WO2018133302A1 - 归芍茶的检测方法 - Google Patents

归芍茶的检测方法 Download PDF

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WO2018133302A1
WO2018133302A1 PCT/CN2017/088029 CN2017088029W WO2018133302A1 WO 2018133302 A1 WO2018133302 A1 WO 2018133302A1 CN 2017088029 W CN2017088029 W CN 2017088029W WO 2018133302 A1 WO2018133302 A1 WO 2018133302A1
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tea
detecting
gas
guilty
group
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刘玥
徐志坚
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刘玥
徐志坚
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation

Definitions

  • the invention relates to the field of tea, in particular to a method for detecting guinea tea.
  • tea is generally divided into “tea”, “Chen tea” and “old tea”, of which "tea” generally refers to new tea or storage time and oxidation level of no more than three years. Tea; “Chen tea” generally refers to tea with storage time and oxidation degree between three and twenty years; “old tea” refers to tea with storage time and oxidation degree of more than 20 years. There is no strict standard above. .
  • the invention unintentionally finds that some "old-aged tea leaves” are in the prevention and treatment of gastric cancer, and particularly have the remarkable effect of inhibiting the growth of gastric cancer tumor cells and prolonging the tumor-bearing life.
  • the present invention has carried out a large number of creative experimental screenings, and found that "old-aged tea leaves” having "baked fragrant scent” (also known as “mixed taste of Chinese pharmacy” by those skilled in the art) have the most remarkable effect on the prevention and treatment of gastric cancer.
  • the present invention analyzes the composition and efficacy of "old tea leaves” having "baked fragrant", and names and defines a new tea, Angelica and Radix Paeoniae Alba Tea, ART, and A method for detecting guilty tea is given.
  • a method for detecting guilty tea includes the following steps:
  • Step 1 pulverize the tea sample to be tested into a powder, place it in a detection container, and use a gas purge to capture the purge gas;
  • Step 2 The content of 4-terpineol, ⁇ -pinene, ⁇ -phellandene, ⁇ -myrcene and ⁇ -ocimene in the purge gas is detected by gas chromatography.
  • the guinea tea must contain volatile gas components such as 4-terpineol, ⁇ -pinene, ⁇ -phellandene, ⁇ -myrcene, and ⁇ -ocimene.
  • the method for detecting guinea tea further comprises the step of detecting the content of the following eighteen volatile gas components in the purge gas: 6-methyl-5-heptene-2- Ketone, ⁇ -limonene, ⁇ -terpinene, ⁇ -phellandene, Vitispirane, tridecane, 2,6,10-trimethyldodecane, 6,10-dimethyl-2-undecane Ketone, rohan cedar, hydroxy dihydroergoline, 2,6-di-tert-butyl-1,4-benzoquinone, ⁇ -decene, decyl cyclohexane, methyl-2-endo-acetylamino [2.2.1] Heptyl-2-exo-carboxylate, 2-methylpentadecane, 5,6-dihydro-5,6-dimethylbenzo[c]porphyrin, 3-methyl Hexadecane and nonadecane.
  • the guilty tea also needs to contain other volatile gas components which are newly emerging or significantly increased in content, such as the above-mentioned 6-methyl-5-heptene-2- Ketone, ⁇ -limonene, ⁇ -terpinene, ⁇ -phellandene, Vitispirane, tridecane, 2,6,10-trimethyldodecane, 6,10-dimethyl-2-undecane Ketone, rohan cedar, hydroxy dihydroergoline, 2,6-di-tert-butyl-1,4-benzoquinone, ⁇ -decene, decyl cyclohexane, methyl-2-endo-acetylamino [2.2.1] Heptyl-2-exo-carboxylate, 2-methylpentadecane, 5,6-dihydro-5,6-dimethylbenzo[c]porphyrin, 3-methyl At least nine of eight
  • the method for detecting glutinous tea further comprises detecting the following ten kinds of waves in the purge gas Step of content of hair gas component: linalool, ⁇ -terpineol, dihydro kiwi lactone, methyl salicylate, ⁇ -ionone, 2-methylpropionic acid-3-hydroxy-2,2 -Dimethyl-1-(2-hydroxy-1-methylethyl)pentyl ester, methyl-N-anthranilic acid methyl ester, ⁇ -junepine, 1,1-diphenyl-2 -Methyl propylene and 2,3-dihydro-1-methyl-3-phenyl-1H-indole.
  • the volatile gas component which is lost in the tea or which is significantly reduced in content is required to satisfy at least the above ten volatile gas components. At least five.
  • the method for detecting glutinous tea further comprises the following steps:
  • Preparing a test solution pulverizing the tea sample to be tested into a powder, adding to the container, adding the extract to oscillate and leaching, and then centrifuging, collecting the supernatant, and filtering the filtrate to obtain the test solution;
  • the content of caffeine and theophylline in the test solution was measured using high performance liquid chromatography.
  • the method for detecting glutinous tea further comprises the step of performing odor detection after brewing the tea sample to be tested.
  • the tea After the brewing, the tea has a strong mixed smell of angelica and white peony.
  • the powder has a particle size of no greater than 20 mesh; the extract contains 70% methanol, 29.7% water, and 0.3% acetic acid; and the filtration is filtered using a 0.45 ⁇ m filter.
  • the conditions for the high performance liquid chromatography detection are: a column of 250 mm ⁇ 4.6 mm, a C18 column of 5 ⁇ m, a mobile phase A: 0.2% acetic acid acetonitrile solution; a mobile phase B: 0.2% aqueous acetic acid;
  • the gradient elution method was performed at a flow rate of 1.0 mL/min, a column temperature of 35 ° C, and a detection wavelength of 270 mm.
  • the step 1 is specifically: pulverizing the tea sample to be pulverized into a powder of not more than 20 mesh, placing it in a closed container, and placing the sealed container in a constant temperature water bath, the glue of the closed container
  • the opening is provided with two openings, the purge nitrogen is introduced into the bottom of the bottle from one of the openings, the nitrogen flow rate is adjusted to 150 mL/min by the flow meter, and the other opening is used for the discharge of the purge gas, and the activated carbon tube is connected at the discharge port for sampling.
  • the tube is used for trapping the purge gas, and the dynamic headspace is separated.
  • the activated carbon in the sampling tube is taken out and then the carbon disulfide solution is added for analysis.
  • the conditions for GC/MS detection are: suctioning carbon disulfide analytical solution, manual injection; gas phase inlet mode is split mode, split ratio 5:1; inlet temperature 250 ° C; carrier gas He, loading The gas is in constant current mode, the column flow rate is 1.0mL/min, the average line speed is 37cm/sec; the analytical column is 30m ⁇ 250 ⁇ m ⁇ 0.25 ⁇ m capillary quartz column; the heating program is the initial temperature of 60°C, kept for 3min, at 5°C/ Min rose to 150 ° C, held for 10 min, then increased to 5 ° C / min to 250 ° C, held for 9 min, a total of 60 minutes; ion source temperature of 230 ° C, mass spectrometry transmission line temperature of 250 ° C, mass scan range of 30-550 amu.
  • the method for detecting guilt tea further comprises the step of performing the same detection as the tea sample to be tested using fresh tea of the same type as the tea sample to be tested.
  • the invention proposes and defines guinea tea by studying and analyzing the composition of the old tea leaves, and finds that it has the effect of inhibiting the growth of gastric cancer cells and prolonging the survival time of the tumor, and thus can be used for preparing the treatment.
  • gastric cancer drugs or foods it provides new methods and ideas for the treatment of gastric cancer.
  • the present invention also conducts an analysis of the intestinal flora of the present invention, and finds that it has a good effect of regulating the intestinal flora, and thus can be applied to the preparation of a drug or food for regulating the intestinal flora.
  • Figure 1 shows the results of GC/MS test showing that Guiqi Tea has significant differences. Among them, ART 1-4 is different from Guiqi Tea, and New Tea 1,2 is the same kind of new tea in the same year.
  • Figure 2 shows the tumor volume of different groups on days 1, 3, 5, and 7.
