A method for rapid identification of wild ginseng and garden ginseng using terahertz frequency domain spectroscopy

Abstract

The present invention provides a method for identifying forest mountain ginseng and garden ginseng by using terahertz frequency domain spectroscopy, including a step of obtaining terahertz fingerprints of forest mountain ginseng and garden ginseng: a step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum; comparing the terahertz frequency domain spectra of forest mountain ginseng and garden ginseng, it is found that there is a difference between 8.25-9.50THz, taking 8.25-9.50THz as the horizontal coordinate, taking the first-order derivative of the spectrum data of the two ginsengs, making the maximum value Ymax and the minimum value Ymin, ΔY=Ymax-Ymin if: ΔY>0.15, it is garden ginseng; ΔY<0.15, it is forest mountain ginseng. The present invention utilizes terahertz spectroscopy technology with strong sensitivity and simple operation compared with traditional known methods, which can achieve the purpose of quickly and accurately identifying and distinguishing forest mountain ginseng and garden ginseng.

Description

Technical field:

[0001] The invention belongs to the field of Chinese medicine identification and detection, and relates to a method for identifying wild ginseng and garden ginseng under forests, and specifically is a method for identifying wild ginseng and garden ginseng under forests by using terahertz frequency domain spectroscopy. Background technology:

[0002] Ginseng (Panax ginseng CAMey) is a perennial herb that has been one of the world's best-selling natural health products due to its anti-aging, anti-cancer, anti-stress, anti-fatigue and anti-anxiety effects.

[0003] Ginseng can be divided into three categories according to the different growing environments and cultivation methods: garden ginseng, forest ginseng and wild ginseng. Wild ginseng takes a long time to grow and is currently in extremely short supply. Garden ginseng refers to ginseng cultivated artificially in gardens, which is generally planted for 4-6 years. Forest ginseng refers to ginseng that grows naturally in the wild by sowing or transplanting to the mountains. It has higher medicinal and edible value than ordinary garden ginseng, and its market price is much higher than ordinary ginseng.

[0004] Currently, forest ginseng and garden ginseng can be identified by observing their appearance as whole plants, and professionals can identify them using the traditional ginseng "five types" method. However, it is very challenging to distinguish the varieties of ginseng slices, powder, or processed products.

[0005] The national standard "Wild Ginseng Identification and Grading Quality GB / T 18765-2015" uses physical and chemical methods to identify the content of ginsenosides through high-performance liquid chromatography. It describes the composition of ginsenosides in wild ginseng, such as Rb1 content greater than 0.40%, Re and Rg1 content sum greater than 0.60%, and total saponin content greater than 4.40%. This content standard requires complex sample preparation and processing. It often takes 6-8 hours to detect the extracted ginsenosides with the help of a liquid-mass mass spectrometry platform. Since the number of wild ginseng in the world is extremely limited, the "wild ginseng" circulating on the market is mainly forest ginseng that grows for many years. The national standard does not give the main saponin content of garden ginseng, nor does it give the method of using saponin content to distinguish between forest ginseng and garden ginseng.

[0006] The national standard method provides a technique for identifying ginseng species by using a liquid-mass spectrometry technology platform to confirm the difference in saponin content in the identification of physical and chemical properties. This technique is suitable for the identification of mountain ginseng and garden ginseng. It is subject to complex experimental operations and processes such as sample powdering, chemical reagent extraction, liquid-mass spectrometry instrument injection, and standard material comparison. It cannot be widely used in actual market circulation. Other reported methods include the use of Raman spectroscopy, near-infrared spectroscopy, and other optical methods, which are simple to operate, but have low sensitivity and specificity, and have not been verified for consistency between standard methods and spectroscopic methods on the same samples.

[0007] Terahertz (THz) is an electromagnetic wave with a specific wavelength between microwaves and infrared. Its unique band frequency is in the range of 0.1-10THz and the wavelength is in the range of 0.03-3m. The principle of using terahertz in the field of modern analysis is: using terahertz to pass through the object to be detected, you will get the terahertz wave after passing through the detection material or the terahertz wave signal reflected by the object to be detected. These signals contain the spectral absorption characteristics of the object to be detected in the terahertz band. After removing the background signal from the obtained time domain signal, the spectral time domain signal of the object to be detected can be obtained.

