CN105106254A - Anti-influenza loosestrife extract - Google Patents
Anti-influenza loosestrife extract Download PDFInfo
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- CN105106254A CN105106254A CN201510560415.9A CN201510560415A CN105106254A CN 105106254 A CN105106254 A CN 105106254A CN 201510560415 A CN201510560415 A CN 201510560415A CN 105106254 A CN105106254 A CN 105106254A
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Abstract
The invention discloses an anti-influenza loosestrife extract. A preparation method of the extract includes: (a), performing heat reflux extraction on dried loosestrife by the aid of 70% of ethanol solution, combing extracting solution, and concentrating the extracting solution until alcohol taste disappears to obtain ethanol extraction concentrated solution; (b), diluting the ethanol extraction concentrated solution obtained in the step (a) with water, performing extraction by the aid of petroleum ether, ethyl acetate and water-saturated n-butyl alcohol sequentially, and performing vacuum concentration to obtain a petroleum ether extract, an ethyl acetate extract and an n-butyl alcohol extract respectively; (c), dissolving the n-butyl alcohol extract with water, filtering, gathering active ingredients by macroporous resin, washing eight column volumes with 15% of ethyl alcohol to remove high polarity components, eluting ten column volumes with 70% of ethyl alcohol, collecting 70% of eluent, and performing vacuum concentration to obtain the loosestrife extract. The loosestrife extract has anti-influenza effect and can be developed into an anti-influenza drug.
Description
Technical field
The present invention relates to Chinese medicine extract field, be specifically related to a kind of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract and the pharmaceutical preparation containing this extract, this extract can be applied to resisiting influenza virus field, treatment and prevention influenza.
Background technology
From natural product, find the bioactive substance of novel structure or pharmacological action uniqueness, and find that new drug has been one of important means of generally acknowledged new drug development as lead compound by methods such as structure of modification.Ratify in the 1073 kinds of small-molecule drugs gone on the market between 1981-2010, have 50% to derive from natural product or relevant with natural guide structure.From last century, the medicine of many extensive uses clinically is all excavated out, as arteannuin, paclitaxel etc. from natural product.
China uses natural drug to have a long history, and defines oneself unique theory of medicine, and much traditional Chinese medicinal formulae also shows good effect so far in clinical practice.Meanwhile, China region is wide, various places Meteorological difference great disparity, natural product aboundresources, and of a great variety, natural resources of Chinese medicinal materials reaches kind more than 12000 especially.Therefore, along with natural drug more and more comes into one's own, active chemical components is extracted from Chinese herbal medicine, its curative effect is determined through pharmacology, clinical trial, then various preparation is made for Clinical practice, become after China joined WTO, found independent intellectual property right new drug, grown the important means of China's medicinal industry.
Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) LysimachiaclelhroidesDuby is Primulaceae Lysimachia plant, has another name called Herba Lysimachiae Clethroids, Arisaema balansae Engl. grass etc.All herbal medicine, is distributed widely in each provinces and regions on the south North China and the Changjiang river, has the effects such as heat-clearing and toxic substances removing, promoting blood flow to regulate menstruation, inducing diuresis to remove edema, being among the peoplely used for the treatment of edema, pyretic stranguria, yellow cellulitis, leukorrhagia, amenorrhea, injury and bone fracture, mastalgia treat drag, venom etc.Modern science grinds to make internal disorder or usurp proves that Lysimachia main chemical compositions is flavone and triterpene saponin, also has benzene a pair of horses going side by side lactone, quinones, phenolic acid, alkaloid, steroidal saponin and lignanoid etc., mainly has the multiple biological activitys such as cell toxicant, anti-inflammation, immunomodulating.