  • Figure 3 shows the effect of the subject.
  • Figure 4 shows that the inhibition of different doses of Guiqi Tea has an interaction with time (P ⁇ 0.05).
  • Figure 5 shows that the difference in tumor volume before and after administration of Guiqicha was statistically significant (P ⁇ 0.05).
  • Figure 7 is a diversity analysis in which 7a is the Chao analysis and 7b is the Shannon analysis.
  • Figure 9 is a species classification analysis, in which 9a is a cluster map, 9b is a heat map, and 9c is a classification and phylogenetic information visualization map drawn by GraPhlAn.
  • Figure 11 shows the PCoA analysis for the unweighted analysis and found that the normal and middle dose groups were most similar.
  • Figure 12 shows the difference in bacterial abundance between the groups (P ⁇ 0.05).
  • Figure 13 is a functional analysis of the flora based on COG and KEGG.
  • Figures 14a and 14b show a comparison of differences in functional groups between groups based on COG and KEGG, indicating significant changes in intestinal flora metabolism (P ⁇ 0.05), with 14a being a COG-based result and 14b being a KEGG-based result.
  • BC is the normal group (ie, the blank control group)
  • NC is the tumor group (ie, the negative control group)
  • M is the middle dose group
  • L is the low dose group
  • H is the high dose group.
  • the guilty tea refers to the detection of volatile gas components by gas chromatography or the like using the same type of new tea as a control, wherein newly emerging or significantly increased components must include 4-terpineol, ⁇ -pinene, --phellandene, ⁇ -myrcene and ⁇ - ⁇ Leyne.
  • newly emerging or significantly increased components must include 4-terpineol, ⁇ -pinene, --phellandene, ⁇ -myrcene and ⁇ - ⁇ Leyne.
  • the content of caffeine and theophylline in the guilty tea was significantly reduced.
  • the scented tea and the tea broth after brewing have a strong mixed taste of angelica and white peony.
  • Guiqi tea also includes some other newly emerged or significantly increased ingredients, due to the variety of tea trees (large, medium and small leaf species), origin (Jiangsu, Zhejiang, Yungui, Huguang), production process ( The degree of oxidation varies, the storage environment and time, and the degree of oxidation vary.
  • the following ingredients are the components that significantly change the main contents of the tea after combining different varieties, origins, production processes, etc., and need to satisfy at least nine of the following eighteen components: 6-methyl-5-heptene- 2-keto, ⁇ -limonene, ⁇ -terpinene, ⁇ -phellandene, Vitispirane (a sesquiterpenoid), tridecane, 2,6,10-trimethyldodecane, 6, 10-dimethyl-2-undecyl ketone, rhodamine, hydroxy dihydroergoline, 2,6-di-tert-butyl-1,4-benzoquinone, ⁇ -decene, fluorenylcyclohexane Alkane, methyl-2-endo-acetylamino[2.2.1]heptane-2-exocarboxylate (Methyl 2-endo-Acetamidobicyclo[2.2.1]heptane-2-exo-carboxylate
  • the following ingredients are the ingredients that significantly change the main inclusions of tea after combining different varieties, origins, production processes, etc., and need to meet at least five of the following ten ingredients: linalool, ⁇ -terpineol, two Hydrogen kiwi lactone, methyl salicylate, ⁇ -ionone, 3-hydroxy-2,2-dimethyl-1-(2-hydroxy-1-methylethyl)penta-2-methylpropanoate Base ester, methyl-N-anthranilic acid methyl ester, ⁇ -jussonene, 1,1-diphenyl-2-methylpropene and 2,3-dihydro-1-methyl-3-benzene Base-1H- ⁇ .
  • the significant increase and significant reduction mentioned above means that the P value in the statistical indicator is less than 0.05.
  • the same type of new tea is the above-mentioned tea with a storage time and an oxidation degree of not more than 3 years.
  • the present invention has found that the above-mentioned guilty tea can be used in the preparation of a medicament or a food for treating gastric cancer and/or regulating intestinal flora, and the effect is remarkable.
  • a medicament or food for treating gastric cancer and/or regulating intestinal flora which contains the guinea tea.
  • the food products include, but are not limited to, solid foods, semi-solid foods, liquid beverages, and the like.
  • Such drugs include, but are not limited to, dry creams, powders, tablets, liquids, and the like.
  • the guilty tea is added in the form of an extract thereof
  • the extraction method of the extract comprises the steps of: crushing the guilty tea into boiling water, keeping it slightly boiled, and after filtering, obtaining The extract and the tea residue are respectively added to boiling water, kept slightly boiled, separately filtered, and the extract is repeatedly extracted according to the above steps, and the extract is combined and concentrated under reduced pressure with a vacuum concentrator.
  • the extract is a dry paste.
  • the invention proposes and defines guinea tea by studying and analyzing the composition of the old tea leaves, and finds that it has the effect of inhibiting the growth of gastric cancer cells and prolonging the survival time of the tumor, and thus can be used for preparing the treatment.
  • gastric cancer drugs or foods it provides new methods and ideas for the treatment of gastric cancer.
  • the present invention also conducts an analysis of the intestinal flora of the present invention, and finds that it has a good effect of regulating the intestinal flora, and thus can be applied to the preparation of a drug or food for regulating the intestinal flora.
  • the Chinese tea company Guangxi Branch “China Tea Brand” has been used for more than 20 years in Guangxi Tea Brick, Yunnan Ouhai Chunfu Run Tea Company “Spring Furun Brand” for more than 30 years, Yunnan Tea Brick, Guangdong Shengfan Trade
  • the tea sample was pulverized into 20 mesh, and 50 g of tea powder was placed in a self-made experimental device (one 200 mL sealed glass bottle with a rubber stopper was taken, two ports were opened on the rubber stopper, and a purge nitrogen gas was introduced from one of the bottles to the bottom of the bottle, and the other was used.
  • the activated carbon tube sampling tube is connected to the discharge port for trapping the purge gas
  • the sealed glass bottle is fixed in a constant temperature water bath of 80 ° C, purged with nitrogen, and adjusted by a flow meter.
  • the nitrogen flow rate was 150 mL/min, and the outlet end was connected to an activated carbon sampling tube, and the dynamic headspace was separated for 2 hours. After the activated carbon in the sampling tube was taken out, 0.5 mL of a carbon disulfide solution was added for analysis.
  • 1.2 GC/MS conditions 1.0 ⁇ L of carbon disulfide solution, manual injection; GC inlet mode is split mode, split ratio 5:1; inlet temperature 250°C; carrier gas is He, carrier gas is constant current mode
  • the column flow rate is 1.0 mL/min, the average line speed is 37 cm/sec;
  • the analytical column is Thermo TG-5MS capillary quartz column (30 m ⁇ 250 ⁇ m ⁇ 0.25 ⁇ m); the heating program is the initial temperature of 60 ° C, kept for 3 min, at 5 ° C /min was raised to 150 ° C for 10 min and then raised to 250 ° C at 5 ° C / min for 9 min for a total of 60 minutes.
  • the ion source temperature was 230 ° C
  • the mass spectrometry transmission line temperature was 250 ° C
  • the mass scanning range was 30-550 amu.
  • the tea sample was pulverized into 20 mesh, 10 g of tea powder was placed in a 100 mL Erlenmeyer flask, 50 mL of extract (70% methanol, 29.7% water and 0.3% acetic acid) was added, and the mixture was vortexed at 70 cc for 30 min, and cooled to room temperature. After centrifugation at 3500 r/min for 10 min at room temperature, the supernatant was decanted, filtered through a 0.45 ⁇ m filter, and the obtained filtrate was used as a test solution. Weigh accurately the appropriate amount of each reference substance, placed in a 10mL volumetric flask, add the extract to dissolve, and prepare a 0.5mg/mL reference stock solution.
  • the column is a C18 column (250 mm ⁇ 4.6 mm, 5 ⁇ m, such as Agilent's C18 column, etc.), mobile phase A: 0.2% acetic acid acetonitrile solution; mobile phase B: 0.2% aqueous acetic acid solution.