[0008] Compared with other spectral technologies, terahertz technology has the following advantages in identifying traditional Chinese medicine:

[0009] 1: Measure the time domain spectrum and transient characteristics of traditional Chinese medicine, with terahertz pulse width ranging from picoseconds to femtoseconds;

[0010] 2: The photon energy radiated by Taiheziji is very low (about 4.1meV), which will not cause any harm to the tested Chinese medicine;

[0011] 3: The required sample volume can be as low as a few milligrams, which will not cause waste of precious Chinese medicinal materials in testing;

[0012] 4: Verifiability: The terahertz band is between microwaves and infrared waves, and can be verified and supplemented with other spectral technologies used in traditional Chinese medicine identification;

[0013] 5. Adapt to practical applications. Terahertz technology is easy to operate, usually just powdering and tableting;

[0014] 6: Terahertz spectroscopy has faster detection efficiency and stronger signal-to-noise ratio; Summary of the invention:

[0015] In view of the complex problem of ginseng identification process in the prior art, combined with the advantages of terahertz spectroscopy, the present invention provides a method for identifying wild ginseng and garden ginseng using terahertz frequency domain spectroscopy, which solves the technical problems of the complexity and long time of the methods for identifying wild ginseng and garden ginseng in the prior art. Compared with the results of distinguishing and identifying by physical and chemical properties in the existing national standards, the results are reliable and have good consistency.

[0016] The steps include:

[0017] A method for identifying wild ginseng and garden ginseng using terahertz spectroscopy, comprising the following steps:

[0018] 1) A step of obtaining the fingerprint of the ginseng sample to be tested:

[0019] a) taking the dried root tissue of the ginseng sample to be tested, processing it through a grinder to make ginseng powder, taking 20-200 mg, and drying it;

[0020] b) Mixing the dried ginseng powder to be tested with a light-transmitting auxiliary material (the light-transmitting auxiliary material is a material with weak optical absorption in the terahertz-specific band frequency range of 0.1-10THz)

[0021] c) grinding and mixing the mixture in step b), and pressing into flakes;

[0022] d) testing the thin slice sample in step c) using terahertz spectroscopy to obtain a full-frequency-domain absorption data table of the terahertz spectrum of the sample to be tested;

[0023] 2) A step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum;

[0024] a) Import the collected absorption data table of the full-frequency domain of the terahertz spectrum of the sample to be tested into Origin 14.1 to generate a spectral absorption diagram, with the X-axis being the terahertz frequency domain and the Y-axis being the optical absorption value. It is found that there is a difference in the 8.25-9.50 THz segment.

[0025] b) taking the first-order derivative of the absorption spectrum value of the Y axis corresponding to the X axis between 8.25 and 9.50 THz, taking the 8.25-9.50 THz spectrum region as the abscissa X, and the first-order derivative of the terahertz optical absorption data of the sample to be tested in this interval as the ordinate Y, taking the maximum value of the Y value in this interval as Ymax, and the minimum value as Ymin;

[0026] c) Let ΔY=Ymax-Ymin if:

[0027] If ΔY>0.15, it is garden ginseng;

[0028] If ΔY<0.15, it is forest ginseng;

[0029] As described above, a method for identifying wild ginseng and garden ginseng using terahertz time-domain spectroscopy is used, wherein in a terahertz fingerprint spectrum of a ginseng sample to be tested is obtained, the sample to be tested and a light-transmitting auxiliary material are mixed, preferably in a ratio of 1:1 to 1:10.

[0030] As described above, a method for identifying wild ginseng and garden ginseng using terahertz time-domain spectroscopy, wherein the light-transmitting auxiliary material is a polyethylene polymer compound.

[0031] As described above, a method for identifying wild ginseng and garden ginseng using terahertz time-domain spectroscopy is used, wherein in a step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum, the garden ginseng has specific absorption at 9.2 THz, while the wild ginseng has no absorption peak.

[0032] More specifically, the method comprises the following steps:

[0033] 1) A step to obtain the terahertz fingerprint of wild ginseng and garden ginseng:

[0034] a) taking root tissues of sun-dried wild ginseng and garden ginseng samples respectively, processing them through a grinder to make fine ginseng powder, taking 20-200 mg, drying, and confirming that the water is completely evaporated by constant weight weighing method;

[0035] b) fully mixing the ginseng powder of the fully dried wild ginseng and garden ginseng samples with the auxiliary materials respectively;

[0036] c) grinding and mixing the mixture in step b) respectively, and pressing into flakes;

[0037] d) transferring the thin slice sample in step c) to a terahertz spectrum for detection, and obtaining a full-frequency-domain absorption data table of the terahertz spectrum of the forest wild ginseng and the garden ginseng samples;

[0038] 2) a step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum;

