CN114588184B - Herba Saururi extract, and preparation method and application thereof - Google Patents
Herba Saururi extract, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a herba leonuri extract, a preparation method and application thereof. The coleus extract of the invention comprises active sites such as flavonoid compounds, organic acid compounds, ester compounds and the like. The herba leonuri extract disclosed by the invention can obviously promote cell proliferation, enhance cell energy metabolism to synthesize ATP, and activate CAT enzyme to prevent oxidative damage generated in the energy metabolism process. Therefore, the coleus extract of the invention can be used as a functional component of medicines and daily chemical products for promoting cell proliferation and enhancing cell energy metabolism.
Description
Technical Field
The invention belongs to the field of chemistry, and particularly relates to separation of an active site of coleus blumea and application of the active site in promoting cell proliferation.
Background
The genus Botrytis is a large genus of fern, and its basic plants mainly comprise Botrytis cinerea, sphaeroides angustifolia, sphaeroides petiolatus, etc. The whole herb of the herba polygoni orientalis can be used as a medicine, has the functions of clearing heat and detoxicating, and relieving swelling and pain, is widely applied to people in folk, and is generally used as tea to treat various inflammations. Studies show that the cole grass can accelerate the body temperature of fevers and shorten the course of diseases when being used for treating respiratory tract infection diseases. The herba leonuri is rich in various organic acids, esters and flavonoid compounds, and has certain activities of resisting neuroinflammation, cancer and the like.
Without cell proliferation, there are no multicellular organisms on earth. Not only are large amounts of amino acids, carbohydrates and nucleotides required during cell proliferation, but also a large amount of energy is required, with Adenosine Triphosphate (ATP) being one of the major sources of energy required for some vital activities. Normally most multicellular organisms acquire ATP by respiration. The peroxide that is produced during this process is scavenged by Catalase (CAT) and protects the cells from peroxidative damage.
Therefore, the research on the active site and the efficacy of the cole grass has wide application prospect.
Disclosure of Invention
In order to achieve the aim of the invention, the invention provides a herba leonuri extract, and a preparation method and application thereof.
The term "herb" in the present invention refers to a plant species of the genus Pelargonium (Ophioglossaum Linn.) of the family Pelargoniaceae, including Pelargonium (Ophioglossum vulgatum L.), sphaerocarpum angustum (O.thermale Kom.) and Pelargonium cardiale (O.reiciculatum Linn.), further including Pelargonium (O.petulosum Desv.), pelargonium blunt (O.petulosum hook.), pelargonium graveolens (O.parvifolia et hook.), and other species of the genus Pelargonium, including O.pendulum, O.ailchisoni, O.pusillum, O.palmatum, ophioglossum californicum, O.crotophylloides Walter and 84.
In a first aspect, the invention provides a herba leopariae extract, which comprises 39.98% -54.40% of total flavonoid compounds, 17.11% -69.92% of total organic acid compounds and 13.16% -38.09% of ester compounds by total weight.
In some embodiments, the coleus extract contains 40% -52%, preferably 45% -50% total flavonoids based on total weight.
Further, the coleus extract contains 14.70% -30.92% of flavonoid glycoside compounds by total weight.
In some embodiments, the coleus extract contains 20% -60%, preferably 30% -45% total organic acid compounds by total weight.
Further, the coleus extract contains 1.30% -17.95% of fatty acid compounds by total weight.
In some embodiments, the coleus extract contains 15% -35%, preferably 20% -30% total ester compounds by total weight.
In a second aspect, the present invention provides a method of preparing a coleus extract, the method comprising the steps of: reflux-extracting herba Saussureae Involueratae with ethanol water solution, concentrating the extractive solution, extracting with ether solvent, discarding ether extractive solution, extracting with ester solvent, collecting ester extractive solution, and drying to obtain herba Saussureae Involueratae extract.
In some embodiments, the method further comprises the step of drying and pulverizing prior to extracting the coleus.
Preferably, the drying temperature is from 35℃to 50℃and more preferably from 40℃to 45 ℃.
Preferably, the powder is crushed to 40 to 80 mesh, more preferably 60 to 80 mesh.