  • the flow rate was 1.0 mL/min
  • the column temperature was 35 ° C
  • the detection wavelength was 270 mm.
  • the tea cake of 700g of glutinous tea was crushed, added to 8 times of boiling water, kept slightly boiled for 20 min, and filtered.
  • the first extract was extracted once with 5 times the amount of boiling water.
  • the tea residue was soaked in 5 times boiling water for 20 min and filtered.
  • the third extract was soaked in boiling water.
  • the extracts were combined and concentrated under reduced pressure at 58 ° C using a reduced pressure concentrator to obtain a dry paste.
  • IVC squirrel cages were used in independent aerated animal cages, fed in single cages, 3-4 nude mice per cage. Before use Autoclave sterilization, once a week, or as needed to ensure that the environment is clean and dry as standard; the padding uses a milled corn pole and is steam sterilized.
  • Feeding environment SPF level; indoor temperature 26-28 ° C, indoor humidity 40%-60%; ventilation and ventilation 10-15 times per hour; daily maintenance of 10 hours of light, 14 hours of matt light and dark cycle, use Artificial lighting.
  • Feed and drinking water are used after autoclaving; the water bottle is changed every other day, and the water bottle and drinking water pipe must be cleaned between use.
  • BGC-823 human gastric cancer cell line was cultured in vitro, and the medium was high glucose DMEM of 10% fetal bovine serum.
  • the equivalent dose of Guiqi tea low dose 0.1g / only, medium dose 0.5g / only, high dose 1.0g / only.
  • Dry paste usage low dose 10mg / only / day, medium dose 50mg / only / day, high dose 100mg / only / day.
  • the dry paste was dissolved in deionized water and configured to use a concentration: low dose 0.025 g/ml, medium dose 0.125 g/ml, high dose: 0.250 g/ml.
  • the solution was sterilized at 121 ° C for 30 min.
  • BGC-823 human gastric cancer cells were trypsinized and arranged into a single cell suspension of 3 ml with high glucose DMEM, and the total amount of cells was about 2 ⁇ 10 8 .
  • ear tags were used to place ear tags on the right ear of the nude mice near the scalp.
  • BGC-823 cell suspension 0.1 ml/only was injected into the right lower extremity of 27 nude mice near the right lower extremity, and the remaining 4 were Nude mice were injected with the same amount of normal saline at the same site.
  • the condition of the nude mice was observed daily, weighed, and the length and diameter of the tumor were measured with an electronic vernier caliper, and the data were recorded.
  • the diameter/volume of the tumor was the first factor, and the body weight of the nude mice was divided into two groups: 7 in the high-dose dry paste group and 7 in the dry-dose medium-dose group. There were 7 low-dose groups, 6 negative control groups (tumor-bearing saline group), and 4 blank control groups. The average diameter of each group of tumors was close, and each group had large and small tumors.
  • a 1 ml syringe was used to absorb 0.4 ml of the dry cream solution, and the needle was placed on the No. 9 gavage needle.
  • the nude mice of each group were intragastrically administered.
  • the negative control group was given deionized water, and the blank control group was not gavage.
  • the stomach was administered once every 24 hours, and the stomach was continuously administered until the nude mice died naturally.
  • the body weight of the nude mice and the length and length of the tumor were recorded daily, and the mental state and activity of each group of nude mice were observed.
  • the feces of the nude mice were collected daily with an EP tube, marked, and stored in a refrigerator at -80 °C.
  • the feces of the gut microbiota were sequenced on the day before the death of all nude mice, and the amplified region was v3-v4.
  • the experimental results were analyzed by SPSS13.0 or 19.0, and the survival time was analyzed by rank sum test. Analysis of variance was performed to analyze the difference in tumor volume before and after gastric administration, and the relationship between time and intragastric dose was analyzed by repeated measurements.
  • the main components of gas chromatography-mass spectrometry detection and significant increase mainly include: 4-terpineol, 6-methyl-5-hepten-2-one, ⁇ -limonene, ⁇ -terpinene, ⁇ -myrcene, --ocimene, ⁇ -pinene, ⁇ -phellandene, ⁇ -phellandene, Vitispirane, tridecane, 2,6,10-trimethyldodecane, 6,10-dimethyl-2 -undecyl ketone, rhodamine, hydroxy dihydroergoline, 2,6-di-tert-butyl-1,4-benzoquinone, ⁇ -decylene, decylcyclohexane, Methyl2-endo-Acetamidobicyclo [2.2.1]heptane-2-exo-carboxylate, 2-methylpentadecane, 5,6-dihydro-5,6-
  • the taxonomic comparison of the samples at each classification level can be known, including the species and relative abundance of the microorganisms contained in the sample. It can be seen from Fig. 8 and Fig. 9a that the microbial species and abundance of the normal group and the middle dose group are most similar, followed by the low dose group, and the negative control group and other groups are different, indicating that the normal group is in the intestinal tract after the tumor implantation. The microbial species and abundance of the flora changed significantly. After the gavage of the tea (dry paste), the microbial species and abundance recovered, and the middle dose group basically returned to normal. The classification and phylogenetic information visualization by heat map 9b, GraPhlAn can be seen in Figure 9c.
  • the top 100 species of abundance are: BACTEROIDETES, FIRMICUTES, PROTEOBACTERIA and VERRUCOMICROBIA.
  • the most abundant bacteria include: Bacteroides, Falsiporphyromonas, Coprobacter, Prevotella, Clostridium XlVa, Alistipes, and the like.
  • BC was the normal group (ie, the blank control group)
  • NC was the tumor group (ie, the negative control group)
  • M was the middle dose group
  • L was the low dose group.
  • BC was the normal group (ie, the blank control group)
  • NC was the tumor group (ie, the negative control group)
  • M was the middle dose group
  • L was the low dose group.
  • the normal group and the middle dose group are most similar.
  • further PCoA analysis also showed that the normal and middle dose groups were most similar.
  • the red color was P ⁇ 0.05, and the bacterial population was statistically significant.
  • the genus of genus that is significantly different between BC and NC is Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus.
  • the significant difference between NC and M is Comamonas.
  • Significant differences between NC and L are Prevotella, Pseudoflavonifractor, Flavonifractor, Butyrivibrio, and Methanothrix.
  • BC and M are Neisseria, Chelativorans, Cellulomonas, Dyella, Gp7.
  • Significant differences between BC and L are Pseudomonas, Nitrososphaera, Gp7, Cellulomonas, Neisseria, Methanospirillum, Terrimonas, Pseudoxanthomonas, Dyella, Pseudolabrys.
  • L and M We believe these bacteria are cancer suppressor bacteria.
  • the abundance of the Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus populations was significantly down-regulated (P ⁇ 0.05).
  • the flora of the M group and the NC significantly changed compared with the NC group. Changes in statistical differences, but slightly reduced.
  • the reason for tumor growth in the NC group was that the abundance of Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus was significantly down-regulated and Comamonas was basically unchanged.
  • Comamonas (P ⁇ 0.05) was significantly increased in the M group, and the abundance of the Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus in the BC group was significantly higher than that in the NC group. Both are up, although not statistically significant. It is speculated that M group inhibited tumor growth by significantly up-regulating the abundance of the Comamonas and Responsive BC groups significantly higher than the NC group Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus.
  • the Comamonas remained unchanged and did not significantly up-regulate like the M group, so the reason for the rapid growth of the L group tumors was that the Comamonas abundance was unchanged and the BC group was significantly higher than the NC group Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, The abundance of Methanomassiliicoccus and Haemophilus is lowered.
  • the group L was significantly up-regulated in the L group compared with the NC group Prevotella, Pseudoflavonifractor, Flavonifractor, Butyrivibrio, Methanothrix, but did not return to the BC level, and these groups did not have statistically significant changes in the M group, so it is speculated that these groups can be extended Tumor survival time.
  • the significant down-regulation of Nitrososphaera, Pseudomonas, Gp7, Terrimonas, Cellulomonas, Methanomassicicoccus, and Haemophilus can promote the occurrence and growth of tumors, which is equivalent to the genus of tumor suppressor.
