CN112903876A - Method for extracting root soil and root allelochemical substances of stellera chamaejasme and detecting content of root allelochemical substances - Google Patents
Method for extracting root soil and root allelochemical substances of stellera chamaejasme and detecting content of root allelochemical substances Download PDFInfo
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Abstract
The invention relates to a method for extracting root soil and root allelochemicals of stellera chamaejasme, which comprises the following steps: adding a methanol solution into a stellera chamaejasme rhizosphere soil sample, and carrying out ultrasonic extraction for three times; filtering to remove impurities, and rotary steaming to obtain allelochemical crude extract B; weighing 50g of roots of stellera chamaejasme plants, crushing, adding methanol, performing ultrasonic extraction for three times, and performing rotary steaming to obtain a allelochemicals crude extract F; thirdly, respectively weighing 5-10 mg of the crude allelochemicals B and F, respectively adding chromatographic grade methanol solutions to prepare 2mg/mL solutions, and removing impurities through an organic filter membrane to respectively obtain a solution C to be detected and a solution D to be detected. Meanwhile, the invention also discloses a method for detecting the contents of the root soil and root allelochemicals of the stellera chamaejasme. The method has the advantages of high extraction efficiency of the root soil and root allelochemical substances of the stellera chamaejasme, simple operation, quick, sensitive and accurate quantitative detection and the like.
Description
Technical Field
The invention relates to the technical field of plant allelochemical substance analysis, in particular to a method for extracting and detecting the content of the allelochemical substances in rhizosphere soil and roots of stellera chamaejasme.
Background
Stellera chamaejasme is a perennial herb of stellera of the family of stellera chamaejasme, and is widely distributed in provinces of Gansu, Xinjiang, Ningxia and the like in China. In recent years, stellera chamaejasme has become one of the marked plants for the deterioration of Chinese grassland due to strong ecological adaptability and high competitiveness. Research shows that the stellera chamaejasme can change the rhizosphere microbial community structure and the soil enzyme activity through root exudates and can also generate allelochemicals for inhibiting the growth of other plants, thereby creating a soil ecosystem beneficial to self survival and multiplication. Therefore, the method for accurately measuring the root soil and allelochemicals in the roots of the stellera chamaejasme has important significance for controlling the invasion of the stellera chamaejasme grassland and treating the grassland degeneration problem caused by the stellera chamaejasme.
Nowadays, research on stellera chamaejasme mostly focuses on pharmacological activity, chemical substances, ecology and the like, and related research and reports are few at present regarding extraction and detection methods of allelochemicals in stellera chamaejasme rhizosphere soil and roots. For the reports of the stellera chamaejasme allelochemicals, more experiments focusing on the extraction and activity determination of single allelochemicals are carried out, and the reports of carrying out comprehensive detection on the content of the allelochemicals in rhizosphere soil in different growth periods and different coverage degrees and carrying out content determination on the content of the stellera chamaejasme root allelochemicals in different growth periods and different coverage degrees are not provided.
Therefore, in order to effectively control the invasion of the stellera chamaejasme, a new method capable of comprehensively detecting and reasonably and quantitatively analyzing allelochemicals in the stellera chamaejasme rhizosphere soil and roots is urgently needed to be established.
Disclosure of Invention
The invention aims to solve the technical problem of providing the method for extracting the root soil and root allelochemical substances of the stellera chamaejasme with high efficiency and simple operation.
The invention aims to solve another technical problem of providing a method for quantitatively detecting the contents of root soil and root allelochemicals of stellera chamaejasme.
In order to solve the problems, the method for extracting the root soil and root allelochemicals of the stellera chamaejasme comprises the following steps:
adding not less than 300mL of methanol solution into a stellera chamaejasme rhizosphere soil sample according to every 100g of soil sample, carrying out ultrasonic extraction for not less than 30min, and repeating for three times; after suction filtration and impurity removal, a crude extraction solution A is obtained; removing methanol from the crude extract solution A by using a rotary evaporator to obtain a allelochemical crude extract B;
weighing 50g of roots of stellera chamaejasme plants, crushing the roots by a powder sample machine, adding 150mL of methanol, ultrasonically extracting for 30min, and repeating for three times to obtain a crude extraction solution E; removing methanol from the crude extract solution E by using a rotary evaporator to obtain a allelochemical crude extract F;
thirdly, respectively weighing 5-10 mg of the crude allelochemicals B and F, respectively adding chromatographic grade methanol solutions to prepare 2mg/mL solutions, and removing impurities through a 0.22-micrometer organic filter membrane to respectively obtain a solution C to be detected and a solution D to be detected.