[0039] a) The collected absorption data of the full frequency domain of the terahertz spectrum of the wild ginseng and the garden ginseng samples were imported into Origin 14.1 (Origin is a scientific drawing and data analysis software developed by OriginLab) to generate the spectral absorption diagram of the two ginseng species, with the X-axis being the terahertz frequency domain and the Y-axis being the optical absorption value. By comparing the terahertz absorption peak diagrams of the wild ginseng and the garden ginseng samples, it was found that there was a difference in the 8.25-9.50THz section between the garden ginseng and the wild ginseng;

[0040] b) Locate the frequency domain interval between 8.25-9.50 THz, and after polynomial fitting smoothing, standard normal variation, linear subtraction, first-order derivative, and second-order derivative processing, compare and select the algorithm that can significantly amplify the differential absorption in this frequency domain segment; confirm that the data model for the first-order derivative of the absorption spectrum between 8.25-9.50 THz can effectively distinguish and identify the wild ginseng and the ordinary garden ginseng;

[0041] c) Taking the 8.25-9.50THz spectral region as the horizontal coordinate X, the first-order derivative of the terahertz optical absorption data of the two ginsengs in this interval as the vertical coordinate Y, taking the maximum value of the Y value in this interval as Ymax, and the minimum value as Ymin;

[0042] d) Let ΔY = Ymax - Ymin if:

[0043] ΔY>0.15, it is garden ginseng;

[0044] If ΔY<0.15, it is forest ginseng.

[0045] Furthermore, in obtaining the terahertz fingerprints of wild ginseng and garden ginseng, the ginseng powder of the wild ginseng and garden ginseng samples are fully mixed with auxiliary materials respectively, and the preferred mixing range is that the mass ratio of ginseng powder to auxiliary materials is between 1:1 and 1:10.

[0046] Furthermore, in a step of obtaining the terahertz fingerprint of wild ginseng and garden ginseng, the auxiliary material is preferably a polyethylene polymer compound.

[0047] Furthermore, in a step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum, it was found that the garden ginseng had specific absorption at 9.2 THz, while the wild ginseng under the forest had no absorption peak.

[0048] The present invention provides a method for distinguishing common garden ginseng from wild ginseng in the forest by using the absorption peak of terahertz spectrum. The inventors measure two different types of ginseng samples, use common garden ginseng samples purchased from multiple places, and focus on collecting wild ginseng samples of different years from 7 to 20 years for testing. By analyzing the absorption values ​​of different frequency domains, the spectral absorption bands of wild ginseng and garden ginseng are found. After multiple analyses and mathematical modeling, a mathematical model for effectively distinguishing the two ginsengs in the specific frequency domain is obtained. At the same time, according to the method for analyzing saponin components in ginseng recommended in the national standard "Identification and Grading Quality of Wild Ginseng GB / T 18765-2015", the contents of ginsenoside components Rg1, Re and Rb1 in wild ginseng and garden ginseng are measured, and a method for distinguishing wild ginseng and garden ginseng by using saponin content is found. Comparison shows that the two methods have good consistency. It is shown that the mathematical simulation established based on the specific absorption signal value of terahertz spectrum can be developed into a method for rapid identification of wild ginseng and garden ginseng in the forest.

[0049] Compared with the existing technology, the present invention has significant technical progress. The terahertz spectrum in the spectral technology is applied to the precious Chinese medicinal material ginseng, and a method for non-destructive identification of forest mountain ginseng and ordinary garden ginseng is established. At the same time, the method of distinguishing the two types of ginseng by ginsenosides is comparable with the existing chromatographic technology, and the consistency of the two is good. Description of the drawings:

[0050] Figure 1 :Sample photos of forest ginseng aged 7 to 20 years.

[0051] Figure 2 :A Liquid chromatogram of wild ginseng in the forest, B Liquid chromatogram of ginseng in the garden

[0052] Figure 3 : Terahertz fingerprints of 7-20-year-old wild ginseng.

[0053] Figure 4 : Differential absorption diagrams of stems and whiskers of common garden ginseng and 8-year and 20-year-old wild ginseng at 9.2THz.

[0054] Figure 5 :The method of distinguishing wild ginseng and garden ginseng at 8.25THz-9.50THz.

[0055] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Specific implementation method:

[0056] Example 1

[0057] The 20 samples include:

[0058] 8 samples of ginseng taproot powder (4-6 years old), weigh 500mg-1g of powder for later use

[0059] Samples of 12 wild ginseng plants (7-20 years old) Figure 1 , whole ginseng sample, cut the main root part and grind it into powder, weigh 500mg-1g of powder for later use

[0060] 1. Use the high performance liquid chromatography method recommended by the National Pharmacopoeia to detect the content of common ginsenosides Rg1, Re, and Rb1 in the samples. The two types of ginseng can be distinguished by comparing the ratio of (Rg1+Re) / Rb1.