  • Significant up-regulation of Comamonas can inhibit tumor growth and is also equivalent to the genus Tumor.
  • Significantly upregulated Prevotella, Pseudoflavonifractor, Flavonifractor, Butyrivibrio, and Methanothrix can prolong the survival time of tumors, which is equivalent to the genus Tumor.
  • the flora function is mainly concentrated in Carbohydrate transport and metabolism carbohydrate transport and metabolism, Amino acid transport and metabolism amino acid transport and metabolism, Membrane transport membrane transport, Replication, recombination and repair replication, recombination and repair, Transcription transcription, Translation, ribosomal Structure and biogenesis translation, ribosome structure and biotransformation, Cell wall/membrane/envelope biogenesis, cell wall/membrane/envelope biogenesis, general function prediction only, general function prediction, function unknown unknown function.
  • the high expression of the NC group is Carbohydrate Metabolism, Transcription transcription, and Membrane Transport membrane transport, indicating that the bacteria can promote tumorigenesis and growth by increasing carbohydrate metabolism, transcription and membrane transport.
  • L group The functions of high expression of L group are Coenzyme transport and metabolism, post-translational modification, environmental adaptation, Metabolism of Cofactors and Vitamins, and Folding, Sorting and Degradation. And degradation, metabolism of Terpenoids and Polyketides, metabolism of Glycan Biosynthesis and Metabolism, and biosynthesis and metabolism, indicating that the flora increases coenzyme transport and metabolism, post-translational modification, environmental adaptation, coenzymes and vitamins Metabolism, folding, sorting and degradation, metabolism of polyketides and terpenoids, polysaccharide biosynthesis and metabolism, to prolong the survival time of tumors.
  • German scientist Otto Warburg discovered in the 1920s that cancer cells absorb glucose more efficiently than other cells to promote their rapid growth.
  • these glucoses are mainly in the cell through the glycolysis pathway.
  • Normal cells only undergo glycolysis in the absence of oxygen, and tumor cells preferentially carry out glycolysis even in the absence of oxygen, consuming more glucose and producing more lactic acid.
  • the study found that the Warburg effect is related to the vigorous growth of tumor cells. Glycolysis not only provides energy for the proliferation of tumor cells, but also provides raw materials for the synthesis of fatty acids and nucleic acids. The results of our bacterial function differences are in full compliance with the characteristics of tumor metabolism.

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Abstract

提供了一种归芍茶的检测方法。通过对"经久老茶叶"进行成分及药效研究分析，命名并定义了一种新的茶即归芍茶，归芍茶的主要挥发性气体成分跟当归和白芍的主要挥发性气体成分相同较多，主要挥发性气体成分跟当归相同的有β-蒎烯、β-水芹烯、β-月桂烯、β-罗勒烯，跟白芍相同的有β-蒎烯、β-月桂烯、β-罗勒烯，因此根据其气味近似程度，把具有"焙药香"的"经久老茶叶"命名为归芍茶。通过对归芍茶的药效研究发现，其具有抑制胃癌细胞生长和延长带瘤生存期的效果，因而可用于制备治疗胃癌药物中，从而为胃癌的治疗提供了新的方法和思路。

Description

归芍茶的检测方法 技术领域
本发明涉及茶叶领域，尤其是涉及一种归芍茶的检测方法。
背景技术
中国是世界的产茶大国，各种茶的种类繁多。中国人饮茶的历史悠久。茶具有多种保健功效。根据茶的储存时间和氧化程度的不同，以前一般将茶分为“茶叶”、“陈茶”以及“经久老茶叶”，其中“茶叶”一般指新茶或储存时间和氧化程度不超过三年的茶；“陈茶”一般指储存时间和氧化程度在三年到二十年之间的茶；“经久老茶叶”一般指储存时间和氧化程度在二十年以上的茶，以上没有严格的标准。1973年陈椽提出按制茶时茶多酚氧化程度分为白、绿、黄、青、红、黑茶六种。“经久老茶叶”在长时间存放中发生缓慢的氧化反应所产生的物质，可能具有多种功效，目前对“经久老茶叶”的成分及其药效研究，尚属空白。
发明内容
本发明是在无意中发现部分“经久老茶叶”在胃癌的防治，特别是对抑制胃癌肿瘤细胞的生长以及带瘤生存期的延长具有显著的效果。对此，本发明进行了大量的创造性实验筛选，发现具有“焙药香”(本领域技术人员又称之为“中药房混合味”)的“经久老茶叶”对胃癌的防治效果最为显著。进一步，本发明对具有“焙药香”的“经久老茶叶”进行成分及药效研究分析，命名并定义了一种新的茶，即归芍茶(Angelica and Radix PaeoniaeAlba Tea，ART)，并给出了一种归芍茶的检测方法。