The crude allelochemicals B and F comprise the following 7 allelochemicals: umbelliferone, chamaejasmone, daphnoretin, isostellarine A, neostellarine B, dihydrodaphnoretin B, and 7-methoxyneostellarine A.
The structural formula of each allelochemicals is as follows:
the method for detecting the content of the root soil and root allelochemicals of the stellera chamaejasme, which is obtained by the method, is characterized by comprising the following steps: firstly, respectively establishing standard curves of seven stellera chamaejasme allelochemicals including umbelliferone, stellera chamaejasme chromone, daphnetin, isoneochamaejasmine A, neochamaejasmine B, dihydrodaphnetin B and 7-methoxy neochamaejasmine A by adopting High Performance Liquid Chromatography (HPLC); respectively detecting the solution C to be detected and the solution D to be detected by adopting High Performance Liquid Chromatography (HPLC), and determining the retention time of allelochemicals by an external standard method; and finally, converting the content of the allelochemicals by comparing the peak surface of a standard curve of the allelochemicals.
The chromatographic conditions for the High Performance Liquid Chromatography (HPLC) were as follows: a chromatographic column: a Symmetry μ C18 reversed phase column with particle size of 250 mm × 4.6 mm and 5 μm; flow rate of mobile phase: 1 mL/min; mobile phase: phase A is acetonitrile, phase B is water containing 0.2% acetic acid; sample introduction amount: 20 mu L of the solution; elution gradient: 0-6 min: 20% -30% of A; 6-8 min: 30% -35% of A; 8-15 min: 35-45% of A; 15-18 min: 45-60% of A; 18-20 min: 60-80% of A; 20-25 min: 80% of A.
Compared with the prior art, the invention has the following advantages:
1. the invention can realize the full extraction of 7 allelochemicals by carrying out ultrasonic extraction on the stellera chamaejasme rhizosphere soil and allelochemicals in roots by methanol and repeating the ultrasonic extraction for three times, and has simple operation and high extraction efficiency.
2. The invention adopts the high performance liquid chromatography to effectively separate the A regions of stellera chamaejasme chromone, daphnetin and isoneochamaejasmine with similar structures and consistent molecular formulas in seven allelochemicals of stellera chamaejasme, so that the seven allelochemicals have independent peaks, and the problems of difficult peak appearance and nonstandard peak types of the allelochemicals are effectively solved.
3. The method can accurately measure five allelochemicals in rhizosphere soil and seven allelochemicals in roots (the contents of umbelliform lactone and daphnoretin in roots are less), and has the advantages of high detection speed, high sensitivity, good selectivity and high accuracy.
4. The method can detect and quantify the allelochemicals with full types and accurate content, has important significance for effectively controlling the invasion of the stellera chamaejasme, and can be widely applied and popularized.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a chromatogram of seven allelochemicals of stellera chamaejasme L. Wherein: peak 1 is umbelliferone; peak 2 is stellera chamaejasme chromone; peak 3 is daphnetin; peak 4 is isostellerin a; peak 5 is neochamaejasmine B; peak 6 is dihydrodaphnetin B; peak 7 is 7-methoxy neochamaejasmine A.
FIG. 2 is a graph showing the content of chromone in the rhizosphere soil of stellera chamaejasme with different growth periods and different coverage degrees.
FIG. 3 is a graph showing the content of chamaejasmine A in the rhizosphere soil of stellera chamaejasme with different growth periods and different coverage degrees.
FIG. 4 shows the content of neochamaejasmine B in the rhizosphere soil of stellera chamaejasme with different growth periods and different coverage degrees.
FIG. 5 shows the content of dihydrodaphnetin B in the rhizosphere soil of stellera chamaejasme at different growth stages and different coverage degrees.
FIG. 6 shows the content of 7-methoxyneochamaejasmine A in the rhizosphere soil of stellera chamaejasme at different growth stages and different coverage degrees.
FIG. 7 is a graph showing the lipid content of the umbrella-shaped flower in stellera chamaejasme root with different coverage of 50g at different growth stages.
FIG. 8 is a graph showing the content of chamaejasme chromone in stellera chamaejasme root with different coverage of 50g at different growth stages.
FIG. 9 is a graph showing the content of daphnetin in daphne chamaejasme root of 50g coverage at different growth stages.
FIG. 10 is a graph showing the content of stellerin A in stellera chamaejasme roots of different growth periods with a coverage of 50 g.