Summary of the invention
The object of the present invention is to provide a kind of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract, its preparation method and liquid phase analysis method, the pharmaceutical preparation containing this extract and utilize this extract to prepare the purposes of anti-influenza virus medicament.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
A kind of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract, this extract is prepared by following methods:
A (), by the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) of drying 70% alcoholic solution circumfluence distillation, merge extractive liquid, is concentrated into and obtains ethanol extraction concentrated solution without alcohol taste; B (), by step (a) gained ethanol extraction concentrated solution dilute with water, is used petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, concentrating under reduced pressure, is obtained petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; C () n-butyl alcohol extract water dissolution, filters, uses macroporous resin enrichment active component, first remove large polar component with 15% alcohol flushing, 8 column volumes, then use 70% ethanol elution, 10 column volumes, collect 70% eluent, concentrating under reduced pressure, obtains Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract.
Further, macroporous resin described in step (c) is AB-8 type macroporous resin.
In order to be controlled the differences between batches of extract prepared by different batches by the method setting up HPLC finger printing, the liquid phase analysis method of described extract is:
Chromatographic column: AgilentZorbaxSB-C18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.4% phosphoric acid solution;
Gradient elution program: 0.01 ~ 10min, A10% → 15%; 10 ~ 30min, A15% → 48%; 30 ~ 55min, A48% → 78%; 55 ~ 65min, A78% → 10%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 260nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Pharmaceutical preparation, the described extract containing treatment effective dose and pharmaceutically acceptable carrier.
The application of described extract in the medicine preparing resisiting influenza virus.
The application of described pharmaceutical preparation in the medicine preparing resisiting influenza virus.
When extract of the present invention is used as medicine, directly can uses, or use in the form of a pharmaceutical preparation.
Described pharmaceutical preparation contains the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract of the present invention for the treatment of effective dose, and all the other are acceptable on materia medica, nontoxic to humans and animals and pharmaceutically suitable carrier of inertia and/or excipient.
Described pharmaceutically suitable carrier or excipient are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation adjuvant.Pharmaceutical preparation of the present invention is used with the form of per weight dose.Extract of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into aqueous or oily solution, aseptic powder injection, liposome or the Emulsion etc. of sterilizing.
Advantage of the present invention: the present invention by extracting Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis), remove impurity, enrichment process, the extract with resisiting influenza virus effect can be obtained; Liquid phase analysis method provided by the invention may be used for the finger printing setting up this extract, for controlling the differences between batches of extract prepared by different batches.
Accompanying drawing explanation
Fig. 1 is Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract HPLC chromatograms.
Detailed description of the invention
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: prepared by extract
Crude drug source: Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) purchased from Hui nationality's Chinese Medicinal Materials Markets, Hebei, the place of production.
Main agents: food-grade ethanol is purchased from Shanghai Ling Feng chemical reagent company limited; Pharmaceutical grade AB-8 macroporous resin is purchased from sky tunami letter resin company limited; Acetonitrile is HPLC level, is purchased from TEDIA; 85% phosphoric acid is HPLC level, is purchased from TEDIA; Chromatographic grade pure water is heartily pure water.
Preparation method: by the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) of 10kg drying 70% alcoholic solution circumfluence distillation (25L × 3 time), merge extractive liquid, is concentrated into and obtains ethanol extraction concentrated solution (2.5L) without alcohol taste; Gained ethanol extraction concentrated solution is diluted with water to 4L, petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) is used to extract successively, concentrating under reduced pressure, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract 258g respectively; (c) n-butyl alcohol extract 1.5L water dissolution, medical absorbent cotton filters, with AB-8 macroporous resin (2kg, column volume 1.5L) deposition activity composition, first use 15% alcohol flushing, 8 column volumes (12L) to remove large polar component, then use 70% ethanol elution, 10 column volumes (15L), collect 70% eluent, concentrating under reduced pressure, obtains Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract 123g.
Embodiment 2: liquid-phase chromatographic analysis
Need testing solution is prepared: in the brown volumetric flask of extract 5mg to 50mL that Example 1 method is obtained, add 30mL10% acetonitrile solution ultrasonic dissolution, after being cooled to room temperature, continue to add 10% acetonitrile solution standardize solution.