一种归芍茶的检测方法，包括如下步骤：
步骤一：将待测茶样粉碎成粉料，置于检测容器中，使用气体吹扫，捕集吹扫气体；
步骤二：使用气质联用检测所述吹扫气体中的4-松油醇、β-蒎烯、β-水芹烯、β-月桂烯以及β-罗勒烯的含量。
归芍茶中必须同时含有4-松油醇、β-蒎烯、β-水芹烯、β-月桂烯以及β-罗勒烯等挥发性气体成分。
在其中一个实施例中，所述归芍茶的检测方法还包括检测所述吹扫气体中的如下十八种挥发性气体成分的含量的步骤：6-甲基-5-庚烯-2-酮、α-柠檬烯、α-松油烯、α-水芹烯、Vitispirane、十三烷、2，6，10-三甲基十二烷、6，10-二甲基-2-十一烷酮、罗汉柏烯、羟基二氢鸡蛋果素、2，6-二叔丁基-1，4-苯醌、β-桉叶烯、壬基环己烷、甲基-2-内-乙酰氨基[2.2.1]庚烷基-2-外-羧酸酯、2-甲基十五烷、5，6-二氢-5，6-二甲基苯并[c]噌啉、3-甲基十六烷以及十九烷。
进一步，除上述必须含有的挥发性气体成分外，优选的，归芍茶还需要含有其他新出现或含量显著增加的其他挥发性气体成分，如上述6-甲基-5-庚烯-2-酮、α-柠檬烯、α-松油烯、α-水芹烯、Vitispirane、十三烷、2，6，10-三甲基十二烷、6，10-二甲基-2-十一烷酮、罗汉柏烯、羟基二氢鸡蛋果素、2，6-二叔丁基-1，4-苯醌、β-桉叶烯、壬基环己烷、甲基-2-内-乙酰氨基[2.2.1]庚烷基-2-外-羧酸酯、2-甲基十五烷、5，6-二氢-5，6-二甲基苯并[c]噌啉、3-甲基十六烷以及十九烷等十八种挥发性气体成分中的至少九种。
在其中一个实施例中，所述归芍茶的检测方法还包括检测所述吹扫气体中的如下十种挥 发性气体成分的含量的步骤：芳樟醇、α-松油醇、二氢猕猴桃内酯、水杨酸甲酯、β-紫罗兰酮、2-甲基丙酸-3-羟基-2，2-二甲基-1-(2-羟基-1-甲基乙基)戊基酯、甲基-N-邻氨基苯甲酸甲酯、δ-杜松烯、1，1-二苯基-2-甲基丙烯及2，3-二氢-1-甲基-3-苯基-1H-茚。
更进一步，除上述必须含有以及新出现或含量显著增加的挥发性气体成分外，优选的，归芍茶中消失的或含量显著降低的挥发性气体成分需要至少满足上述十种挥发性气体成分中的至少五种。
在其中一个实施例中，所述归芍茶的检测方法还包括如下步骤：
制备供试品溶液：将待测茶样粉碎成粉料，加入容器中，加入提取液进行震荡浸提，后离心，收集上清液，过滤后得到的滤液作为所述供试品溶液；
使用高效液相色谱检测所述供试品溶液中咖啡因和茶碱的含量。
归芍茶中的咖啡因和茶碱的含量需要显著降低。
上述显著增加和显著降低指相对于同类新茶，满足统计学指标P＜0.05。
在其中一个实施例中，所述归芍茶的检测方法还包括对所述待测茶样冲泡后进行气味检测的步骤。
归芍茶在冲泡后，具有浓烈的当归和白芍为主的混合气味。
在其中一个实施例中，所述粉料的粒径不大于20目；所述提取液含有70％甲醇、29.7％水和0.3％乙酸；所述过滤是使用0.45μm的滤膜过滤。
在其中一个实施例中，所述高效液相色谱检测的条件：色谱柱为250mm×4.6mm、5μm的C18柱，流动相A：0.2％乙酸乙腈溶液；流动相B：0.2％乙酸水溶液；采用梯度洗脱的方法，流速为1.0mL/min，柱温35℃，检测波长为270mm。
在其中一个实施例中，所述步骤一具体是将待测茶样粉碎成不大于20目的粉料，置于密闭容器中，并将密闭容器置于恒温水浴锅中，所述密闭容器的胶塞开设有两个开口，将吹扫氮气从其中一开口引入瓶底，用流量计调节氮气流量为150mL/min，另一开口用于吹扫气体的排出，在该排出口连接活性碳管采样管，用于捕集吹扫气体，动态顶空分离，将采样管中活性炭取出后加入二硫化碳溶液进行解析。
在其中一个实施例中，气质联用检测的条件：吸取二硫化碳解析液，手动进样；气相进样口模式为分流模式，分流比5：1；进样口温度250℃；载气He，载气为恒流模式，柱流速1.0mL/min，平均线速37cm/sec；分析柱为30m×250μm×0.25μm的毛细石英管柱；升温程序为初始温度60℃，保持3min，以5℃/min升至150℃，保持10min，再以5℃/min升至250℃，保持9min，共计60分钟；离子源温度230℃，质谱传输线温度250℃，质量扫描范围30-550amu。
在其中一个实施例中，所述归芍茶的检测方法还包括使用与待测茶样同类的新茶进行与所述待测茶样同样检测的步骤。
本发明通过对经久老茶叶的成分研究分析，提出并定义了归芍茶，并通过对其的药效研究发现，其具有抑制胃癌细胞生长和延长带瘤生存期的效果，因而可用于制备治疗胃癌药物或食品中，从而为胃癌的治疗提供了新的方法和思路。此外，本发明还对其进行肠道菌群的分析研究，发现其具有良好的调节肠道菌群的功效，因而可以应用于制备调节肠道菌群的药物或食品中。
附图说明
图1为气质联用检测结果显示归芍茶具有显著差异成分，其中，ART 1-4是不同的归芍茶，新茶1，2是当年同类新茶。
图2为第1、3、5、7天不同分组的肿瘤体积情况。
图3为受试者的影响情况，图中的time*group分组中看第一行(p＝0.007)，表示有统计学意义，说明剂量的效应随着时间的改变而改变，即剂量和时间有交互作用。
图4为归芍茶不同剂量的抑制作用与时间有交互作用(P＜0.05)，中剂量组可抑制肿瘤生长，第7天抑制效果最显著(H，M，L的n＝7，NC的n＝6，BC的n＝4)。
图5为归芍茶灌胃前后肿瘤体积差异有统计学意义(P＜0.05)，中剂量组肿瘤体积差异最小(H，M，L的n＝7，NC的n＝6，BC的n＝4)，说明中剂量对肿瘤生长抑制效果最显著，低剂量体积差最大，说明低剂量促进肿瘤体积增大，高剂量体积差为0，因为高剂量1天后即死亡。
图6为各组生存时间有显著差异，低剂量组显著延长生存时间(P＜0.05)(H，M，L的n＝7，NC的n＝6，BC的n＝4)。
图7为多样性分析，其中，7a为Chao分析，7b为Shannon分析。
图8为采用热和聚类图进行物种分类分析，结果表明归芍茶可逆转癌症引起的肠道菌群失调，A为门，B为属，(M，L和BC的n＝2，NC的n＝4)。
图9为物种分类分析，其中9a为聚类图，9b为热图，9c为GraPhlAn绘制的分类和系统发育信息可视化图。
图10为PCA分析表明归芍茶可逆转癌症引起的肠道菌群失调(M，L和BC的n＝2，NC的n＝4)，发现正常组和中剂量组最相似。
图11为PCoA分析，为unweighted分析，发现正常组和中剂量组最相似。
图12为菌群丰度差异分析表明各组间细菌存在显著差异(P＜0.05)。
图13为基于COG和KEGG进行菌群的功能分析。
图14a和14b为基于COG和KEGG进行的分组间菌群功能差异比较，表明肠道菌群代谢显著改变(P＜0.05)，其中14a为基于COG的结果，14b为基于KEGG的结果。
上述图中其中BC是正常组(即空白对照组)，NC是肿瘤组(即阴性对照组)，M是中剂量组，L是低剂量组，H是高剂量组。
具体实施方式
为了便于理解本发明，下面将参照相关附图及具体实施例对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明所述的归芍茶，是指以同类新茶作为对照，通过气质联用等检测挥发性气体成分，其中新出现的或显著增加的成分一定包括4-松油醇、β-蒎烯、β-水芹烯、β-月桂烯以及β-罗 勒烯。同时，所述归芍茶中咖啡因以及茶碱的含量都显著减少。而且，所述归芍茶在冲泡后香气和茶汤具有浓烈的当归和白芍为主的混合味道。
进一步，以同类新茶作为对照，归芍茶还包括一些其他的新出现的或显著增加的成分，由于茶树的品种(大、中、小叶种)、产地(江浙、云贵、湖广)、制作工艺(氧化程度不同)、储存环境和时间以及氧化程度的不同，茶叶的内含成分特点有所不同。下列成分是综合了不同的品种、产地、制作工艺等因素后，茶叶主要内含物显著改变的成分，需要满足如下十八种成分中的至少九种：6-甲基-5-庚烯-2-酮、α-柠檬烯、α-松油烯、α-水芹烯、Vitispirane(一种倍半萜类物质)、十三烷、2，6，10-三甲基十二烷、6，10-二甲基-2-十一烷酮、罗汉柏烯、羟基二氢鸡蛋果素、2，6-二叔丁基-1，4-苯醌、β-桉叶烯、壬基环己烷、甲基-2-内-乙酰氨基[2.2.1]庚烷基-2-外羧酸酯(Methyl 2-endo-Acetamidobicyclo[2.2.1]heptane-2-exo-carboxylate)、2-甲基十五烷、5，6-二氢-5，6-二甲基苯并[c]噌啉、3-甲基十六烷以及十九烷；