FIG. 11 is a graph showing the amount of neochamaejasmine B in stellera chamaejasme roots of different growth stages with a coverage of 50 g.
FIG. 12 is a graph showing the content of dihydrodaphnetin B in stellera chamaejasme root of different coverage of 50g at different growth stages. Wherein: LE is the leaf-spreading period of stellera chamaejasme, FB is the blooming period, SM is the maturing period, and DP is the resting period; 0% is the growth coverage of stellera chamaejasme 0, and 25%, 52% and 89% are respectively the growth coverage of stellera chamaejasme 25%, 52% and 89%.
FIG. 13 is a graph showing the content of 7-methoxyneochamaejasmine A in stellera chamaejasme roots of different growth stages with a coverage of 50 g. Wherein: LE is the leaf-spreading period of stellera chamaejasme, FB is the blooming period, SM is the maturing period, and DP is the resting period; 0% is the growth coverage of stellera chamaejasme 0, and 25%, 52% and 89% are respectively the growth coverage of stellera chamaejasme 25%, 52% and 89%.
Detailed Description
The method for extracting root soil and root allelochemicals of stellera chamaejasme comprises the following steps:
adding not less than 300mL of methanol solution into a stellera chamaejasme rhizosphere soil sample according to every 100g of soil sample, carrying out ultrasonic extraction for not less than 30min, and repeating for three times; after suction filtration and impurity removal, a crude extraction solution A is obtained; removing methanol from the crude extract solution A by using a rotary evaporator to obtain a allelochemical crude extract B;
weighing 50g of roots of stellera chamaejasme plants, crushing the roots by a powder sample machine, adding 150mL of methanol, ultrasonically extracting for 30min, and repeating for three times to obtain a crude extraction solution E; removing methanol from the crude extract E by using a rotary evaporator to obtain a allelochemical crude extract F;
thirdly, respectively weighing 5-10 mg of crude allelochemicals B and F, respectively adding chromatographic grade methanol solution to prepare 2mg/mL solution, and removing impurities through a 0.22 mu m organic filter membrane to respectively obtain solution C to be detected and solution D to be detected.
Wherein: the crude allelochemicals B and F comprise the following 7 allelochemicals: umbelliferone, chamaejasmone, daphnoretin, isostellarine A, neostellarine B, dihydrodaphnoretin B, and 7-methoxyneostellarine A.
The content detection method of stellera chamaejasme rhizosphere soil and root allelochemicals comprises the following steps:
firstly, High Performance Liquid Chromatography (HPLC) is adopted to respectively establish a standard curve of seven stellera chamaejasme allelochemicals including umbelliferone, stellera chamaejasme, isostellera chamaejasme A, stellera chamaejasme B, dihydrostellera chamaejasme B and 7-methoxy chamaejasme A. Respectively detecting the solution C to be detected and the solution D to be detected which are obtained by the extraction method by adopting High Performance Liquid Chromatography (HPLC), and determining the retention time of allelochemicals by an external standard method; and finally, converting the content of the allelochemicals by comparing the peak surface of a standard curve of the allelochemicals. The specific process is as follows:
screening high performance liquid chromatography conditions:
preparing seven allelochemicals of stellera chamaejasme standard substances into 1mg/mL mother liquor respectively, diluting the mother liquor into 100 mu g/mL solution, mixing the mother liquor with equal amount respectively, and performing condition groping.
Firstly, methanol and water are used as mobile phases of liquid chromatography, but the peak-to-peak type is not standard, and the designed flow gradient cannot generate an independent peak, so that the peak-to-peak type has larger fluctuation; and the addition of 0.2% acetic acid in the aqueous phase did not change the peak problem either.
Changing the mobile phase into acetonitrile and a water phase, and setting the gradient to be 0-6 min and 20-30% of B; 6-8 min, 30-35% of B; 8-15 min, 35-50% of B; 15-18 min, 50-60% B; for 18-20 min, 60-80% of B; 20-25 min, 80% B; at this time, seven separate peaks are also not present, daphnetin and allochamaejasmine a are still overlapped, but the peak patterns are more standard; changing the elution gradient into 0-6 min: 20% -30% of A; 6-8 min: 30% -35% of A; 8-15 min: 35-45% of A; 15-18 min: 45-60% of A; 18-20 min: 60-80% of A; 20-25 min: 80% of A; daphnetin and isostellerin A still partially overlap.
The seven allelochemicals can generate independent and standard peak patterns by adding 0.2 percent of acetic acid into the water phase.
