divided into 5 groups: the high, medium and low dosage of patchouli oil Group；Positive drug control group；Solvent negative control group.Every group mouse 6.

2.2.3 drug dose and administration route

This laboratory once carried out mouse peritoneal drug administration by injection acute toxicity testing to patchouli oil, as the result is shown patchouli oil LD is injected intraperitoneally₅₀For 4.441mg/10g^【88】.According to above-mentioned experimental studies results, patchouli oil high dose is set as by this experiment 80mg/kg (about LD₅₀1/5), middle dosage 40mg/kg (about LD₅₀1/10), low dosage 20mg/kg (about LD₅₀1/ 20)；Solvent negative control group uses 0.5% Tween-80 solution, and every injection volume is 0.1ml/10g, one time a day.Positive drug Dose of paclitaxel is 25mg/kg according to the common therapeutic dose conversion of clinic, and every injection volume is 0.1ml/10g, 1 times a week.It will Conventional 75% alcohol disinfecting in mouse web portion injection site, is administered using intraperitoneal injection.Continue the raising of SPF environment, daily timing Supplement feed, moisture, replacement padding, continuously treat 21d.

The observation of 2.3 experimental index and detection

2.3.1 animal observation and data collection

Every active state, diet situation, figure, survival state and the death condition of 2d observation mouse, weight is weighed, is surveyed It measures tumour length and width diameter (mm), calculate gross tumor volume (V) and records.

Gross tumor volume (tumor volume, TV) calculation formula are as follows: V (mm³)=long (mm) × wide²(mm²)×0.5。

Relative tumour volume (relative tumor volume, RTV), calculation formula are calculated according to the result of measurement Are as follows: RTV=V_t /V₀.Wherein V₀(d when being administered for grouping₀) measurement gross tumor volume.V_tTumour body when to measure each time Product.Thus Relative tumor proliferation rate T/C (%) is calculated: for the antitumor activity evaluation index of PC3 cell transplantation model.It calculates Formula is as follows:

T/C%=T_RTV/C_RTV× 100%.Wherein T_RTV: treatment group RTV；C_RTV: negative control group RTV.

2.3.2 stripping tumor collects tumor specimen

After administration 21 days, cervical vertebra Pulling escape, which is concentrated, puts to death each experimental mice, removes tumour in aseptic working platform, removes more Remaining adipose tissue, filter paper remove tumor surface blood, and last time surveys knurl footpath, claim knurl weight, take pictures.

2.4 statistical procedures

It is for statistical analysis using SPSS19.0 statistical software.Data withIt indicates, compares use between multisample mean One-Way ANOVA is examined, and variance is then examined using LSD together, and heterogeneity of variance then uses Tamhane ' s T2 to examine；Ranked data Comparison among groups are carried out using non-parametric test；It examines to think that its group difference has conspicuousness when P < 0.05.

3 experimental results

Ordinary circumstance after 3.1 nude mice tumor formations and administration

There is a degree of absorption in injection site to the nude mice of hypodermic injection injection in 1 week after inoculating cell suspension, and 1 week After there is subcutaneous tumor and be gradually increased, tumor mass average external volume > 60mm after 3 weeks³.Cauliflower-shaped is presented in dissection discovery tumor mass, Matter is soft, section such as flesh of fish shape.

Each group nude mice is tested during administration, does not occur death.Compared with negative solvent control group, patchouli oil experiment The activity of group nude mice, dietary amount have no significant change, and the activity of taxol group nude mice and dietary amount are declined.

3.2 nude mice weight situations of change

During administration, each dosage group nude mice weight of patchouli oil has different degrees of reduction, after administration 21 days, weight between group Difference is unobvious (P > 0.05).Taxol group nude mice weight loss is most obvious, the weight ratio between each dosage group of patchouli oil experiment There is statistical significance (P < 0.05) compared with difference.For above-mentioned each group compared with negative solvent control group, weight differences have statistics Meaning (P < 0.01).See Fig. 4；Table 7.

Table 7 tests each group nude mice body weights

Note: compared with negative control group, * P < 0.05；Compared with positive controls, ▲ P < 0.05；Comparison among groups, ● P > 0.05.