In some embodiments, reflux extraction with 60% to 95% aqueous ethanol; preferably, reflux-extracting for 2-3 times; it is further preferred that each extraction is carried out for 0.5 to 1.5 hours. Preferably, the ratio of the volume of the 60% -95% ethanol aqueous solution to the mass of the coleus extract is (10-30): 1.
In some embodiments, the volume ratio of extraction solvent to concentrate is (1-2): 1, preferably 1:1.
In some embodiments, each extraction solvent is extracted 3 to 5 times.
In some embodiments, the ethereal solvent is selected from one or more of methyl ether, ethyl ether, and petroleum ether; petroleum ether is preferred.
In some embodiments, the ester solvent is selected from one or more of ethyl formate, ethyl acetate, ethyl propionate, or ethyl butyrate; ethyl acetate is preferred.
In some embodiments, the method of preparing the coleus extract of the present invention comprises the steps of: (1) Taking dried whole herb of the herba leonuri, crushing the whole herb to 40-80 meshes, adding 60-95% ethanol water solution, carrying out micro-boiling reflux extraction twice for 0.5-1.5 hours each time, combining the extracting solutions, and carrying out reduced pressure rotary evaporation on the filtrate to obtain coarse concentrate of the herba leonuri; (2) Adding petroleum ether into the concentrated solution according to the volume of 1:1 for extraction for 3-5 times, and discarding the extract; (3) Adding ethyl acetate into the concentrated solution subjected to petroleum ether extraction for 3-5 times according to the volume of 1:1, combining the extracts, and performing reduced pressure rotary evaporation to obtain the dried herba Saururi extract.
In a third aspect, the present invention provides a coleus extract prepared by the method.
The active site of the extract of the coleus blume obtained by the preparation method is not a single compound, but contains the combination of all the compounds in the extract obtained by the preparation method.
Preferably, the herba leopariae extract of the invention contains 39.98-54.40% of total flavonoid compounds, 17.11-69.92% of total organic acid compounds and 13.16-38.09% of ester compounds by total weight.
In some embodiments, the coleus extract contains 40% -52%, preferably 45% -50% total flavonoids based on total weight.
Further, the coleus extract contains 14.70% -30.92% of flavonoid glycoside compounds by total weight.
In some embodiments, the coleus extract contains 20% -60%, preferably 30% -45% total organic acid compounds by total weight.
Further, the coleus extract contains 1.30% -17.95% of fatty acid compounds by total weight.
In some embodiments, the coleus extract contains 15% -35%, preferably 20% -30% total ester compounds by total weight.
In a fourth aspect, the invention provides an application of the herba leoniae chinensis extract in preparing medicines and daily chemical products for promoting cell proliferation.
In some embodiments, the promoting cell proliferation comprises promoting proliferation of HaCaT and/or HOEC cells, and/or increasing transcription of structural proteins COL1A1 and FN1 and cytokine TNF- α in cells.
In a fifth aspect, the invention provides the use of the herb extract in the manufacture of a medicament, daily chemical product for enhancing cellular energy metabolism.
In some embodiments, the enhancing cellular energy metabolism comprises increasing intracellular ATP concentration and/or increasing intracellular CAT enzyme activity.
In a sixth aspect, the present invention provides the use of the coleus extract in the manufacture of a medicament for promoting hair growth or regeneration and/or eyelash growth, a daily chemical product.
In a seventh aspect, the invention provides the use of the coleus extract in the preparation of a medicament for promoting wound healing, a daily chemical product.
The herba leoniae extract of the invention can be prepared into various dosage forms such as capsules, tablets, powder, liniment, soft capsules, oral liquid, pills, gel, emulsion, essential oil, sustained release agent and the like which are mainly prepared from the herba leoniae extract with auxiliary materials approved in medicaments, daily chemical products and foods by modern mixing and preparation technologies.
The beneficial effects of the invention are as follows: the coleus extract of the invention can be 1: promoting cell energy metabolism, and showing that the intracellular ATP content and CAT enzyme activity are increased; 2: promoting cell proliferation, exhibiting an increase in cell number, an increase in transcription of cytokine TNF-a associated with cell proliferation, and structural proteins FN1 and COL1A1, can be used as functional components of medicines and daily chemical products for promoting cell proliferation such as hair growth, wound healing, eyelash growth, etc.