更进一步，以同类新茶作为对照，归芍茶中有部分挥发性气体成分消失或显著降低，由于茶叶的品种(大、中、小叶种)、产地(江浙、云贵、湖广)、制作工艺(发酵程度不同)、储存环境和时间以及氧化程度的不同，茶叶的成分特点有所不同。下列成分是综合了不同的品种、产地、制作工艺等因素后，茶叶主要内含物显著改变的成分，需要满足以下十种成分中的至少五种：芳樟醇、α-松油醇、二氢猕猴桃内酯、水杨酸甲酯、β-紫罗兰酮、2-甲基丙酸-3-羟基-2，2-二甲基-1-(2-羟基-1-甲基乙基)戊基酯、甲基-N-邻氨基苯甲酸甲酯、δ-杜松烯、1，1-二苯基-2-甲基丙烯及2，3-二氢-1-甲基-3-苯基-1H-茚。
上面提到的显著增加及显著减少指满足统计学指标中的P值小于0.05。
所述同类新茶即上述存放时间和氧化程度不超过3年的茶叶。
本发明经过研究发现，上述归芍茶可以应用在制备治疗胃癌和/或调节肠道菌群的药物或食品中，效果显著。如一种治疗胃癌和/或调节肠道菌群的药物或食品，其含有所述归芍茶。所述食品包括但不限于固体食品、半固体食品及液体饮料等。所述药物包括但不限于干膏、粉剂、片剂、水剂等。
优选的，所述归芍茶是以其提取物的形式添加，所述提取物的提取方法包括如下步骤：将所述归芍茶敲碎后加入沸水中，保持微沸，过滤后，将得到的提取液及茶渣分别加入沸水中，保持微沸，分别过滤，按照以上步骤重复提取多次后合并提取液，用减压浓缩器进行减压浓缩即得。
进一步优选的，所述提取物是干膏。
本发明通过对经久老茶叶的成分研究分析，提出并定义了归芍茶，并通过对其的药效研究发现，其具有抑制胃癌细胞生长和延长带瘤生存期的效果，因而可用于制备治疗胃癌药物或食品中，从而为胃癌的治疗提供了新的方法和思路。此外，本发明还对其进行肠道菌群的分析研究，发现其具有良好的调节肠道菌群的功效，因而可以应用于制备调节肠道菌群的药物或食品中。
以下为具体实施例部分。
1.气质联用(即气相色谱与质谱联用)和高效液相色谱检测归芍茶
根据“焙药香”的标准筛选中国茶叶公司广西分公司“中茶牌”20年以上广西茶砖、云南勐海春福润茶叶公司“春福润牌”30年以上云南茶砖、广东圣凡贸易公司“中茶牌”30年以上云南茶砖(中国茶叶公司云南分公司)、广西悟州钎源绿叶茶叶公司“农家牌”30年以上 广西茶砖等4个厂家的经久老茶叶共4种产品众多个子款，以冲泡后气味和味道带有明显浓烈的“焙药香”茶为感观标准筛选16款，也即每种产品筛选4个子款，送中科院广州分析测试中心和南方医科大学中医药学院广东省制剂重点实验室两个机构用气质联用和高效液相色谱进行检测分析，并以中国茶叶公司广西分公司“中茶牌”2015年生产的广西茶砖、中国茶叶公司云南分公司“中茶牌”2015年生产的云南茶砖等2种当年同类新茶的不同子款作对照。
1.1 气质联用检测样品处理
将茶样粉碎成20目，取50g茶叶粉置于自制实验装置(取带橡胶塞的200mL密闭玻璃瓶一个，在橡胶塞上开两口，将吹扫氮气从其中一口引入瓶底，另一口用于吹扫气体的排出，在排出口连接活性碳管采样管，用于捕集吹扫气体)中，将密封玻璃瓶固定于80℃恒温水浴锅中，通入吹扫氮气，用流量计调节氮气流量为150mL/min，出口端连接活性碳采样管，动态顶空分离2小时。将采样管中活性炭取出后加入0.5mL二硫化碳溶液进行解析。
1.2 气质联用条件：吸取二硫化碳解析液1.0μL，手动进样；GC进样口模式为分流模式，分流比5：1；进样口温度250℃；载气为He，载气为恒流模式，柱流速1.0mL/min，平均线速37cm/sec；分析柱为Thermo公司TG-5MS毛细石英管柱(30m×250μm×0.25μm)；升温程序为初始温度60℃，保持3min，以5℃/min升至150℃，保持10min，再以5℃/min升至250℃，保持9min，共计60分钟。离子源温度230℃，质谱传输线温度250℃，质量扫描范围30-550amu。
1.3 气质联用数据分析：按上述气质联用条件进样得到样品挥发性气味总离子流图，用面积归一化法获得各化合物的相对含量。使用Wiley275、NIST14数据库对积分的色谱峰进行自动及人工检索，最后鉴定化学成分。
1.4 高效液相色谱检测样品处理
将茶样粉碎成20目，取10g茶叶粉置于100mL锥形瓶中，加入50mL提取液(70％甲醇、29.7％水和0.3％乙酸)，在70cc下震荡浸提30min，冷却至室温，在3500r/min下常温离心10min，倒出上清液，用0.45μm滤膜过滤，所得滤液作为供试品溶液。精密称取各对照品适量，置于10mL容量瓶中，加提取液溶解，配制成0.5mg/mL的对照品储备液。
1.5 高效液相色谱条件：色谱柱为C18柱(250mm×4.6mm，5μm，如Agilent公司的C18柱等)，流动相A：0.2％乙酸乙腈溶液；流动相B：0.2％乙酸水溶液。采用梯度洗脱的方法，流速为1.0mL/min，柱温35℃，检测波长为270mm。
1.6 高效液相色谱检测数据分析：测定各成分的峰面积，并以峰面积(y)对进样量(X，mg/mL)进行线性回归分析。
2.归芍茶的干膏提取
将700g归芍茶的茶饼敲碎，加入到8倍量沸水中，保持微沸20min，过滤。将第一次提取液用5倍量沸水同法提取1次。茶渣用5倍量沸水浸泡20min，过滤。同法将第三次提取液用沸水浸泡处理。合并提取液，用减压浓缩器在58℃下进行减压浓缩，得干膏。
3.归芍茶干膏药效学试验
3.1 动物及饲养条件
3.1.1 实验动物：5周龄SPF级雄性裸鼠31只，平均体重20g/只。由南方医科大学动物实验中心提供，广东省实验动物质量合格证明编号：No.44002100008649。
3.1.2 饲养设备：使用独立通气动物笼IVC鼠笼，单笼喂养，每笼3-4只裸鼠。使用前经 高压蒸汽灭菌，每周清洁一次，或视需要处理，以保证环境清洁、干燥为标准；垫料选用研磨过的玉米杆，使用需经高温蒸汽灭菌。
3.1.3 饲养环境：SPF级；室内温度26-28℃，室内湿度40％-60％；每小时通风换气10-15次；每日维持10小时光照，14小时无光的明暗周期，使用人工光照。
3.1.4 供食及供水饲料、饮水均在高压灭菌后使用；隔天换水瓶，水瓶和饮水管在两次使用之间必须清洗。
3.2 细胞株
体外培养BGC-823人胃癌细胞株，培养基为10％胎牛血清的高糖DMEM。
3.3 原料及试剂
3.3.1 原料：归芍茶提取的干膏
3.3.2 剂量
归芍茶使用等效剂量：低剂量0.1g/只，中剂量0.5g/只，高剂量1.0g/只。干膏使用量：低剂量10mg/只/天，中剂量50mg/只/天，高剂量100mg/只/天。干膏采用去离子水溶解，配置成使用浓度：低剂量0.025g/ml，中剂量0.125g/ml，高剂量：0.250g/ml。溶液121℃灭菌30min。
3.4 建立胃癌模型
3.4.1 配置细胞悬液
将BGC-823人胃癌细胞用胰蛋白酶消化，用高糖DMEM配置成单细胞悬液3ml，细胞总量约为2×108个。
3.4.2 皮下注射胃癌细胞
在无菌操作台中，用耳标钳在裸鼠右耳靠近头皮处打耳号，在27只裸鼠的背部右下方靠近右下肢处注射BGC-823细胞悬液0.1ml/只，剩余4只裸鼠在同样部位注射等量生理盐水。
3.5 分组及给药
3.5.1 分组
建模后，每日观察裸鼠状态，称重，用电子游标卡尺测量肿瘤长短径，记录数据。待肿瘤直径达到8mm以上时(约2周)，以肿瘤直径/体积为第一要素，裸鼠体重为第二要素进行分组：干膏高剂量组7只，干膏中剂量组7只，干膏低剂量组7只，阴性对照组(荷瘤生理盐水组)6只，空白对照组4只。每组肿瘤直径平均值接近，每组都有大、小肿瘤。
3.5.2 给药
1ml注射器吸取干膏溶液0.4ml，换上9号灌胃针头，对各组裸鼠进行灌胃，其中阴性对照组灌胃去离子水，空白对照组不灌胃。每隔24h灌胃一次，连续灌胃至裸鼠自然死亡。
3.6 移植瘤的观察
每日记录裸鼠体重及肿瘤长短径，观察每组裸鼠精神状态及活动程度。
4.肠道菌群检测
