CN104807925A - Method for extracting and separating allelopathic autotoxicity substances from panax notoginseng rhizosphere soil - Google Patents
Method for extracting and separating allelopathic autotoxicity substances from panax notoginseng rhizosphere soil Download PDFInfo
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- CN104807925A CN104807925A CN201510136116.2A CN201510136116A CN104807925A CN 104807925 A CN104807925 A CN 104807925A CN 201510136116 A CN201510136116 A CN 201510136116A CN 104807925 A CN104807925 A CN 104807925A
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Abstract
The invention discloses a method for extracting and separating allelopathic autotoxicity substances from panax notoginseng rhizosphere soil. The method comprises steps as follows: soil samples are collected and treated, 20 g of the treated soil samples are weighed, extracted with methanol and subjected to ultrasonic processing, and the allelopathic autotoxicity substances are separated with an high performance liquid chromatography method; the chromatographic conditions are as follows: the chromatographic instrument adopts Agilent 1260, the chromatographic column adopts specifications of AgilentZorbax SB-C18, 250mm*4.6mm and 5 mu m, the mobile phase A is an H3PO4 aqueous solution with mass fraction of 0.1%, the mobile phase B is methanol, the flow speed is 1.0ml/min, the column temperature is 25 DEG C, the detecting wavelength is 203 nm, and gradient elution is performed. An experiment proves that the maximum varieties of the allelopathic autotoxicity substances are obtained through extraction and separation with the method; the method is simple and convenient to implement, and disturbance and loss caused by an extraction solvent to target compounds in samples as a result of a complicated extraction technology in the sample extraction process are reduced.
Description
Technical field
The invention belongs to the method in medicinal plant planting technology field, be specifically related to a kind ofly from pseudo-ginseng rhizosphere soil, extract the method for allelopathic from poisonous substance matter.
Technical background
Pseudo-ginseng [Panax notoginseng (Burk) F.H.Chen] is araliaceae ginseng plant, is used as medicine with root, root-like stock, has the effects such as promoting blood circulation and removing blood stasis, detumescence ding-tong.At present, the application of pseudo-ginseng is very extensive, and statistics shows only domesticly just have 1300 many enterprises to be raw material with pseudo-ginseng, and obtain 3207 and produce certifications, produce more than 400 product, wherein Chinese patent drug kind has exceeded 300, almost covers all herbal pharmaceutical enterprises.
Pseudo-ginseng originates from ancient tropical At Southwest Mountainous District in China in the tertiary period before 2,500 ten thousand, and distribution range is only confined to the tang near southwest China height above sea level 1500 ~ 1800m, 23.5 ° of N.Now do not find wild population, be cultigen.Wenshan Prefecture of Yunnan Province is medicinal material pseudo-ginseng original producton location and Genuine producing area, is the main producing region of current pseudo-ginseng, and its cultivated area and output all account for the whole nation more than 90%.Within 2013, pseudo-ginseng agriculture value reaches 13,000,000,000 yuan, and the market share is also in swift and violent expansion, and the demand of market to pseudo-ginseng is growing on and on.Therefore, pseudo-ginseng is the most successful kind of the current bio-resource exploitation in Yunnan Province.
The same with other Panax medicinal plants, it is extremely strong that notoginseng planting avoids ground property, there is serious Soil-sickness Problem.It is generally acknowledged that the plot of planting pseudo-ginseng needs to plant the crops such as corn, upland rice, clover continuously and again could plant pseudo-ginseng in more than 10 years.The driving of the market demand, continuous cropping obstacle causes the suitable soil in pseudo-ginseng tradition producing region nervous gradually without effective overcoming method temporarily in addition, and the continuous extension of growing area, the genuineness of production of crude drugs is difficult to ensure.
Now it is generally acknowledged, the continuous cropping obstacle origin cause of formation mainly contains: the change of (1) soil microbial community, and pathogenic microorganism increases, and beneficial microbe reduces, and soil-borne disease insect pest aggravates; (2) Auto toxicity that causes of allelochemical; (3) soil salinization and acid pickling increase the weight of and the change etc. (Chen Changbao, ginseng allelopathy and avoid continuous cropping Mechanism Study, Ph.D. Dissertation, Jilin Agriculture University, 2006,2 – 9) of the physicochemical character such as soil nutrient.Soil environment catastrophe or to cancerate be that pseudo-ginseng can not the main cause of continuous cropping, certainly may be also relevant with the adaptive faculty of pseudo-ginseng self.