Drawings
FIG. 1 is a flow chart of the preparation of the extract of Botrytis cinerea of the present invention.
FIG. 2 is a chart of UPLC-Q/TOF-MS analysis of the extract of Botrytis cinerea of the present invention.
FIG. 3 shows the activity of the extract of Botrytis cinerea of the present invention in promoting cell proliferation.
FIG. 4 shows that the extract of Botrytis cinerea of the present invention promotes transcription of COL1A1 and FN1 and TNF-a.
FIG. 5 shows that the extract of Botrytis cinerea promotes the synthesis of cellular ATP.
FIG. 6 shows that the extract of Botrytis cinerea of the present invention enhances CAT enzyme activity.
FIG. 7 is a diagram of the promotion of hair regeneration in the dehairing area of a mouse by using the Bolbostemma niveum of the present invention.
Detailed Description
The invention will be further described with reference to specific examples for further illustration of the invention. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
In the present invention, unless otherwise specified, the raw materials, reagents and equipment employed are commercially available or commonly used in the art.
Example 1
This example provides a coleus extract prepared by a process comprising the steps of:
(1) The collected whole herb of the cole grass (Ophioglossum vulgatum L.) is dried by a baking oven at 45 ℃, crushed to 60 meshes and stored in a cool and ventilated place.
(2) 5g of the herba leonuri powder is taken and placed in a 200mL round bottom flask, 75vol% ethanol is added according to the mass volume ratio of 20 times, the mixture is subjected to micro-boiling reflux extraction twice, each time lasts for 1 hour, and the extracting solutions are combined.
(3) Concentrating the extract by rotary evaporation under reduced pressure until no alcohol smell exists, adding water until the volume of the concentrate reaches 100mL, adding petroleum ether with equal volume according to the volume ratio of 1:1 for extraction, discarding the extract, and continuously extracting for 3 times until the extract has no color.
(4) Extracting with equal volume of ethyl acetate for 3 times until the extract is colorless, and mixing the extracts.
(5) And (5) performing reduced pressure rotary evaporation to dry the extract liquid to obtain the herba Saururi extract, and placing the herba Saururi extract in a dryer for standby.
The process flow is summarized in fig. 1.
The extraction efficiency of the extract prepared from the dried powder of the coleus blumei is 1.67+/-0.77% (g/g of the coleus blumei powder).
The obtained herba Saururi extract has total flavonoids content of 46.47+ -3.50%, and further contains flavonoid glycoside compounds content of 22.70+ -4.53%; the content of the total organic acid is 41.07+/-3.93%, and further, the content of the fatty acid compound is 13.85+/-3.46%; the content of the ester compound is 23.83 +/-5.30%.
Example 2
This example provides a coleus extract prepared by a process comprising the steps of:
(1) Collected whole grass of Peucedanum praeruptorum (O.pedunculosum Desv.) is dried in an oven at 35deg.C, pulverized to 40 mesh, and stored in a cool and ventilated place.
(2) 5g of the herba leonuri powder is taken and placed in a 200mL round bottom flask, 95vol% ethanol is added according to the mass volume ratio of 10 times, the mixture is subjected to micro-boiling reflux extraction for three times, each time lasts for 0.5 hour, and the extracts are combined.
(3) Concentrating the extract by rotary evaporation under reduced pressure until no alcohol smell exists, adding water until the volume of the concentrate reaches 100mL, adding petroleum ether according to the volume ratio of 1:1.5 for extraction, discarding the extract, and continuously extracting for 3 times until the extract has no color.
(4) Ethyl acetate is added for extraction according to the volume ratio of 1:1.5, extraction is carried out for 3 times continuously until the extract liquid has no color, and the extract liquid is combined.
(5) The extract is dried by reduced pressure rotary evaporation to obtain the extract of the herba Saururi.
The extraction efficiency of the extract prepared from the dried powder of the coleus blumei is 0.80+/-0.91% (g/g of the coleus blumei powder).
The obtained herba Saururi extract has total content of 44.21 + -4.23% and total content of flavonoid glycoside compounds 17.75+ -3.05%; the content of the total organic acid is 23.70+/-3.70%, and further, the content of the fatty acid compound is 5.56+/-4.26%; the content of the ester compound is 18.98+/-3.98 percent.