裸鼠开始灌胃后，每日用EP管收集裸鼠的粪便，做好标记，置于-80℃冰箱保存。实验结束后，将所有裸鼠死亡前一天的粪便进行肠道菌群的宏基因组16sDNA测序，扩增区域为v3-v4。
5.统计分析
采用SPSS13.0或19.0对实验结果进行分析，其中采用秩和检验对生存时间进行分析， 方差分析对灌胃前后肿瘤体积差进行分析，重复测量对时间和灌胃剂量的关系进行分析。
6.结果
6.1 气质联用检测16款“焙药香”的“经久老茶叶”挥发性气体成分，与其他中药挥发性气体成分比较后发现，主要成分跟当归和白芍相同较多，主要成分跟当归相同的有β-蒎烯、β-水芹烯、β-月桂烯、β-罗勒烯，跟白芍相同的有β-蒎烯、β-月桂烯、β-罗勒烯，因此根据其气味近似程度，把具有“焙药香”的“经久老茶叶”命名为归芍茶。
6.2 采用气质联用和高效液相色谱对4种归芍茶和2种当年同类新茶进行检测发现，每种归芍茶中各子款的图谱数据基本一致，并且每种当年同类新茶中各子款的图谱数据也基本一致。图1示出了每种茶的其中一个子款的图谱，每种茶的其他子款的图谱数据也基本一致。
请结合图1和表1，相对于当年同类新茶，归芍茶的主要成分共同新出现和显著增加23种，共同消失和显著减少12种，其中大多数是萜烯及其衍生物，特别是单萜。
其中气质联用检测新出现和显著增加主要成分主要包括：4-松油醇、6-甲基-5-庚烯-2-酮、α-柠檬烯、α-松油烯、β-月桂烯、β-罗勒烯、β-蒎烯、α-水芹烯、β-水芹烯、Vitispirane、十三烷、2，6，10-三甲基十二烷、6，10-二甲基-2-十一烷酮、罗汉柏烯、羟基二氢鸡蛋果素、2，6-二叔丁基-1，4-苯醌、β-桉叶烯、壬基环己烷、Methyl2-endo-Acetamidobicyclo[2.2.1]heptane-2-exo-carboxylate、2-甲基十五烷、5，6-二氢-5，6-二甲基苯并[c]噌啉、3-甲基十六烷及十九烷；消失和显著减少主要成分主要包括：芳樟醇、α-松油醇、二氢猕猴桃内酯、水杨酸甲酯、β-紫罗兰酮、2-甲基丙酸-3-羟基-2，2-二甲基-1-(2-羟基-1-甲基乙基)戊基酯、甲基-N-邻氨基苯甲酸甲酯、δ-杜松烯、1，1-二苯基-2-甲基丙烯及2，3-二氢-1-甲基-3-苯基-1H-茚。高效液相色谱检测显著减少主要成分包括：咖啡因和茶碱。
表1 气质联用和高效液相色谱检测的归芍茶及新茶的相应成分的含量
Figure PCTCN2017088029-appb-000001
Figure PCTCN2017088029-appb-000002
6.3 选择4款归芍茶中新出现和显著增加成分含量最高的1款(云南勐海春福润茶叶公司“春福润牌”30年以上云南茶砖)，即图1中ART 3，减压浸提，得到干膏79克，出膏率为11.3％。
6.4 在裸鼠建立胃癌模型，采用归芍茶干膏溶液灌胃
6.4.1 归芍茶不同剂量的抑制作用与时间有交互作用(P＜0.05)，中剂量组具有显著抑制肿瘤生长的作用，第7天抑制效果最显著，结果如图2、3和4所示。
6.4.2 归芍茶灌胃前后肿瘤体积差异有统计学意义(P＜0.05)，中剂量组肿瘤体积差异最小，低剂量组肿瘤增长最迅速，如表2、表3和图5所示。
表2 各组裸鼠自然死亡时和开始灌胃时肿瘤的体积差
Report
肿瘤体积差
Figure PCTCN2017088029-appb-000003
表3 肿瘤体积差方差分析结果
ANOVA
肿瘤体积差
Figure PCTCN2017088029-appb-000004
注：表中的看第一行Between Groups(p＝0.004)，表示有统计学意义，说明各组间肿瘤体积差有统计学差异。
6.4.3生存时间分析表明，各组生存时间有显著差异，其中低剂量组存活时间最长，说明低剂量可以延长生存时间(P＜0.05)，高剂量组1天后所有裸鼠死亡，说明高剂量组具有急性毒性作用，结果如图6、表4、表5和表6所示。
表4 各组生存时间
Report
给药后生存时间(天)
Figure PCTCN2017088029-appb-000005
表5 各组生存时间秩和检验结果
Ranks
Figure PCTCN2017088029-appb-000006
表6 秩和检验结果
Test Statisticsa，b
Figure PCTCN2017088029-appb-000007
a.Kruskal Wallis Test
b.Grouping Variable：group
注：表中的看第一行Asymp.Sig(p＝0.000)，表示有统计学意义，说明各组存活时间有统计学差异。6.4.4总之，高剂量组灌胃一天后全部死亡，说明归芍茶高剂量具有急性毒性作用。中剂量组可以显著抑制肿瘤生长。低剂量组可以显著延长带瘤生存时间。
6.5 肠道菌群
6.5.1 首先研究群落生态学中微生物多样性，通过单样品的多样性分析(Alpha多样性)，可以反映微生物群落的丰度和多样性。其中Chao说明群落分布丰度，Shannon说明群落分布多样性，由图7可看出，正常组菌落丰度和多样性在种瘤后明显减少，种瘤后再灌胃归芍茶干膏，菌落丰度和多样性回复，其中，中浓度组菌落丰度和多样性基本回复到正常水平，低浓度组菌落丰度和多样性高于正常水平。
接着进行物种分类分析，可以得知样品在各分类水平上的分类学比对情况，包括样品中含有的微生物种类和相对丰度。由图8和图9a可以看出，正常组和中剂量组的微生物种类和丰度最相似，其次是低剂量组，阴性对照组和其他几组均不同，说明正常组在种瘤后肠道菌群的微生物种类和丰度发生明显改变，灌胃归芍茶(干膏)后，微生物种类和丰度发生回复，其中，中剂量组基本回复到正常。由热图9b、GraPhlAn绘制的分类和系统发育信息可视化图9c可知，丰度前100个物种集中的门为：BACTEROIDETES、FIRMICUTES、PROTEOBACTERIA和VERRUCOMICROBIA，丰度最高的细菌的属包括：Bacteroides、Falsiporphyromonas、Coprobacter、Prevotella、Clostridium XlVa、Alistipes等。
6.5.2 然后进行PCA分析，其中BC是正常组(即空白对照组)，NC是肿瘤组(即阴性对照组)，M是中剂量组，L是低剂量组。由图10可知，正常组和中剂量组最相似。如图11所示，进一步进行PCoA分析，也说明正常组和中剂量组最相似。
6.5.3 如图12所示，
然后进行菌群丰度差异分析，发现各组间菌群差异如下，红色为P＜0.05具有显著统计学差异的菌群。BC和NC间显著差异的属的菌群为Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus。NC和M显著差异为 Comamonas。NC和L显著差异为Prevotella、Pseudoflavonifractor、Flavonifractor、Butyrivibrio、Methanothrix。BC和M显著差异为Neisseria、Chelativorans、Cellulomonas、Dyella、Gp7。BC和L显著差异为Pseudomonas、Nitrososphaera、Gp7、Cellulomonas、Neisseria、Methanospirillum、Terrimonas、Pseudoxanthomonas、Dyella、Pseudolabrys。L和M间没有差异。我们认为这些细菌是抑癌细菌。
结果说明：
NC组和BC组相比，Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus菌群丰度显著下调(P＜0.05)，M组和NC相比显著改变的菌群Comamonas虽然在NC组没有统计学差异的改变，但轻微下调。推测NC组肿瘤生长的原因是Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus菌群丰度显著下调和Comamonas基本不变。
M组中菌群丰度显著升高的是Comamonas(P＜0.05)，BC组显著高于NC组的菌群Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus的丰度基本在M组中都上调，虽然没有统计学意义。推测M组抑制肿瘤生长是通过显著上调Comamonas和回复BC组显著高于NC组的菌群Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus的丰度实现的。