② determining the chromatographic conditions of High Performance Liquid Chromatography (HPLC) as follows: a chromatographic column: a Symmetry μ C18 reversed phase column with particle size of 250 mm × 4.6 mm and 5 μm; flow rate of mobile phase: 1 mL/min; mobile phase: phase A is acetonitrile, phase B is water containing 0.2% acetic acid; sample introduction amount: 20 mu L of the solution; elution gradient: 0-6 min: 20% -30% of A; 6-8 min: 30% -35% of A; 8-15 min: 35-45% of A; 15-18 min: 45-60% of A; 18-20 min: 60-80% of A; 20-25 min: 80% of A.
Establishing seven allelochemical standard curves y = ax + b; wherein: x is the concentration of the sample to be measured and the unit is mu g/mL; y is a peak area detected by a high performance liquid chromatography apparatus, and is shown in Table 1.
Under the selected conditions, the standard curve R of seven allelochemicals of stellera chamaejasme2All reach above 0.999. The condition can better detect and quantify the stellera chamaejasme allelochemicals.
TABLE 1 Standard Curve of the allelopathic substances of stellera chamaejasme
And fourthly, respectively detecting the solution C to be detected and the solution D to be detected which are obtained by the extraction method by adopting High Performance Liquid Chromatography (HPLC), and determining the retention time of the allelochemicals by an external standard method.
Namely: in the standard curve making process, the peak time of each allelochemicals is determined by repeatedly measuring the peak time of the allelochemicals with the same concentration and different concentrations. And after the solution C to be detected and the solution D to be detected are measured by the high performance liquid chromatograph, comparing the peak appearance time of the obtained spectrum with that of a standard product, and determining the retention time of allelochemicals in the sample. The peak appearance and retention time of each allelochemicals are shown in FIG. 1.
Converting the allelochemicals content by comparing the peak surface of the standard curve of the allelochemicals according to the following formula:
mass M = v.a (y-b)/(a.A) of allelochemicals in crude extract1），A1=V•wIn the formula: v is the volume of each sample injection and the unit is mu L; a is the total mass of the crude extract, unit g; a. the1The unit mg is the consumed sample mass of each sample introduction;wthe unit is mg/mL for the concentration of the sample.
The content of lipid in the umbrella-shaped flower extracted from the root is taken as an example for explanation:
total crude extract mass of root a =17.0916 g; volume of each injection sample V =20 μ L, injection concentrationw=2mg/mL, the sample mass consumed A per injection1=V•w=0.02 mL×2mg/mL =0.04mg。
The standard curve for umbelliferone is: y =49295x +28694 (i.e., y = ax + b); y is peak area, x is the concentration of the sample to be measured, and the unit is mu g/mL; a for 49295 and b for 28694.
Measuring the area y =46876 of the umbelliferone lactone peak by a high performance liquid chromatograph; then the measured sample concentration x = (46876-28694)/49295 =0.3688 μ g/mL, i.e., x = (y-b)/a.
Then the mass M of the medium umbrella-shaped flower lactone is injected into the sample of 20 mu L1= v.x =20 μ L × 0.3688 μ g/mL =0.0074 μ g; i.e., 0.02 mL. times.x.
Since the total crude extract mass measured is a =17.0916g, then there is an umbelliferone mass M = a. M1/ A1=17.0916 g÷0.04 mg×0.0074 μg=3.1619 mg。
Thus, the mass of umbelliferone in the total crude extract, M = a (y-b)/2 a.
Example 1 analysis and comparison of the content of allelopathic substances in the rhizosphere soil of stellera chamaejasme at different growth stages and different coverage degrees:
and respectively measuring the contents of allelochemicals in rhizosphere soil in the leaf expanding period, the flowering period, the maturing period and the dormancy period of the stellera chamaejasme, wherein the detected allelochemicals are shown in figures 2-6. As can be seen from the figure, the content of allelochemicals in the root soil of the stellera chamaejasme approximately shows an increasing trend along with the increase of the growth coverage; the content of allelochemicals in the leaf expanding period has the largest change trend; in the full-bloom stage, the highest allelochemicals content is 52% coverage. The allelochemicals in the seed setting period and the dormancy period are less. Therefore, for the prevention and treatment of stellera chamaejasme, the stellera chamaejasme should be in the leaf-spreading period or the blooming period.