Example 3
This example provides a coleus extract prepared by a process comprising the steps of:
(1) The collected whole grass of narrow She Pinger grass (o.thermal kon.) was dried in an oven at 50 c, pulverized to 80 mesh, and stored in a cool and ventilated place.
(2) 5g of the herba leonuri powder is taken and placed in a 200mL round bottom flask, 60vol% ethanol is added according to the mass volume ratio of 30 times, the mixture is subjected to micro-boiling reflux extraction twice, each time lasts for 1.5 hours, and the extracts are combined.
(3) Concentrating the extract by rotary evaporation under reduced pressure until no alcohol smell exists, adding water until the volume of the concentrate reaches 100mL, adding petroleum ether according to the volume ratio of 1:1 for extraction, discarding the extract, and continuously extracting for 4 times until the extract has no color.
(4) Ethyl acetate is added for extraction according to the volume ratio of 1:1, extraction is carried out for 4 times continuously until the extract liquid has no color, and the extract liquid is combined.
(5) The extract is dried by reduced pressure rotary evaporation to obtain the extract of the herba Saururi.
The extraction efficiency of the extract prepared from the dried powder of the coleus blumei is 2.64+/-0.132% (g/g of the coleus blumei powder).
The obtained herba Saururi extract has total content of 46.30 + -5.70% and total content of flavonoid glycoside compounds 26.63+ -4.29%; the content of the total organic acid is 39.45 +/-3.36%, and further, the content of the fatty acid compound is 12.42+/-5.53%; the content of the ester compound is 26.80+/-3.20%.
Experimental example 1
1. Activity verification of the extract of coleus blumea for enhancing cell energy metabolism and promoting cell proliferation.
1.1 Experimental materials
HaCaT cells (human immortalized keratinocytes) and HOEC cells (human oral epithelial cells) were purchased from a national laboratory cell resource sharing platform. Organic reagents such as DMSO (from eastern china medicine); fetal bovine serum and DMEM high sugar medium (Sigma aldrich); ATP kit, CAT kit (Nanjing build); cDNA synthesis kit, PCR reaction system and SYBR GREEN (Dalianbao organism); COL1A1, FN-B1, GAPDH upstream and downstream primers (Shanghai).
1.2 Experimental drugs
The extract of Botrytis cinerea prepared in the method of example 1 was dissolved in DMSO to prepare a stock solution of 10 mg/mL.
1.3 laboratory apparatus
HH-2 digital display constant temperature water bath (Jiangsu gold altar Honghua Instrument works); WH-2 micro vortex mixer (Shanghai Xie analytical instruments Co.); BP211D electronic analytical balance (Sartorius AG); water high performance liquid chromatograph; ultra-micro ultraviolet spectrophotometry (Quawell Q5000); rotary evaporator (western security super jie).
1.4 Experimental methods
UPLC-Q/TOF-MS analysis: dissolving herba Saururi extract in methanol, and analyzing the compound composition of 4 μl methanol solution of herba Saururi extract with concentration of 1mg/mL by UPLC-Q-TOF-MS system. Chromatographic conditions: the chromatographic column is ACQUITY UPLC BEH C (150×2.1mm,1.7 μm), the mobile phase is a complex system of solution A (acetonitrile containing 0.1% formic acid) and solution B (0.1% formic acid water) mixed according to a certain concentration gradient along time, and the specific conditions are as follows: 15% -20% of A in 0-5 min; 20-40% A for 5-32 min; 32-37 minutes 40-95% A. The flow rate was 0.3mL/min. The chemical composition of the coleus extract was analyzed using a turboion spray ion source and ESI anion and cation scanning mode. The specific conditions are as follows: ion source Gas1 (Gas 1): 55, ion source Gas2 (Gas 2): 55, curtain gas (Cur): 35, source temperature: ion phase voltage float (ISV F) at 60 ℃): 5500v/4500v; TOFMS scans m/z range: 50-1500 da, production scan m/z range: 50-1000 da, MS scanning accumulation time of 0.25 s/spectrum, and product ion scanning accumulation time of 0.05 s/spectrum; secondary mass spectrometry adopts IDA and a high sensitivity mode, and attenuation potential (DP): 60v (positive and negative ion mode). The obtained molecular weight is matched with a secondary data spectrum comprising more than 1500 Chinese herbal medicines through SCIEXOS software, and analysis and identification of the compound are completed.