But the driving factors of soil environment catastrophe derive from again pseudo-ginseng itself, namely the plantation of pseudo-ginseng changes soil environment, and the soil environment changed has surmounted adaptation and the adjustment capability of pseudo-ginseng itself, thus makes pseudo-ginseng can not continuous cropping.Therefore, the soil environmental background value studying notoginseng driving is the effective way of resolving continuous cropping obstacle Forming Mechanism.Current great majority are all find answer from soil environmental background value about the research of notoginseng formational cause of obstruction.And allelopathic is the core reasons causing soil catastrophe from poisonous substance matter, allelopathic from poisonous substance matter refer to certain plants by misty rain leaching, root system secretion and stubble degraded one class of release to same stubble or the more of the same race or equal plant of stubble produce a compounds of growth inhibition effect.Although these compound molecular weights, structure is simple, but is discharged into after in soil environment, not only can change the physicochemical property of plant rhizosphere soil, biological community structure in rhizosphere soil is impacted simultaneously, finally cause plant growth to be suppressed or directly cause its death.Therefore, explore method that allelopathic in a kind of fast and effectively extraction and analysis pseudo-ginseng rhizosphere soil form from poisonous substance matter to probe into notoginseng obstacle product because of, and alleviate and overcome notoginseng Effects and be of great importance.
Summary of the invention
The one that the object of this invention is to provide from poisonous substance matter, and carries out the method that is effectively separated from pseudo-ginseng rhizosphere soil extract allelopathic to its extract.
Main contents of the present invention comprise: the collection of pseudo-ginseng rhizosphere soil, the process of pedotheque, the extraction of pedotheque and the separation of allelochemical, and concrete technical scheme is as follows:
From pseudo-ginseng rhizosphere soil, extraction and isolation allelopathic is from a method for poisonous substance matter, it is characterized in that:
(1) collecting soil sample: carry out soil collecting by intersection five-spot in the plantation pseudo-ginseng soil of 3 years, get pseudo-ginseng plant rhizosphere soil sample;
(2) pedotheque process: the pedotheque that step (1) gathers is taken back indoor, air-dry, removal of impurities, crosses 1-2mm sieve, loads in valve bag, labelled, stand-by;
(3) take the pedotheque 20g processed through step (2), be placed in 200ml methyl alcohol, described methyl alcohol is pure for analyzing, ultrasonic process 30min; With Filter paper filtering, collect filtrate, collected filtrate is extracts the allelopathic that obtains from poisonous substance matter extract; In 4 DEG C of preservations, to be measured;
(4) filtrate that step (3) is preserved is separated by high performance liquid chromatography, chromatographic condition is: chromatograph is Agilent 1260, chromatographic column be Agilent Zorbax SB-C18,250mm × 4.6mm, 5 μm, mobile phase A to be massfraction be 0.1% H
3pO
4aqueous solution, Mobile phase B is methyl alcohol, described methyl alcohol is chromatographically pure, flow velocity 1.0ml/min, column temperature 25 DEG C, determined wavelength 203nm, and carry out gradient elution by being described in table 1 below Parameters of gradient elution, namely the material that the single chromatographic peak that separation obtains is corresponding represents the individualized compound with allelopathic autotoxicity
Table 1 Parameters of gradient elution table
| Time (min) | Mobile phase A solution v/v (%) | Mobile phase B solution v/v (%) |
| 0 | 90 | 10 |
| 20 | 80 | 20 |
| 65 | 60 | 40 |
| 70 | 40 | 60 |
| 75 | 40 | 60 |
。
The invention has the beneficial effects as follows:
Prove through experiment, the extract (liquid) adopting the present invention to extract with pure methyl alcohol and the technical scheme of separation parameter, be separated the allelopathic of acquisition from poisonous substance matter most species, and this method is simple, decrease Extraction solvent that extraction process complicated in sample extraction process is caused to the interference of target compound in sample and loss.And relative to other feasibility organic extraction solvent, pure methyl alcohol toxicity is minimum, extraction efficiency is the strongest, and applicability compound is the widest, is best Extraction solvent.
Accompanying drawing explanation
Fig. 1 is soil extract thing HPLC (high performance liquid chromatography) chromatogram that the pure methyl alcohol of the present invention extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the milli absorbance (mAu) of separating obtained material.
Fig. 2 is soil extract thing HPLC (high performance liquid chromatography) chromatogram that 80% methyl alcohol extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Fig. 3 is soil extract thing HPLC (high performance liquid chromatography) chromatogram that 70% methyl alcohol extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Fig. 4 is holard extract HPLC (high performance liquid chromatography) chromatogram that water extraction extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Fig. 5 is soil extract thing HPLC (high performance liquid chromatography) chromatogram of acetone extraction, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Fig. 6 is soil extract thing HPLC (high performance liquid chromatography) chromatogram that alkaline extraction extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Fig. 7 is soil extract thing HPLC (high performance liquid chromatography) chromatogram that ethyl acetate extracts, and in figure, horizontal ordinate is sample retention time (min), and ordinate is the absorbance log (mAu) of separating obtained material.