The situation of change of 3.3 nude mouse tumor volumes

During administration, each dosage group nude mice mean tumour volume of patchouli oil has a different degrees of increase, but with negative solvent Control group mean tumour volume increasing degree compares, and tumour growth has the trend delayed.And taxol group mean tumour volume It is on a declining curve.After administration 21 days, patchouli oil high dose group mean tumour volume is 128.39 ± 17.4mm³；Middle dose group Mean tumour volume is 196.07 ± 57.01mm³；Low dose group mean tumour volume is 233.08 ± 80.82mm³.In, low dose Gross tumor volume comparing difference is not statistically significant (P > 0.05) between amount group；High dose group and in, comparison of tumor body between low dose group Product moment is different to have statistical significance (P < 0.05).With 442.27 ± 80.04mm of negative solvent control group mean tumour volume³Than Compared with each dosage group gross tumor volume is less than normal, and difference has statistical significance (P < 0.05).Taxol positive controls are averagely swollen Knurl product is minimum, is 42.43 ± 14.85mm³, there is statistical significance (P < 0.01) with above-mentioned each group comparing difference.See figure 5；Table 8.

Table 8 tests each group nude mouse tumor volume situation

Note: compared with negative control group, * P < 0.05；Compared with positive controls, ▲ P < 0.05；Comparison among groups, ● P > 0.05.

Influence of 3.4 patchouli oils to nude mice PC3 tumor proliferation rate

Tumor bearing nude mice is continuously injected intraperitoneally 21 days in various dose patchouli oil, and experimental result is shown, low dose group tumour increases Value rate T/C (%) minimum 56.17%, up to 102.38%；Middle dose group tumour appreciation rate T/C (%) is minimum 45.06%, up to 84.13%；High dose group tumour appreciation rate T/C (%) minimum 29.32%, up to 85.71%.It is purple China fir alcohol positive controls tumour appreciation rate T/C (%) minimum 9.57%, up to 88.09%.The minimum tumour appreciation rate of each group T/C (%) be followed successively by from low to high taxol positive drug control group, high dose patchouli oil group, middle dosage patchouli oil group, Low dosage patchouli oil group, comparison among groups have statistical difference (P < 0.05).See Fig. 6；Table 9；Table 10.

Table 9 tests each group nude mice RTV mean value

Table 10 tests each group nude mouse tumor appreciation rate T/C (%；N=7)

Note: comparison among groups, * P < 0.05.

3.5 experiment each group tumor weights

After experiment, removing each group nude mouse tumor weighing.Patchouli oil high dose group average knurl weight 174.17 ± 24.6mg；291.00 ± 52.81mg of middle dose group；422.83 ± 77.89mg of low dose group；Positive drug control group 88.00 ± 16.84mg；Negative 974.00 ± 145.67mg of solvent control group.See Fig. 7.

4 brief summaries

After 4.1 experiments are using Balb/c nude mice by subcutaneous inoculation people's androgen dependent/non-dependent prostate cancer PC3, not with three kinds With dosage patchouli oil intraperitoneal injection, tumour situation of change is observed.After successive administration 21 days, three dosage groups of patchouli oil Nude mice weight has different degrees of decline, but mouse weight there are no significant difference (P > 0.05) between each dosage group, illustrates wide Palchouli oil intraperitoneal injection still has whole body toxic side effect to nude mice, but toxic effect is simultaneously under the concentration gradient of experimental design Without significant difference, this provides the foundation to evaluate the tumor proliferation inhibiting effect of patchouli oil under the conditions of identical toxicity.Experiment Middle positive control medicine taxol group nude mice weight is decreased significantly (P < compared with patchouli oil group and negative solvent control group 0.05) it is big compared with patchouli oil, to illustrate that taxol influences nude mice general toxicity.

4.2 experiment discoveries, high, medium and low dosage patchouli oil can delay PC3 tumour growth, after administration 21 days, Pogostemon cablin Oily high dose group mean tumour volume is 128.39 ± 17.4mm³；Middle dose group mean tumour volume be 196.07 ± 57.01mm³；Low dose group mean tumour volume is 233.08 ± 80.82mm³；Negative solvent control group mean tumour volume is 442.27±80.04mm³；Taxol positive controls mean tumour volume is 42.43 ± 14.85mm³.In patchouli oil, low dose Gross tumor volume comparing difference is not statistically significant (P > 0.05) between amount group, in prompt, low dosage patchouli oil may tumor killing effect Unanimously；High dose group inhibit tumor proliferation effect be better than in, low dose group (P < 0.05).It is average with taxol positive controls Gross tumor volume compares, and high dose group patchouli oil inhibits tumor proliferation effect closest, and tumor killing effect is best.