Cell concentration determination: haCaT cells were cultured at 1.0X10 4 Inoculating with 10% foetal calf serum and 90% DMEM high sugar culture medium 96-well plate at 37deg.C and 5% CO 2 Culturing in a cell culture incubator. The following day, culture was continued for 14-16 hours with freshly prepared medium containing different concentrations of coleus extract (DMSO solution) according to the schedule, with 6 replicates for each drug concentration. Cell concentrations were measured using the MTT kit according to the instructions.
ATP concentration and CAT activity assays: haCaT cells were cultured at 5.0X10 5 The concentration of each mL is inoculated into a 24-well plate according to the phaseCells were cultured and treated in the same manner, and the intracellular ATP content and CAT activity were determined according to the methods provided by the ATP and CAT assay kit.
Real-Time PCR: haCaT cells were cultured at 5.0X10 5 The cells were cultured and treated in the same manner in 24-well plates inoculated at a concentration of individual/mL. After the collected cells were washed with PBS, total RNA was extracted by Trizol-chloroform method, 1. Mu.L of RNA sample was taken and mixed well with 99. Mu.L of DEPC water, and the concentration of RNA in the sample was measured, and a reverse transcription system was prepared based on the concentration of RNA. Preparing 12.5 mu L of real-time PCR amplification system after reverse transcription of RNA; after the reaction system was activated at 95℃for 3min, 45 cycles of denaturation at 95℃for 2 seconds, annealing at 60℃and extension for 30 seconds were performed. The primer sequences used are listed in Table 1 below:
TABLE 1 primer sequences
Western blot: haCaT cells were cultured at 5.0X10 5 The cells were cultured and treated in the same manner in 24-well plates inoculated at a concentration of individual/mL. After the collected cells were washed with PBS solution, the cells were sonicated with PBS solution and centrifuged to remove cell debris. After the protein concentration of the supernatant was quantified by the Bradford method, SDS Loading buffer (Loading buffer) was prepared at a concentration of 40. Mu.g of protein. Proteins were separated by 10% SDS-PAGE, electrotransferred to cellulose acetate membrane, blocked with 2 hours skimmed milk, incubated overnight with antibody, and detected the next day.
1.5 animal treatment and painting experiments
After applying the depilatory cream to the two-ribbed areas of 8-week-old female BALB/c mice, the mice were rinsed with sterile distilled water 2 times for 3 minutes each. The skin was moistened with 1mg/mL of the Pelargonium Graveolentis extract and an equal volume of distilled water, respectively, 6 times per day. The anesthetized mice measured the exposed skin surface area on both sides after 14 days.
1.6 statistics and analysis
Metering data resultsThe inter-group differences are indicated by T-test.
2. Results
FIG. 1 is a flow chart showing the preparation process of the extract of Botrytis cinerea; the extraction efficiency of the extract prepared from the dried powder of coleus blumei was 1.67.+ -. 0.77% (g/g of coleus blumei powder) according to the method of example 1.
FIG. 2 shows the analysis of the extract of Botrytis cinerea of the present invention UPLC-Q/TOF-MS.
By comparison and screening with the primary exact mass number, isotope distribution ratio and MS/MS of the compound TCM MS/MS Library in the secondary database of the chinese medicine with SCIEX OS software, 15 (see table 2) of the peaks were attributed in the positive ion mode (fig. 2A) and 22 (see table 3) of the peaks were attributed in the negative ion mode (fig. 2B).
TABLE 2 chemical composition attributed to the extract of Botrytis cinerea in the positive ion mode
TABLE 3 chemical composition attributed to the extract of Botrytis cinerea under anion mode
FIG. 3 shows that the extract of Botrytis cinerea of the present invention is capable of promoting proliferation of both HaCaT (FIG. 3A) and HOEC (FIG. 3B); the cell concentration in each concentration gradient treatment group was increased when the extract concentration was less than 125. Mu.g/mL, compared to the untreated control group (Ct), with the two concentration effects of 62.5. Mu.g/mL and 125. Mu.g/mL being significant and extremely significant, respectively.
