CN110632162A - A method for identifying local wild Astragalus membranaceus and cultivated Astragalus membranaceus - Google Patents

Abstract

The invention discloses a method for identifying local wild astragalus and cultivated astragalus. Microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS) is used to analyze wild and cultivated astragalus in Shanxi to obtain the content of barium and phosphorus elements, and through binary logistic regression , to get the discriminant formula and discriminant probability P of wild Astragalus membranaceus; under the same conditions, conduct elemental analysis and attribute discrimination on Shanxi Astragalus membranaceus whose planting method is unknown, and determine its attribute as wild or cultivated. The invention is accurate, reliable, simple and feasible; it is especially suitable for the identification of the planting mode of the astragalus in Shanxi.

Description

A method for identifying local wild Astragalus membranaceus and cultivated Astragalus membranaceus

technical field

The invention relates to a method for identifying local wild astragalus and cultivated astragalus, belonging to the field of analytical chemistry.

Background technique

Astragalus is the dried root of Astragalus membranaceus, a leguminous plant. Shanxi is the authentic medicinal material production area of Astragalus membranaceus. In the pharmacological research of Astragalus, the collection of authentic Astragalus is the first and extremely important step. Wild Astragalus membranaceus grows purely naturally and has high efficacy, which has an irreplaceable effect. On the other hand, wild Astragalus membranaceus has few sources and is precious, so the price is high. In order to expand the output, some artificial cultivation methods, such as hillside cultivation and artificial sowing (semi-wild) cultivation, came into being. In view of the large difference in the efficacy of wild Astragalus and cultivated Astragalus, it is a problem worthy of attention to identify wild Astragalus and cultivated Astragalus.

It is difficult to distinguish wild Astragalus membranaceus from cultivated (semi-wild) Astragalus membranaceus in appearance. It is difficult to distinguish whether Astragalus membranaceus is a wild variety only by the appearance of samples and the thickness of roots.

Contents of the invention

The present invention aims to provide a method for identifying local wild Astragalus membranaceus and cultivated Astragalus membranaceus. Combining microwave digestion with ICP-MS analysis method, it is proposed for the first time to use the elements in Astragalus membranaceus as indicators to distinguish the wild and cultivated (semi-wild) attributes of Astragalus membranaceus in Shanxi. Provide a basis for the quality identification of traditional Chinese medicine Astragalus.

The present invention utilizes the ratio of the content of barium and phosphorus in the main root slices of Astragalus membranaceus to discriminate the wild or cultivated attributes of Astragalus membranaceus in Shanxi. Meta-logistic regression analysis was used to discriminate the planting methods of local Astragalus membranaceus.

The invention provides a method for identifying local wild Astragalus membranaceus and cultivated Astragalus membranaceus. Microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS) is used to analyze wild and cultivated Astragalus membranaceus in Shanxi to obtain the content of barium and phosphorus elements. Logistic regression was used to obtain the discriminant formula of wild Astragalus membranaceus and its discriminant probability P (Probability). Under the same conditions, the elemental analysis and attribute discrimination of Shanxi Astragalus membranaceus with unknown planting methods were carried out to determine its wild or cultivated attributes.

The specific steps are:

(1) Collect sliced samples of wild and cultivated Astragalus root slices from Shanxi Province, grind them into powder with a pulverizer, and pass through a 60-mesh sieve;

(2) Weigh 150 mg (accurate to 0.001 g) of astragalus sample/astragalus standard substance sample powder into a polytetrafluoroethylene digestion tube, add 6 mL of concentrated nitric acid, shake gently, mix well and seal it, and place it in a microwave digestion apparatus for Digestion; after completion, wait for the digestion liquid to cool below 70°C, transfer it to a 100 ml volumetric flask, wash the digestion tank with a small amount of ultrapure water for 3 times, combine the washing liquid into the volumetric flask, and dilute to volume with ultrapure water to 100 mL; conduct a blank experiment, that is, no sample is added during the above experiment, and the rest of the operations are the same as above;

The Astragalus sample refers to the collected unknown Astragalus sample to be analyzed; the Astragalus standard substance sample refers to the standard sample whose element content has been verified by the relevant department, which is used as a quality control sample to verify the accuracy of the method;

(3) According to the content of barium and phosphorus in Astragalus, prepare a mixed standard solution of 35000 ng/mL phosphorus and 300 ng/mL barium, and then dilute the mixed standard solution to (V/V) 50% and 10% respectively , 5%, 1%, 0.5%, 0.1%, to obtain standard solutions with a series of concentration gradients;