Analytical method:
High performance liquid chromatograph: Agilent1260, binary pump;
Chromatographic column: AgilentZorbaxSB-C18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.4% phosphoric acid solution;
Gradient elution program: 0.01 ~ 10min, A10% → 15%; 10 ~ 30min, A15% → 48%; 30 ~ 55min, A48% → 78%; 55 ~ 65min, A78% → 10%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 260nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Analyze with the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract of 10 batches of preparation, carry out chromatographic peak coupling, 1 ~ No. 7 peak all occurs in 10 batch sample chromatograms as a result.Therefore demarcate 7 peaks for total chromatographic peak, set up the HPLC quality control collection of illustrative plates of this extract accordingly, the results are shown in Figure 1.This Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract pharmacologically active of 10 batches is similar.
Embodiment 3: extract pharmacological testing
One, materials and methods
1, medicine and reagent:
Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract is by the preparation of embodiment 1 method; Virazole: Zhejiang Asia-Pacific Pharmaceutical Co., Ltd produces, lot number: 130901.
2, animal: Kunming mouse, male and female half and half, body weight 20 ~ 24g, SPF level, is provided by Guangdong Medical Lab Animal Center, the animal quality certification number: check and affirm word 2013A018 in Guangdong.
3, Strain and cell: influenza A virus, influenza virus A-prime Mus lung adapted strain (FM1), purchased from Virology Inst., Chinese Academy of Preventive Medical Science; Mdck cell strain, purchased from Zhongshan University's Experimental Animal Center Cytology Lab.
4, cell culture fluid and maintenance medium: cell culture fluid is containing the penicillin of 100U/mL, the streptomycin of 100 μ g/mL and the DMEM culture fluid of 10% hyclone; Cell maintenance medium except containing except 2% hyclone, the same culture fluid of other compositions.
5, method
(1) Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract is in vitro to the toxicity test of MDCK cultured cell
After Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract mother solution cell culture fluid is done proportional diluted, be added to and grow up in MDCK cell monolayers culture plate, 100 μ L/ holes, each dilution factor medicinal liquid does 4 multiple holes, establishes normal cell controls, virus control and virazole to contrast simultaneously.Culture plate is put 37 DEG C of 5%CO
2cultivate 4d in incubator, every day is observation of cell pathological changes situation under inverted microscope.Half cytotoxic concentration (TC is calculated by Reed-Muench method
50).
(2) the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) cytopathogenic impact of infected by influenza (CPE method) in vitro
Get the culture plate growing up to cell monolayer, outwell culture fluid, add corresponding dilution medicinal liquid, 100 μ L/ holes, each dilution factor is parallel does 4 multiple holes.Put 37 DEG C of 5%CO
2outwell medicinal liquid after acting on 1h in incubator, inoculate 100 times of TCID
50the virus liquid of (median infective dose), 50 μ L/ holes, put 37 DEG C of 5%CO
2outwell virus liquid after adsorbing 1h in incubator, rinse cell 2 times with cell maintenance medium, then add corresponding dilution medicinal liquid, establish virus control, virazole to contrast and normal cell controls simultaneously, put 37 DEG C of 5%CO
2cultivate in incubator, every day is observation of cell pathological changes situation under inverted microscope.Cytopathy degree (Cytopathiceffect, CPE) judges by following 6 grade standards: "-" as Growth of Cells normal, occur without pathological changes; " ± " for cytopathy accounts for whole cell monolayer 0% ~ 10%; "+" for cytopathy accounts for whole cell monolayer 10% ~ 25%; " ++ " for cytopathy accounts for whole cell monolayer 25% ~ 50%; " +++ " for cytopathy accounts for whole cell monolayer 50% ~ 75%; " ++++" accounts for more than 75% of whole cell monolayer for cytopathy.The record experimental result when virus control group cytopathy is " ++++", to compare P<0.05 (CPE for " ++ " below) through rank test and is judged as effective with virus control group.And press Reed-Muench calculating medium effective concentration (IC50) and therapeutic index (TI).TI＝TC
50/IC
50。
(3) impact on mouse influenza virus pneumonia in Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract body
Get Kunming mouse 60, be divided into Normal group, virus-infected controls group, virazole group, basic, normal, high 3 the dosage groups of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract at random, totally 6 groups, often organize 10.Except Normal group, all the other respectively organize mice ether light anesthesia, select 15 LD of influenza virus A-prime Mus lung adapted strain FM1