4.3 pairs of experimental results are further analyzed, and find patchouli oil low dose group tumour appreciation rate at the end of experimental administration T/C (%) is 56.17%；Middle dose group is 45.06%；High dose group is 29.32%.Taxol positive controls tumour increases Value rate T/C (%) is 9.57%.Prompt patchouli oil various dose that there is different degrees of tumor inhibitory effect, and action intensity In dose dependent (P < 0.05).Using≤40% tumor proliferation rate as standard, high dose group patchouli oil has certain tumor suppression Activity has anti-tumor drug potentiality to be exploited although being weaker than taxol tumor-inhibiting action.Last tumor quality result is equally demonstrate,proved This real conclusion.

The anti-tumor activity of comparative example Bai Qiuli alcohol

The discovery of inventor's early-stage study, Bai Qiuli alcohol have inhibition to make androgen-independent prostate cancer cell lines in vitro DU145 With (Cai Jian waits Chinese experimental pharmacology of traditional Chinese medical formulae magazine, the phase of volume 20140,20 10, page 165~169).In addition to containing white in patchouli oil Outside autumn Lee's alcohol, also contain other compounds, as Pogostone, thorn stamen oxalene, guaiene, patchoulene, cloves alkene, great Ye are fragrant Ketone, cedrene, Patchoulene, bulnesene, cedar wood alcohol, spoon leaf eucalyptus oil ketenes etc..The anticancer activity of patchouli oil and Bai Qiuli alcohol it Between have what kind of connection, yet there are no someone research.

Inventor is referring to the method for test example 1, and under equal conditions the anti-tumor activity of dialogue autumn Lee's alcohol is compared, knot Fruit is as follows:

1 Patchoulicalcohol inhibits PC3 cel l proliferation

Solvent control group cell form under light microscopic keeps normal, Patchoulicalcohol experimental group cell during the test There is different degrees of shrinkage, fall off, cell growth is suppressed.MTT testing result further confirms that Patchoulicalcohol is thin to PC3 Born of the same parents have growth inhibition effect, and when action intensity has m- dose dependent.Patchoulicalcohol to PC3 for 24 hours, 48h, 72h most Big inhibition concentration is respectively 650 μ g/ml, 700 μ g/ml and 600 μ g/ml, and inhibiting rate is respectively 80.05%, 79.73% and 87.71%.Be calculated Patchoulicalcohol to PC3 for 24 hours, 48h, 72h IC₅₀Respectively 167.64 μ g/ml, 101.37 μ g/ml, 60.37 μ g/ml, mean value are 109.79 μ g/ml, and group difference has statistical significance (P < 0.05).It the results are shown in Table 11.

11 Patchoulicalcohol of table inhibits test result (x ± s, n=3) to PC3 growth of tumour cell

Note: Patchoulicalcohol acts on PC3 cell for 24 hours, IC after 48h, 72h₅₀Comparison among groups have statistical difference, * P < 0.05.

2 Patchoulicalcohols inhibit DU145 cel l proliferation

Solvent control group cell form under light microscopic keeps normal, Patchoulicalcohol experimental group cell during the test There is different degrees of shrinkage, fall off, cell growth is suppressed.MTT testing result further confirms Patchoulicalcohol to DU145 Cell has growth inhibition effect, and when action intensity has m- dose dependent.Patchoulicalcohol to DU145 for 24 hours, 48h, 72h Maximum suppression concentration be respectively 650 μ g/ml, 650 μ g/ml and 500 μ g/ml, inhibiting rate is respectively 97.39%, 83.37% and 100%.Be calculated Patchoulicalcohol to DU145 for 24 hours, 48h, 72h IC₅₀Respectively 63.31 μ g/ml, 56.88 μ g/ml, 52.45 μ g/ml, mean value are 57.55 μ g/ml, and group difference has statistical significance (P < 0.05).It the results are shown in Table 12.

12 Patchoulicalcohol of table inhibits test result (x ± s, n=3) to DU145 growth of tumour cell

Note: Patchoulicalcohol acts on DU145 cell for 24 hours, IC after 48h, 72h₅₀Comparison among groups have statistical difference, * P < 0.05。

It can be seen that:

(1) patchouli oil to PC3 for 24 hours, the IC of 48h, 72h₅₀Respectively 89.68 μ g/ml, 79.89 μ g/ml, 66.74 μ g / ml, 78.77 μ g/ml of mean value.And Bai Qiuli alcohol to PC3 for 24 hours, 48h, 72h IC₅₀Respectively 167.64 μ g/ml, 101.37 μ g / ml, 60.37 μ g/ml, mean value are 109.79 μ g/ml.Comparison it can be found that patchouli oil to the IC of PC3₅₀Significantly lower than the white autumn Lee's alcohol illustrates that patchouli oil is significantly better than Bai Qiuli alcohol to the inhibitory activity of PC3 cell.