(4) Use microwave digestion-inductively coupled plasma mass spectrometry ICP-MS to analyze the wild and cultivated Astragalus membranaceus in Shanxi. According to the order of concentration from low to high, ICP-MS analysis is performed on the element standard solution in turn, using software or manual calculation. Draw a standard curve; carry out ICP-MS analysis to the Astragalus sample and standard substance at the same time, calculate the content of barium and phosphorus in the Astragalus sample according to the standard curve, and obtain the element ratio barium/phosphorus; Compare to ensure the accuracy of the method;

(5) After performing binary logistic regression analysis on the barium/phosphorus value of Astragalus membranaceus and its wild and cultivated attributes, the regression equation Y=17684.370 barium/phosphorus-48.669 is obtained, and the formula for discriminant probability of the wild category of Astragalus membranaceus is P=1/ (1+e ^-Y ), in the formula, P represents the probability that Astragalus membranaceus is the wild type, and Y is the result calculated based on barium/phosphorus and the regression equation. The closer the P value is to 1, the higher the wild probability of Astragalus membranaceus, and the P value The closer to 0, the higher the cultivation probability.

In the above method, the Astragalus standard substance sample is GBW10028, the barium and phosphorus elements in it are analyzed, and the measurement results are compared with the calibration values to ensure the accuracy of the method. The receiver operating characteristic curve (receiver operating characteristic curve, referred to as ROC curve) was used to judge the specificity and sensitivity of prediction.

In the above method, 1% (V/V) _HNO3 is used as the dilution solution during the dilution process of the mixed standard solution in the step (3).

In the above method, in the step (4), set the ICP-MS instrument conditions as follows: ICP-MS power: 1600 W, plasma argon flow rate: 18 L/min, auxiliary gas flow rate 1.2 L/min, carrier gas flow rate 0.94 L/min, the nebulizer is Meinhard, the residence time is 50 ms, the instrument tuning needs to meet the following technical indicators: routine analysis sensitivity index Be: >2000 cps, In: >40000 cps, U: >30000 cps; background: Bkgd220≤1 cps, the ratio of cerium oxide to cerium ( ¹⁵⁶ CeO ⁺ / ¹⁴⁰ Ce ⁺ ): ≤2.5%, the ratio of cerium double charge to single charge ion ( ⁷⁰ Ce ²⁺ / ¹⁴⁰ Ce ⁺ ): ≤3.0%, the mass number and resolution are based on Li, Mg, In and U at 10% peak height, and the peak width is in the range of 0.65-0.8 u. Before the instrument test, the position of the torch, the flow rate of the nebulizer, the voltage of the quadrupole ion deflector (QID) and the voltage of the detector were optimized, and the dual-mode calibration of the detector was performed; the helium flow rate was set to 4.4 mL/min in the KED mode.

In the above method, all samples were randomly sorted before analysis, crushed, digested and analyzed by ICP-MS in ascending order of random numbers; each Astragalus sample was prepared in three replicates (parallel test), and each preparation was measured once; collected Different batches of stilbene samples with unknown planting methods were used for discriminant verification, and the planting methods were predicted according to the P value.

The technical route of the present invention is: Astragalus samples collected in 2019 from Shanxi (including 8 wild and 8 cultivated types) were used to determine the content of inorganic elements in them by microwave digestion ICP-MS. The analysis showed that wild Astragalus and cultivated The contents of inorganic elements in Astragalus samples had obvious differences. The non-parametric test results showed that among the 53 elements that were stably determined in Astragalus membranaceus, except K, Al, Si, B, Mo, Pb, As, Li, Ta, Hf and Re, the remaining 42 elements were stable between the two Astragalus membranaceus. There are significant differences, and the elements with a content difference of more than 2 times are Ti, Ba, Rb, Co, Cs, La, Ce, Pr, Nd, Sm, Gd, Lu, Te, Ga, W, and barium/phosphorus. Further, binary logistic regression analysis was used to study the correlation between the element content and the wild cultivation attributes of Astragalus membranaceus, and the stepwise method was used to screen the variables, and the variables with large differences included Ba, Ga, Te, barium/phosphorus, phosphorus and Mg, and the synthesis was simple. Finally, the barium/phosphorus variable (see Figure 1) was used to establish the binary logistic regression equation Y, and the discriminant probability of wild Astragalus membranaceus was P=1/(1+e ^-Y ). Use the ROC curve to judge the sensitivity and specificity, collect the second batch of astragalus samples from different sources and unknown planting methods, use the same method to analyze the content of barium and phosphorus elements, calculate the discrimination probability P, and obtain the astragalus Wild or cultivated properties.