50virus concentration, collunarium infecting mouse, every 0.05mL.From the dosage gastric infusion every day 2 times by 0.1mL/10g 1d before infecting, continuous 5d, virus-infected controls group is with equal-volume distilled water gavage.6d dissects after taking Mouse Weight, perusal pulmonary lesion, and the degree of record pulmonary liver sample consolidation, wins full lung and weigh, calculate Lung Exponent value, and obtain lung index with body weight.Computing formula is as follows:
Lung Exponent=[heavy (the g)/body weight (g) of lung] × 100%
Lung index=(virus control group Lung Exponent average-test group Lung Exponent average)/virus control group Lung Exponent average × 100%
(4) Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract infected by influenza causes the impact of dead mouse
Get Kunming mouse 90, be divided into virus-infected controls group, virazole group, basic, normal, high 3 the dosage groups of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract at random, totally 5 groups, often organize 18.Each group of mice with after ether light anesthesia, with 2 LD
50the influenza virus A-prime Mus lung adapted strain FM1 virus liquid collunarium of virus concentration infects, every 0.05mL.By the dosage gastric infusion of 0.1mL/10g from 1d before infecting, every day 2 times, continuous 5d, virus-infected controls group is with equal-volume distilled water gavage.Observe and record the death toll infecting rear mice, calculating mortality rate, protective rate, Average Survival natural law and increase in life span.Computing formula is as follows:
Death prevention rate=(virus-infected controls group mortality rate-test group mortality rate)/virus-infected controls group mortality rate
Increase in life span=(test group Average Survival natural law-virus-infected controls group Average Survival natural law/virus-infected controls group Average Survival natural law) × 100%
(5) statistical method
SPSS10.0 software kit is adopted to carry out statistical analysis, t inspection between the comparison employing group of Lung Exponent value, survival natural law; Protective rate relatively use χ
2inspection.
Two, interpretation of result
1, Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract In Vitro Anti influenza virus effect
Experimental result shows, the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract of larger concentration to the certain toxicity of mdck cell tool, main manifestations be cell proliferation slowly, granule increases, morphologic change, refractivity enhancing etc., its TC
50crude drug is 24.91mg/ml; Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract can suppress the cytopathy caused by influenza virus in vitro, its IC
50crude drug is 3.33mg/ml.Infected by influenza is inhibited in vitro for prompting Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract, and its TI is 7.48.
2, Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract is on the impact of mouse influenza virus pneumonia
From table 1, the Lung Exponent value of Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract basic, normal, high dosage group mice is starkly lower than virus-infected controls group, compare with virus-infected controls group and have significant difference (P<0.01 or 0.05), the mouse lung inflammation that prompting Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract infected by influenza causes has obvious inhibitory action.Note: compare with Normal group,
##p<0.01; Compare with virus-infected controls group,
*p<0.05,
*p<0.01.
Table 1 Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract is on the impact of mouse influenza virus pneumonia
3, Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract infected by influenza causes the protective effect of dead mouse
From table 2, after mouse infection influenza virus, virus-infected controls group 94.4% animal occurs dead, Average Survival 8.1d.And basic, normal, high dosage Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract obviously can suppress the dead mouse caused by influenza infection, and (Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract basic, normal, high dosage group mortality rate compares with virus-infected controls group; P<0.01 or 0.05); and can obviously extend mice Average Survival natural law (Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract basic, normal, high dosage group survival natural law compare with viral group; P<0.01 or 0.05), prompting Chinese medicine Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract infected by influenza causes dead mouse obvious protective effect.Note: compare with virus-infected controls group,
*p<0.05,
*p<0.01.