L组中，BC组显著高于NC组的菌群Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus的丰度不但没有上调，反而下降，虽然没有统计学意义；M组显著高于NC组的Comamonas保持不变，没有像M组那样显著上调，因此推测L组肿瘤生长迅速的原因是Comamonas丰度不变和BC组显著高于NC组的菌群Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus的丰度下调。
L组和NC组相比显著上调的菌群Prevotella、Pseudoflavonifractor、Flavonifractor、Butyrivibrio、Methanothrix，但没有回复到BC水平，而这些菌群在M组没有统计学差异的改变，因此推测这些菌群可以延长带瘤生存时间。
综上所述，Nitrososphaera、Pseudomonas、Gp7、Terrimonas、Cellulomonas、Methanomassiliicoccus、Haemophilus的显著下调，可以促进肿瘤的发生和生长，相当于抑癌菌属。显著上调Comamonas可以抑制肿瘤生长，也相当于抑癌菌属。显著上调Prevotella、Pseudoflavonifractor、Flavonifractor、Butyrivibrio、Methanothrix可以延长带瘤生存时间，相当于抑癌菌属。
6.5.4 如图13所示，接着基于COG和KEGG进行菌群的功能分析，发现菌群功能也是BC和M组最相近，其次为L组，和NC组差别最大。菌群功能最主要集中在Carbohydrate transport and metabolism碳水化合物的运输和代谢、Amino acid transport and metabolism氨基酸运输和代谢、Membrane Transport膜运输、Replication，recombination and repair复制、重组和修复、Transcription转录、Translation，ribosomal structure and biogenesis翻译，核糖体结构和生物转化、Cell wall/membrane/envelope biogenesis细胞壁/膜/信封生源论、General function prediction only通常功能预测、Function unknown未知功能。
6.5.5 如图14a和14b所示，最后基于COG和KEGG进行的分组间菌群功能差异比较，发现P＜0.05的菌群功能如下：
NC组菌群高表达的功能为Carbohydrate Metabolism碳水化合物代谢、Transcription转录、Membrane Transport膜运输，说明菌群通过增加碳水化合物的代谢、转录和膜运输，达到促进肿瘤发生和生长的作用。
L组菌群高表达的功能为Coenzyme transport and metabolism辅酶运输和代谢、Post-translational modification翻译后修饰、Environmental Adaptation环境适应、Metabolism of Cofactors and Vitamins辅酶因子和维生素代谢、Folding，Sorting and Degradation折叠，排序和退化、Metabolism of Terpenoids and Polyketides多酮类化合物和萜类化合物的代谢、Glycan Biosynthesis and Metabolism多糖生物合成和代谢，说明菌群通过增加辅酶运输和代谢、翻译后修饰、环境适应、辅酶因子和维生素代谢、折叠，排序和退化、多酮类化合物和萜类化合物的代谢、多糖生物合成和代谢，达到延长带瘤生存时间的作用。
德国科学家奥托·瓦伯格(Otto Warburg)在上世纪20年代发现：癌细胞比其他细胞以更高的效率吸收葡萄糖来促进自身快速生长，然而这些葡萄糖主要是通过糖酵解途径在细胞内被利用。正常细胞只有在缺氧的情况下进行糖酵解，而肿瘤细胞即使在不缺氧的情况下也优先进行糖酵解，消耗更多的葡萄糖和产生更多的乳酸，这就是著名的Warburg效应。研究发现Warburg效应跟肿瘤细胞旺盛的生长需求有关，糖酵解不仅为肿瘤细胞的增殖提供能量，而且还为其脂肪酸和核酸的合成提供原料。我们的菌群功能差异比较结果完全符合肿瘤代谢的特点。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种归芍茶的检测方法，其特征在于，包括如下步骤：
    步骤一：将待测茶样粉碎成粉料，置于检测容器中，使用气体吹扫，捕集吹扫气体；
    步骤二：使用气质联用检测所述吹扫气体中的4-松油醇、β-蒎烯、β-水芹烯、β-月桂烯以及β-罗勒烯的含量。
  2. 如权利要求1所述的归芍茶的检测方法，其特征在于，还包括检测所述吹扫气体中的如下十八种挥发性气体成分的含量的步骤：6-甲基-5-庚烯-2-酮、α-柠檬烯、α-松油烯、α-水芹烯、Vitispirane、十三烷、2，6，10-三甲基十二烷、6，10-二甲基-2-十一烷酮、罗汉柏烯、羟基二氢鸡蛋果素、2，6-二叔丁基-1，4-苯醌、β-桉叶烯、壬基环己烷、甲基-2-内-乙酰氨基[2.2.1]庚烷基-2-外-羧酸酯、2-甲基十五烷、5，6-二氢-5，6-二甲基苯并[c]噌啉、3-甲基十六烷以及十九烷。
  3. 如权利要求2所述的归芍茶的检测方法，其特征在于，还包括检测所述吹扫气体中的如下十种挥发性气体成分的含量的步骤：芳樟醇、α-松油醇、二氢猕猴桃内酯、水杨酸甲酯、β-紫罗兰酮、2-甲基丙酸-3-羟基-2，2-二甲基-1-(2-羟基-1-甲基乙基)戊基酯、甲基-N-邻氨基苯甲酸甲酯、δ-杜松烯、1，1-二苯基-2-甲基丙烯及2，3-二氢-1-甲基-3-苯基-1H-茚。
  4. 如权利要求3所述的归芍茶的检测方法，其特征在于，还包括如下步骤：
    制备供试品溶液：将待测茶样粉碎成粉料，加入容器中，加入提取液进行震荡浸提，后离心，收集上清液，过滤后得到的滤液作为所述供试品溶液；
    使用高效液相色谱检测所述供试品溶液中咖啡因和茶碱的含量。
  5. 如权利要求4所述的归芍茶的检测方法，其特征在于，还包括对所述待测茶样冲泡后进行气味检测的步骤。
  6. 如权利要求4所述的归芍茶的检测方法，其特征在于，所述粉料的粒径不大于20目；所述提取液含有70％甲醇、29.7％水和0.3％乙酸；所述过滤是使用0.45μm的滤膜过滤。
  7. 如权利要求6所述的归芍茶的检测方法，其特征在于，所述高效液相色谱检测的条件：色谱柱为250mm×4.6mm、5μm的C18柱，流动相A：0.2％乙酸乙腈溶液；流动相B：0.2％乙酸水溶液；采用梯度洗脱的方法，流速为1.0mL/min，柱温35℃，检测波长为270mm。
  8. 如权利要求1所述的归芍茶的检测方法，其特征在于，所述步骤一具体是将待测茶样粉碎成不大于20目的粉料，置于密闭容器中，并将密闭容器置于恒温水浴锅中，所述密闭容器的胶塞开设有两个开口，将吹扫氮气从其中一开口引入瓶底，用流量计调节氮气流量为150mL/min，另一开口用于吹扫气体的排出，在该排出口连接活性碳管采样管，用于捕集吹扫气体，动态顶空分离，将采样管中活性炭取出后加入二硫化碳溶液进行解析。
  9. 如权利要求8所述的归芍茶的检测方法，其特征在于，气质联用检测的条件：吸取二硫化碳解析液，手动进样；气相进样口模式为分流模式，分流比5∶1；进样口温度250℃；载气He，载气为恒流模式，柱流速1.0mL/min，平均线速37cm/sec；分析柱为30m×250μm×0.25μm的毛细石英管柱；升温程序为初始温度60℃，保持3min，以5℃/min升至150℃，保持10min，再以5℃/min升至250℃，保持9min，共计60分钟；离子源温度230℃，质谱传输线温度250℃，质量扫描范围30-550amu。
  10. 如权利要求1～9中任一项所述的归芍茶的检测方法，其特征在于，还包括使用与待测茶样同类的新茶进行与所述待测茶样同样检测的步骤。
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