After studying wild and non-wild samples of Astragalus, the researchers of the present invention found the correlation between the element content and the planting method of Astragalus, in which the phosphorus element is related to the cell wall components of Astragalus, and the content of barium/phosphorus in wild Astragalus is higher than that in cultivated Astragalus . Therefore, barium/phosphorus can be used as an index to distinguish the wild and cultivated attributes of Shanxi Astragalus.

Beneficial effects of the present invention:

Based on the content ratio of barium and phosphorus (barium/phosphorus) in the main root slices of traditional Chinese medicine Astragalus membranaceus, a binary logistic regression equation was established to obtain the discriminant probability value P of wild Astragalus membranaceus, which can distinguish the wild and cultivated types of Astragalus membranaceus in Shanxi. The invention is accurate, reliable, simple and feasible. It is suitable for the identification of the planting methods of Astragalus membranaceus in Shanxi.

Description of drawings

Figure 1 is a boxplot of Barium/Phosphorus in Example 1 (A) Discovery Set and (B) Validation Set.

Figure 2 shows (A) the discriminant probability value P for the discrimination of wild Astragalus membranaceus and cultivated Astragalus membranaceus in the discovery set; (B) the discrimination probability value P for the discrimination of wild Astragalus membranaceus and cultivated Astragalus membranaceus in the verification set.

Figure 3 is (A) the discriminant probability value P used for the ROC curve for the identification of wild Astragalus and cultivated Astragalus in the discovery set, AUC=1; (B) the discriminant probability value P for the ROC curve for the identification of wild Astragalus and cultivated Astragalus in the verification set, AUC =1.

Detailed ways

The present invention is further illustrated by the following examples, but not limited to the following examples.

Example 1:

1. Astragalus sample collection

Collected 16 samples of wild and cultivated Astragalus membranaceus taproot slices (including 8 wild samples and 8 cultivated samples) from Hengshan Mountain, Hunyuan County, Datong City, Shanxi Province, and numbered them immediately as a discovery set. 60 mesh sieve.

2. Microwave digestion pretreatment

Weigh about 150 mg (accurate to 0.001g) of Astragalus sample or Astragalus standard substance powder into a polytetrafluoroethylene digestion tube, add 6 mL of concentrated nitric acid, shake gently, mix well and seal it, and place it in a microwave digestion apparatus for digestion . After completion, when the digestion liquid is cooled to below 70°C, transfer it to a 100 ml volumetric flask, wash the digestion tank with a small amount of ultrapure water for 3 times, combine the washing liquid into the volumetric flask, and dilute to 100 ml with ultrapure water. mL. For the blank sample, except for the sample addition step, the other operations are the same as the sample.

3. The standard curve of the prepared elements:

According to the content of barium and phosphorus in Astragalus membranaceus, a mixed standard solution of 35000 ng/mL phosphorus and 300 ng/mL barium was prepared, and then the mixed standard solution was diluted to 50%, 10%, 5%, 1%, 0.5%, respectively. %, 0.1% (V/V), to obtain a standard solution with a series of concentration gradients. (Use 1% HNO3 as dilute solution).

4. Instrument parameter adjustment:

ICP-MS power: 1600 W, plasma argon flow rate: 18 L/min, auxiliary gas flow rate 1.2 L/min, carrier gas flow rate 0.94 L/min, nebulizer is Meinhard type (Meinhard), residence time 50 ms, instrument tuning needs to meet the following technical indicators: routine analysis sensitivity index Be: >2000 cps, In: >40000 cps, U: >30000 cps; background: Bkgd220≤1cps, ratio of cerium oxide to cerium ( ¹⁵⁶ CeO ⁺ / ¹⁴⁰ Ce ⁺ ): ≤2.5%, the ratio of double-charged to single-charged ions of cerium element ( ⁷⁰ Ce ²⁺ / ¹⁴⁰ Ce ⁺ ): ≤3.0%, the mass number and resolution are based on Li, Mg, In and U The peak width at 10% peak height is in the range of 0.65-0.8u. Before the experiment, the position of the torch, the flow rate of the nebulizer, the voltage of the quadrupole ion deflector (QID) and the voltage of the detector were optimized, and the dual-mode calibration of the detector was performed. In KED mode, set the helium flow rate to 4.4 mL/min.