Table 2 Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract infected by influenza causes the protective effect of dead mouse
Embodiment 4: the preparation of tablet
By embodiment 1 method first obtained extract, add excipient, pelletizing press sheet in itself and excipient weight than the ratio for 1:10.
Embodiment 5: the preparation of oral liquid
By embodiment 1 method first obtained extract, oral liquid method for making makes oral liquid routinely.
Embodiment 6: the preparation of capsule or granule
By embodiment 1 method first obtained extract, add excipient in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 7: the preparation of injection
Obtain extract by embodiment 1 method, inject with water, fine straining, injection is made in embedding sterilizing.
Embodiment 8: the preparation of aseptic powder injection
By embodiment 1 method first obtained extract, be dissolved in sterile water for injection, stirring makes molten, filters with aseptic suction funnel, more aseptic fine straining, and be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains injectable powder.
Claims (6)
1. a Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract, is characterized in that described extract is prepared by following methods:
A (), by the Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) of drying 70% alcoholic solution circumfluence distillation, merge extractive liquid, is concentrated into and obtains ethanol extraction concentrated solution without alcohol taste; B (), by step (a) gained ethanol extraction concentrated solution dilute with water, is used petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, concentrating under reduced pressure, is obtained petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; C () n-butyl alcohol extract water dissolution, filters, uses macroporous resin enrichment active component, first remove large polar component with 15% alcohol flushing, 8 column volumes, then use 70% ethanol elution, 10 column volumes, collect 70% eluent, concentrating under reduced pressure, obtains Herba Lysimachiae Clethroids (Radix Seu Herba Lysimachiae Clethroidis) extract.
2. extract according to claim 1, is characterized in that: macroporous resin described in step (c) is AB-8 type macroporous resin.
3. extract according to claim 1, is characterized in that: the liquid phase analysis method of described extract is:
Chromatographic column: AgilentZorbaxSB-C18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.4% phosphoric acid solution;
Gradient elution program: 0.01 ~ 10min, A10% → 15%; 10 ~ 30min, A15% → 48%; 30 ~ 55min, A48% → 78%; 55 ~ 65min, A78% → 10%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 260nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
4. pharmaceutical preparation, is characterized in that: the extract according to claim 1 containing treatment effective dose and pharmaceutically acceptable carrier.
5. the application of extract according to claim 1 in the medicine preparing resisiting influenza virus.
6. the application of pharmaceutical preparation according to claim 4 in the medicine preparing resisiting influenza virus.
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| CN201510560415.9A CN105106254A (en) | 2015-09-05 | 2015-09-05 | Anti-influenza loosestrife extract |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773578A (en) * | 2016-08-28 | 2018-03-09 | 成都宝科生物科技有限公司 | A kind of common cnidium fruit P.E of resisiting influenza virus |
| CN110297047A (en) * | 2019-07-11 | 2019-10-01 | 江西省药品检验检测研究院 | The method for building up and its finger-print of the HPLC finger-print of Herba lysimachiae capillipedis medicinal material |
-
2015
- 2015-09-05 CN CN201510560415.9A patent/CN105106254A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773578A (en) * | 2016-08-28 | 2018-03-09 | 成都宝科生物科技有限公司 | A kind of common cnidium fruit P.E of resisiting influenza virus |
| CN110297047A (en) * | 2019-07-11 | 2019-10-01 | 江西省药品检验检测研究院 | The method for building up and its finger-print of the HPLC finger-print of Herba lysimachiae capillipedis medicinal material |
| CN110297047B (en) * | 2019-07-11 | 2021-09-21 | 江西省药品检验检测研究院 | Establishment method of HPLC fingerprint of lysimachia capillipes hemsl medicinal material and fingerprint thereof |
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Application publication date: 20151202 |