Embodiment
Embodiment 1 methyl alcohol extracts
(1) collecting soil sample: carry out soil collecting by intersection five-spot in the plantation pseudo-ginseng soil of 3 years, get pseudo-ginseng plant rhizosphere 0-30cm pedotheque.
(2) sample preparation: the pedotheque that step (1) gathers is taken back indoor, air-dry, removal of impurities, cross 1-2mm sieve, be divided into 7 deciles, be distributed in 7 valve bags, labelled, be labeled as sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, sample 7 respectively, sample 1 extracts as follows, and all the other 6 samples extract by embodiment 2, embodiment 3, embodiment 4, embodiment 5, embodiment 6, embodiment 7 mode respectively.
(3) methanol extract liquid preparation method: the pedotheque 1 processed through step (2) is taken 20g with electronic balance, be placed in 200ml methyl alcohol (analyzing pure), ultrasonic process 30min, filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 2 (contrast 1-80% methyl alcohol extracts)
80% methanol extract liquid preparation method: configuration volume fraction is the methanol solution of 80%, and by sample 2 described in embodiment 1, take 20g with electronic balance, being placed in 200ml volume fraction is 80% methyl alcohol, ultrasonic process 30min.Filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, and it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, and 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 3 (contrast 2-70% methyl alcohol extracts)
70% methanol extract liquid preparation method: configuration volume fraction is the methanol solution of 70%, and by sample 3 described in embodiment 1, take 20g with electronic balance, being placed in 200ml volume fraction is 70% methyl alcohol, ultrasonic process 30min.Filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, and it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, and 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 4 (contrast 3-acetone extraction)
Acetone extraction liquid and preparation method thereof: by sample 4 described in embodiment 1, take 20g with electronic balance, is placed in 200ml acetone (analyzing pure) solution, ultrasonic process 30min.Filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, and it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, and 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 5 (contrast 4-ethyl acetate extracts)
Acetic acid ethyl acetate extract preparation method: by sample 5 described in embodiment 1, take 20g with electronic balance, is placed in 200ml ethyl acetate (analyzing pure) solution, ultrasonic process 30min.Filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, and it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, and 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 6 (contrast 5-alkaline extraction)
NaOH extract preparation method: by sample 6 described in embodiment 1,20g is taken with electronic balance, being placed in 200ml massfraction is 80%NaOH solution, ultrasonic process 30min, filter with Medium speed filter paper, collect filtrate, the filtrate of collecting is extracts the allelopathic that obtains from poisonous substance matter extract, filtrate hydrochloric acid regulates its pH to 2.5, with ethyl acetate (analyzing pure) extraction five times, collects extract, it is 5ml that extract Rotary Evaporators carries out being concentrated into filtrate, 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 7 (contrast 6-water extraction)
Aqueous extracts preparation method: by sample 7 described in embodiment 1, take 20g with electronic balance, be placed in 200ml pure water, ultrasonic process 30min.Filter with Medium speed filter paper, collect filtrate, the filtrate of collection is the allelopathic extracting and obtain from poisonous substance matter extract, and it is 5ml that filtrate Rotary Evaporators carries out being concentrated into filtrate, and 4 DEG C of preservations, thickening temperature is 50 DEG C, to be measured.
Embodiment 8 high performance liquid chromatography is separated above-mentioned soil extract thing (liquid)
Extract in above embodiment 1-embodiment 7 after concentration is all respectively separated by following parameter HPLC (high performance liquid chromatography), chromatograph used is Agilent 1260, chromatographic column is Agilent ZorbaxSB-C18,250mm × 4.6mm, 5 μm, adopt gradient elution, flow velocity is 1.0ml/min, and column temperature is 25 DEG C, determined wavelength 203nm, mobile phase A to be massfraction be 0.1% H
3pO
4aqueous solution, Mobile phase B is methyl alcohol, and described methyl alcohol is chromatographically pure, and carries out gradient elution by being described in table 1 below Parameters of gradient elution, and namely the material that the single chromatographic peak that separation obtains is corresponding represents the individualized compound with allelopathic autotoxicity,
Table 1 Parameters of gradient elution table
| Time (min) | Mobile phase A solution v/v (%) | Mobile phase B solution v/v (%) |
| 0 | 90 | 10 |
| 20 | 80 | 20 |
| 65 | 60 | 40 |
| 70 | 40 | 60 |
| 75 | 40 | 60 |
In table 1, v/v (%) represents volume fraction.