5. ICP-MS analysis to obtain the element ratio barium/phosphorus

Under the above instrument conditions, the barium and phosphorus element standard solutions were analyzed by ICP-MS in order of concentration from low to high, and the data was processed by instrument software or manually to draw a standard curve. The standard curve of an element is obtained by plotting the series concentration as the independent variable and the peak intensity corresponding to the series concentration as the dependent variable, that is, the formula (1):

Ai = KiCi (1)

Among them: Ai-the peak intensity of barium/phosphorus element series concentration, Ci-barium/phosphorus element series concentration, Ki-the slope of the standard working curve. After obtaining the standard curve, substitute the barium/phosphorus element peak intensity in the sample into the formula to obtain the concentration of elemental barium/phosphorus. All samples were randomly sorted before analysis, and ICP-MS analysis was performed in ascending order of random numbers to reduce the influence of systematic errors on sample results. Each Astragalus sample was prepared in triplicate, and each preparation was measured once to reduce the influence of random errors. The Astragalus sample and the Astragalus standard substance were analyzed, the contents of barium and phosphorus in the Astragalus sample were calculated, and the element ratio barium/phosphorus was obtained. Six copies of Astragalus standard sample GBW10028 were prepared in parallel, and the content results of barium and phosphorus were compared with the calibration values (see Table 1). The RSDs of barium and phosphorus were 4.1% and 1.5%, respectively, and the recovery rates were 104.5% and 1.5%. 103.3%, the analytical method is accurate and stable.

Table 1 Determination results of barium and phosphorus in Astragalus standard substance GBW10028

6. Establishment of the probability formula for wild and cultivated Astragalus in Shanxi

After performing binary logistic regression analysis on the barium/phosphorus value of Astragalus membranaceus and its wild and cultivated attributes, the regression equation obtained is Y=17684.370 barium/phosphorus-48.669, and the discriminant probability formula of Astragalus membranaceus is P=1/(1 +e ^-Y ), in the formula, the closer the P value is to 1, the higher the wild probability of Astragalus membranaceus, and the closer the P value is to 0, the higher the cultivation probability. It was found that the P values of 8 cultivated samples were close to 0, and the P values of 8 wild samples were close to 1 (see Figure 2A), the area under the ROC curve AUC was 1, and the specificity and sensitivity were both 1.00 (see Figure 3A).

7. Discrimination of different batches of Shanxi Astragalus planting methods

Collect 9 astragalus samples of different batches and unknown planting methods as the prediction set, and use the same processing method as the discovery set described in steps 1 to 5 to obtain the content ratio of barium and phosphorus in the sample, and further calculate the P value , according to the size of the P value to determine the planting method of Astragalus. Among the 9 samples, 2 were identified as wild Astragalus membranaceus, and the remaining 7 were cultivated Astragalus membranaceus (see Figure 2B). The determination results were consistent with the source of Astragalus membranaceus samples, which proved the stability and validity of this discriminant formula. The ROC curve of the prediction set was further drawn, and the specificity and sensitivity of the prediction reached 1.00, and the area under the curve (AUC) was 1 (see Figure 3B).

Table 2 Discrimination of planting methods of Astragalus membranaceus in Shanxi

The discriminant results of Astragalus membranaceus in the discovery set and verification set (see Table 2) illustrate the feasibility and reliability of the method for discriminating between wild and non-wild Astragalus membranaceus in Shanxi proposed by the present invention.

In summary, this paper proposes a method to distinguish the wild and cultivated planting methods of Astragalus in Shanxi by combining barium and phosphorus elements with binary logistic regression. The method is simple and rapid in operation, and the result is accurate and reliable. The method is suitable for the source differentiation and identification of Astragalus membranaceus, and can provide technical support for the follow-up pharmacological research of the traditional Chinese medicine Astragalus membranaceus.

Although the present invention has been described in detail and some specific examples have been confirmed, it is obvious for those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the present invention.

Claims (7)

1. A method for identifying wild astragalus and cultivated astragalus in the ground is characterized by comprising the following steps: analyzing Shanxi wild and cultivated astragalus roots by adopting microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS) to obtain the content of barium and phosphorus elements, and obtaining a discrimination formula and a discrimination probability P of the wild astragalus roots through binary logistic regression; under the same condition, performing element analysis and attribute discrimination on the Shanxi radix astragali with unknown planting mode, and determining the attribute to be wild or cultivated.