(2) patchouli oil to DU145 for 24 hours, the IC of 48h, 72h₅₀Respectively 109.64 μ g/ml, 93.70 μ g/ml, 86.88 μ g/ml, 96.74 μ g/ml of mean value.And Bai Qiuli alcohol to DU145 for 24 hours, 48h, 72h IC₅₀Respectively 63.31 μ g/ Ml, 56.88 μ g/ml, 52.45 μ g/ml, mean value are 57.55 μ g/ml.Although patchouli oil is to the IC of DU145₅₀Compared with Bai Qiuli alcohol Height, however, the Bai Qiuli alcohol amount about 26~40% contained in patchouli oil, you can get it for conversion, patchouli oil IC₅₀Mean value The Bai Qiuli alcohol contained in " 96.74 μ g/ml " is 25.15~38.70 μ g/ml, compared with Bai Qiuli alcohol is applied alone, to DU145 The amount of Bai Qiuli alcohol is substantially less than and Bai Qiuli alcohol is applied alone in patchouli oil when half-inhibitory concentration, concentration when being only applied alone 43.7%~67.2%.Other compositions i.e. in patchouli oil also have certain DU145 inhibiting effect, they and Bai Qiuli alcohol After being applied in combination, the inhibitory activity to DU145 can be improved.

Therefore, in summary two o'clock compares as it can be seen that patchouli oil is to androgen-independent prostate cancer cell lines in vitro (PC3) Inhibitory activity, which is substantially better than, is applied alone Bai Qiuli alcohol；Bai Qiuli determining alcohol is significantly lower than the feelings that Bai Qiuli alcohol is applied alone in patchouli oil Under condition, patchouli oil, which can reach, is applied alone Bai Qiuli alcohol to the inhibiting effect of prostate cancer brain metastes tumour (DU145), specific In use, i.e. patchouli oil can be used directly, without using the higher Bai Qiuli alcohol monomer of purity, hence it is evident that save medication at This.

To sum up, patchouli oil has antineoplastic action, to inhibiting prostate cancer growth effect clear, can treat hero and swashs Plain dependent/non-dependent prostate cancer and its each organ metastasis lesion, and activity is better than Bai Qiuli alcohol monomer compound, cost is less expensive, Market application prospect is good.

Claims (9)

1. purposes of the palchouli oil in the drug that preparation prevented, treated or assisted in the treatment of prostate cancer brain metastes tumour；Described Palchouli oil derives from Pogostemon cablinPogostemon cablin(Blanco) volatile oil that Benth. is extracted.

2. purposes according to claim 1, it is characterised in that: the prostate cancer is Androgen Independent Prostate Cancer Or castration-resistant prostate cancer.

3. purposes according to claim 1 or 2, it is characterised in that: the drug is the drug of cancer cell specific induction of apoptosis.

4. purposes according to claim 1, it is characterised in that: the palchouli oil is derived from Pogostemon cablinPogostemon cablin(Blanco) volatile oil of Benth., patchouli alcohol content are not less than 26%w/w.

5. purposes according to claim 4, it is characterised in that: the palchouli oil is derived from Pogostemon cablinPogostemon cablin(Blanco) volatile oil of Benth., patchouli alcohol content are 26 ~ 40%w/w.

6. purposes described in any one according to claim 1 ~ 5, it is characterised in that: the palchouli oil is as follows Preparation: Pogostemon cablin is takenPogostemon cablin(Blanco) herb of Benth, is ground into coarse powder, adds water, impregnates, using water Steam distillation method extract to get.

7. purposes according to claim 1, it is characterised in that: the drug is using palchouli oil as active constituent, in addition medicine The preparation that acceptable auxiliary material or complementary ingredient are prepared on product.

8. purposes according to claim 7, it is characterised in that: the preparation is liquid preparation, gaseous formulation, solid system Agent, semisolid preparation.

9. purposes according to claim 8, it is characterised in that: the content of palchouli oil is 0.1%~100% in the preparation (w/w).