Example 2 analysis and comparison of allelopathic substance content of roots of stellera chamaejasme at different growth stages and different coverage degrees:
the allelochemical content of the root samples of the stellera chamaejasme plants with different growth periods and different coverage degrees is determined. Shearing 50g of root samples of the stellera chamaejasme plants after drying in the shade, extracting according to the extraction method, and detecting the content by a high performance liquid chromatography method. The allelochemicals detected are shown in Table 2 and FIGS. 7 to 13. As shown in Table 2 and FIGS. 7 to 13, it is now confirmed that seven allelochemicals belonging to stellera chamaejasme were all detected in the root thereof. The contents of the seven allelochemicals are higher in the leaf expanding period and the full-bloom period, and the contents of the seven allelochemicals are obviously reduced in the fructification period and the dormancy period. The seven allelochemicals have high content of chromone and neochamaejasmine B, and low content of umbelliferone, daphnetin and dihydrodaphnetin B. The content of allelochemicals in the roots affects how much they are secreted into the rhizosphere soil. The content determination of allelochemicals in the stellera chamaejasme roots is beneficial to the research on allelochemicals effect in the process of the stellera chamaejasme roots invading grasslands, thereby finding a more effective stellera chamaejasme prevention and treatment method.
TABLE 2 different growth periods and different Gao degree contents (mg) of allelochemicals in stellera chamaejasme root
Claims (4)
1. The method for extracting root soil and root allelochemicals of stellera chamaejasme comprises the following steps:
adding not less than 300mL of methanol solution into a stellera chamaejasme rhizosphere soil sample according to every 100g of soil sample, carrying out ultrasonic extraction for not less than 30min, and repeating for three times; after suction filtration and impurity removal, a crude extraction solution A is obtained; removing methanol from the crude extract solution A by using a rotary evaporator to obtain a allelochemical crude extract B;
weighing 50g of roots of stellera chamaejasme plants, crushing the roots by a powder sample machine, adding 150mL of methanol, ultrasonically extracting for 30min, and repeating for three times to obtain a crude extraction solution E; removing methanol from the crude extract solution E by using a rotary evaporator to obtain a allelochemical crude extract F;
thirdly, respectively weighing 5-10 mg of the crude allelochemicals B and F, respectively adding chromatographic grade methanol solutions to prepare 2mg/mL solutions, and removing impurities through a 0.22-micrometer organic filter membrane to respectively obtain a solution C to be detected and a solution D to be detected.
2. The method for extracting root soil and root allelochemicals of stellera chamaejasme as claimed in claim 1, wherein the extraction method comprises the following steps: the crude allelochemicals B and F comprise the following 7 allelochemicals: umbelliferone, stellera chamaejasme chromone, daphnetin, isostellera chamaejasme A, neostellera chamaejasme B, dihydrodaphnetin B, and 7-methoxy neostellera chamaejasme A.
3. The method for detecting the content of allelochemicals in the rhizosphere soil and roots of stellera chamaejasme as claimed in claim 1, wherein the method comprises the following steps: firstly, respectively establishing standard curves of seven stellera chamaejasme allelochemicals, including umbelliferone, stellera chamaejasme chromone, daphnetin, isoneochamaejasmine A, neochamaejasmine B, dihydrodaphnetin B and 7-methoxy neochamaejasmine A by adopting a high performance liquid chromatography; respectively detecting the solution C to be detected and the solution D to be detected by adopting High Performance Liquid Chromatography (HPLC), and determining the retention time of allelochemicals by an external standard method; and finally, converting the content of the allelochemicals by comparing the peak surface of a standard curve of the allelochemicals.
4. The method for detecting the content of allelochemicals in the rhizosphere soil and roots of stellera chamaejasme as claimed in claim 3, wherein the method comprises the following steps: the chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column: a Symmetry μ C18 reversed phase column with particle size of 250 mm × 4.6 mm and 5 μm; flow rate of mobile phase: 1 mL/min; mobile phase: phase A is acetonitrile, phase B is water containing 0.2% acetic acid; sample introduction amount: 20 mu L of the solution; elution gradient: 0-6 min: 20% -30% of A; 6-8 min: 30% -35% of A; 8-15 min: 35-45% of A; 15-18 min: 45-60% of A; 18-20 min: 60-80% of A; 20-25 min: 80% of A.
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| 武雪 等: "HPLC 同时测定瑞香狼毒中瑞香素、伞形花内酯和东莨菪内酯的含量", 《中国现代应用药学》 * |
| 陈俊 等: "高效液相色谱- 二极管阵列检测器法测定瑞香狼毒5种黄酮类成分的含量", 《第二军医大学学报》 * |
| 魏丽 等: "瑞香狼毒药材中伞形花内酯和狼毒色原酮含量的高效液相色谱法测定", 《时珍国医国药》 * |
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