2. The method for identifying locally wild astragalus membranaceus and cultivated astragalus membranaceus as claimed in claim 1, wherein: the method comprises the following steps:

(1) collecting cut samples of wild and cultivated radix astragali main roots produced in Shanxi province, grinding into powder by a grinder, and sieving with a 60-mesh sieve;

(2) weighing 150mg of astragalus sample and astragalus standard substance sample powder, respectively placing the astragalus sample and the astragalus standard substance sample powder into a polytetrafluoroethylene digestion tube, adding 6mL of concentrated nitric acid, slightly oscillating, uniformly mixing, sealing, and placing into a microwave digestion instrument for digestion; after the completion, when the digestion solution is cooled to a temperature lower than 70 ℃, transferring the digestion solution into a 100 mL volumetric flask, washing the digestion tank for 3 times by using a small amount of ultrapure water, combining the washing solutions in the volumetric flask, and fixing the volume to 100 mL by using the ultrapure water; carrying out a blank experiment, namely, adding no sample in the experiment process, and carrying out the same operations as the above steps;

the astragalus sample refers to an unknown astragalus sample to be analyzed; the astragalus standard substance sample is a standard sample with element content verified by relevant departments and is used as a quality control sample for verifying the accuracy of the method;

(3) preparing 35000 ng/mL phosphorus element and 300 ng/mL barium element mixed standard solution according to the content of barium and phosphorus elements in radix astragali, and then diluting the mixed standard solution to 50%, 10%, 5%, 1%, 0.5% and 0.1% respectively to obtain a series of standard solutions with volume concentration gradient;

(4) analyzing wild and cultivated radix astragali in Shanxi by adopting microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS), sequentially analyzing element standard solutions by adopting ICP-MS according to the sequence of concentration from low to high, and drawing a standard curve by adopting software or manual calculation; simultaneously carrying out ICP-MS analysis on the astragalus sample and the standard substance, and calculating the content of barium and phosphorus in the astragalus sample according to a standard curve to obtain the element ratio of barium to phosphorus; comparing the element measurement result of the astragalus standard substance with a calibration value to ensure the accuracy of the method;

(5) performing binary logistic regression analysis on the barium/phosphorus value of the astragalus and the wild and cultivation attributes of the astragalus to obtain a regression equation Y =17684.370 barium/phosphorus-48.669, and further obtaining that the discrimination probability formula of the wild astragalus is P = 1/(1+ e)^-Y) In the formula, P represents the probability of the astragalus root being a wild type, Y is a result obtained by calculation according to barium/phosphorus and a regression equation, the closer to 1 the P value is, the higher the wild probability of the astragalus root is, the closer to 0 the P value is, and the higher the cultivation probability is.

3. The method for identifying locally wild astragalus and cultivated astragalus as claimed in claim 2, wherein: the astragalus standard substance sample is GBW10028, barium and phosphorus elements in the astragalus standard substance sample are analyzed, and a measurement result is compared with a calibration value to ensure the accuracy of the method.

4. The method for identifying locally wild astragalus and cultivated astragalus as claimed in claim 2, wherein: using HNO with the volume concentration of 1 percent in the dilution process of the mixed standard solution in the step (3)₃As a dilute solution。

5. The method for identifying locally wild astragalus and cultivated astragalus as claimed in claim 2, wherein: in the step (4), the ICP-MS instrument conditions are set as follows: ICP-MS power: 1600W, plasma argon flow rate: 18L/min, the flow rate of auxiliary gas is 1.2L/min, the flow rate of carrier gas is 0.94L/min, the atomizer is a Meinhard type, the residence time is 50 ms, and the instrument tuning needs to meet the following technical indexes: conventional analytical sensitivity index Be:>2000 cps，In：>40000 cps，U：>30000 cps; background: bkgd220 is less than or equal to 1 cps, and the ratio of cerium oxide to cerium¹⁵⁶CeO⁺/¹⁴⁰Ce⁺: not more than 2.5%, and ratio of double electric charge to single electric charge ion of cerium element⁷⁰Ce²⁺/¹⁴⁰Ce⁺: less than or equal to 3.0 percent, and the mass number and the resolution are In the range of 0.65-0.8U according to the peak width of Li, Mg, In and U at 10 percent of peak height.

6. The method for identifying locally wild astragalus and cultivated astragalus as claimed in claim 5, wherein: before the instrument test, optimizing the position of a rectangular tube, the flow of an atomizer, the voltage of a quadrupole ion deflector and the voltage of a detector, and performing dual-mode correction on the detector; helium flow was set at 4.4 mL/min in KED mode.

7. The method for identifying locally wild astragalus and cultivated astragalus as claimed in claim 2, wherein: all samples are randomly sequenced before being analyzed, and crushing, digestion and ICP-MS sample injection analysis are carried out according to the ascending sequence of random numbers; each astragalus sample was prepared in duplicate in 3 portions, one for each preparation assay.

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