As can be seen from Figure 1, by carrying out HPLC separation to the sample of embodiment 1, obtaining 14 compounds altogether, identifying through external standard method, analyze and draw 7 compounds, be respectively catechol, neck benzenediol, cinnamic acid, Panax Notoginseng saponin R
1, syringic acid, vanillic aldehyde and p-Coumaric Acid, these compounds can cause enzymatic activity in plant rhizosphere soil to change, and provide energy substance for growing of pathogenic microorganism.Its retention time is as following table 2:
The separating obtained compound of soil extract thing that table 2 embodiment 1 methyl alcohol extracts and retention time thereof
| Compound | Retention time RT (min) |
| Catechol | 11.147 |
| Neck benzenediol | 14.943 |
| Cinnamic acid | 18.933 |
| Panax Notoginseng saponin R 1 | 24.231 |
| Syringic acid | 29.312 |
| Vanillic aldehyde | 34.088 |
| P-Coumaric Acid | 35.682 |
And identical pedotheque, the extract of acquisition is got respectively by 80% methyl alcohol extraction described in embodiment 2, embodiment 3, embodiment 4, embodiment 5, embodiment 6, embodiment 7,70% methyl alcohol extraction, acetone extraction, ethyl acetate extraction, 80%NaOH extract, water extraction, be separated by same HPLC method, the chromatographic peak (namely representing individualized compound) that its extraction and isolation obtains is respectively 7,8,7,10,9 and 11, the amount of material that the methanol extraction being all less than the present invention's employing extracts, namely 14.Refer to Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7.
Claims (1)
1. from pseudo-ginseng rhizosphere soil, extraction and isolation allelopathic, from a method for poisonous substance matter, is characterized in that:
(1) collecting soil sample: carry out soil collecting by intersection five-spot in the plantation pseudo-ginseng soil of 3 years, get pseudo-ginseng plant rhizosphere soil sample;
(2) pedotheque process: the pedotheque that step (1) gathers is taken back indoor, air-dry, removal of impurities, crosses 1-2mm sieve, loads in valve bag, labelled, stand-by;
(3) take the pedotheque 20g processed through step (2), be placed in 200ml methyl alcohol, described methyl alcohol is pure for analyzing, ultrasonic process 30min; With Filter paper filtering, collect filtrate, collected filtrate is extracts the allelopathic that obtains from poisonous substance matter extract, in 4 DEG C of preservations, to be measured;
(4) filtrate that step (3) is preserved is separated by high performance liquid chromatography, chromatographic condition is: chromatograph is Agilent 1260, chromatographic column be Agilent Zorbax SB-C18,250mm × 4.6mm, 5 μm, mobile phase A to be massfraction be 0.1% H
3pO
4aqueous solution, Mobile phase B is methyl alcohol, described methyl alcohol is chromatographically pure, flow velocity 1.0ml/min, column temperature 25 DEG C, determined wavelength 203nm, and carry out gradient elution by being described in table 1 below Parameters of gradient elution, namely the material that the single chromatographic peak that separation obtains is corresponding represents the individualized compound with allelopathic autotoxicity
Table 1 Parameters of gradient elution table
。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108072750A (en) * | 2017-12-07 | 2018-05-25 | 甘肃农业大学 | A kind of alfalfa-Wheat/Maize wheel makees the test method to Influence To Soil |
| CN109781872A (en) * | 2018-12-25 | 2019-05-21 | 湖北省烟草科学研究院 | The extracting method and detection method of allelochemical in tobacco-growing soil |
-
2015
- 2015-03-26 CN CN201510136116.2A patent/CN104807925A/en active Pending
Non-Patent Citations (3)
| Title |
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| MIN YANG，ET AL: "Autotoxic Ginsenosides in the Rhizosphere Contribute to the Replant Failure of Panax notoginseng", 《PLOS ONE》 * |
| 吴立洁等: "三七根际土壤中酚酸类物质的鉴定及含量测定", 《世界科学技术-中医药现代化》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108072750A (en) * | 2017-12-07 | 2018-05-25 | 甘肃农业大学 | A kind of alfalfa-Wheat/Maize wheel makees the test method to Influence To Soil |
| CN109781872A (en) * | 2018-12-25 | 2019-05-21 | 湖北省烟草科学研究院 | The extracting method and detection method of allelochemical in tobacco-growing soil |
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