FIG. 4 shows that the extract of Botrytis cinerea of the present invention promotes transcription of structural proteins COL1A1 and FN1 and cell proliferation controlling factor TNF- α in cells; transcription of structural proteins COL1A1 and FN1 in cells was extremely significantly elevated (p < 0.01) at concentrations between 15 μg/mL and 60 μg/mL of the Wilsoniana extract. COL1A1 and FN1 are the coding genes of type I collagen and fibronectin respectively, and the expression of the two proteins indicates that the proliferation of cells is vigorous. The cell proliferation regulating factor TNF-alpha is often involved in the proliferation of cells, and the intracellular TNF-alpha transcription is extremely obviously increased when the concentration of the extract of the Botrytis cinerea is between 15 mug/mL and 60 mug/mL, which is also an important evidence of vigorous cell proliferation.
FIG. 5 shows that the extract of Botrytis cinerea of the present invention can promote cellular ATP synthesis; the intracellular ATP concentration was very significant (p < 0.001) and significantly elevated (p < 0.05) at 7.5 μg/mL and 15 μg/mL for the coleus extract, whereas the ATP concentration was elevated but not significant at concentrations greater than 30 μg/mL.
FIG. 6 shows that the extract of Botrytis cinerea of the present invention enhances the activity of the cell CAT enzyme; the CAT enzyme activity in the cells was very pronounced (p < 0.001) and significantly elevated (p < 0.05) at 30 μg/mL and 60 μg/mL of the coleus extract concentration. This activity decreases with increasing concentration.
FIG. 7 shows that the extract of Botrytis cinerea of the present invention promotes hair regeneration in the dehairing zone of mice; application of the dehairing area of the mice (right side) at a concentration of 1mg/mL of the coleus extract significantly promoted hair regrowth in the dehairing area (P < 0.05), see table 4.
TABLE 4 extract of Botrytis cinerea of the invention for promoting hair regeneration in the dehairing zone of mice
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Hangzhou to ink Biotech Co., ltd
<120> Bolus extract, and preparation method and application thereof
<130> RYP2010606.5
<160> 8
<170> SIPOSequenceListing 1.0
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<213> GAPDH-F
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<213> Fn-R
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<213> TNFα-F
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ctcttctgcc tgctgcactt tg 22
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Claims (2)
1. The herba leonuri extract is characterized by comprising 39.98-52% of total flavonoid compounds, 20-45% of total organic acid compounds and 15-30% of ester compounds by total weight;
the coleus extract is prepared by the following steps: reflux-extracting herba Saussureae Involueratae with 60% -95% ethanol water solution for 2-3 times, each time for 0.5-1.5 hr, concentrating the extractive solution, extracting with ether solvent, discarding ether extractive solution, extracting with ester solvent, collecting ester extractive solution, and drying to obtain herba Saussureae Involueratae extract;
the method also comprises the steps of drying and crushing before extracting the coleus blume; wherein the drying temperature is 35-50 ℃ and/or the powder is crushed to 40-80 meshes;
the ether solvent is selected from one or more of methyl ether, diethyl ether and petroleum ether; and/or the ester solvent is selected from one or more of ethyl formate, ethyl acetate, ethyl propionate or ethyl butyrate.
2. Use of the coleus extract of claim 1 for the preparation of a medicament for promoting hair growth or regeneration and/or eyelash growth, and a daily chemical product.
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| KR20110134612A (en) * | 2010-06-09 | 2011-12-15 | 한불화장품주식회사 | Skin external composition containing bottle isocho fermentation |
| CN111096985A (en) * | 2018-10-09 | 2020-05-05 | 兟齐综合事业有限公司 | Use of blood pressure and blood sugar lowering composition and one-leaf clover extract |
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| KR20110134612A (en) * | 2010-06-09 | 2011-12-15 | 한불화장품주식회사 | Skin external composition containing bottle isocho fermentation |
| CN111096985A (en) * | 2018-10-09 | 2020-05-05 | 兟齐综合事业有限公司 | Use of blood pressure and blood sugar lowering composition and one-leaf clover extract |
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| Title |
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| 狭叶瓶尔小草化学成分的研究;张帼威;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20090115(第01期);第E057-153页 * |
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