[0061] Instruments and reagents used in the experiment:

[0062]

[0063]

[0064] 1) Preparation of test solution:

[0065] Take 100 mg of sample into a 2 mL EP tube, add 1 mL of 75% methanol solution by volume, seal the tube with a sealing film, ultrasonicate for 1 hour, and filter out the solid residue with a 0.22 wn polytetrafluoroethylene disposable filter to obtain about 600 ul of a clear light yellow ginsenoside solution.

[0066] 2) Preparation of reference solution: Accurately weigh the reference substances of Rg1, Re and Rb1, respectively, and dissolve them in methanol to obtain a reference substance stock solution of about 0.50 mg / ml. Then, take an appropriate amount of the stock solution and dilute it with methanol to obtain a mixed standard solution of appropriate concentration.

[0067] 3) Chromatographic and gradient injection conditions

[0068] High pressure liquid chromatography was performed using a binary solvent system of ACQUITY UPLC / Synapt G2, MS system (Waters) with a PDA detector and a Waters ACQUTTY HSS T3 column (100 mm*2.1 mm, 1.8 mm).

[0069] The mobile phase is selected from A) 0.1% by volume formic acid solution, B) 0.1% by volume formic acid solution and acetonitrile. The gradient elution scheme is as follows:

[0070]

[0071] The elution rate was set at 0.4 ml / min, the column temperature and the air temperature were maintained at 40 °C and 8 °C, respectively, and the detected chromatographic peaks were analyzed using MassLynx V4.2 software.

[0072] 4) Through the reference substance, the time of obtaining Re+Rg1 is 6.75 minutes to 7.10 minutes, and the peak time of Rb1 is 10.98 minutes to 11.30 minutes. Figure 2 As shown. The corresponding chromatographic peaks were processed using MassLynx V4.2 software to obtain the absorption peak area. The ratio of (Re+Rg1) / Rb1 was calculated. The result of (Re+Rg1) / Rb1<1 determined that the sample was a garden ginseng, and the result of (Re+Rg1) / Rb1>1 determined that the sample was a wild ginseng.

[0073] 2. Obtaining the terahertz optical signal spectra of wild ginseng and garden ginseng and performing data processing and analysis

[0074] 1) 100 mg of powder sample was dried and weighed to a constant weight to confirm that the water had evaporated completely;

[0075] 2) The ginseng powder of fully dried wild ginseng and garden ginseng samples were fully mixed with auxiliary materials, and the mass ratio of ginseng powder to auxiliary materials was 1:5;

[0076] 3) The above mixtures were pressed into thin sheets, transferred to the terahertz spectrum for detection, and the full-frequency absorption data chart of the terahertz spectrum of the sample was obtained. Among them, the terahertz frequency domain spectra of 12 wild ginsengs from forests aged 7 to 20 years were as follows: Figure 3

[0077] 4) Import the above data table into Origin 14.1 to obtain the spectral absorption diagrams of the two ginseng species. It is found that there is a difference between the 8.25-9.50THz section of the garden ginseng and the forest mountain ginseng. The garden ginseng has an obvious absorption peak at 9.2THz, while the forest mountain ginseng has no absorption. Among them, the spectral difference absorption diagrams of the 8-year and 20-year forest ginseng and the garden ginseng are shown as follows Figure 4

[0078] 5) Take the optical signal value in the frequency domain between 8THz and 10THz and take the first-order derivative. In the range of 8.25-9.50THz, the maximum value is Ymax and the minimum value is Ymin.

[0079] Calculate the ΔY (Ymax-Ymin) value of each sample to make a judgment such as Figure 5

[0080] The accuracy and consistency of the two different methods for identifying understory mountain ginseng and garden ginseng are shown in the following table

[0081] Table 1: HPLC-MS / MS detection results of ginsenoside content samples:

[0082]

[0083]

[0084] Table 2: Optical absorption of different species in the terahertz spectrum in the 8.25Thz-9.50Thz region:

[0085]

[0086] The accuracy of the two methods in identifying forest ginseng and garden ginseng:

[0087] LC-MS accuracy: 90% THz spectroscopy accuracy: 85%

[0088] The processing and identification time of each sample using terahertz spectroscopy technology is about 20 minutes, and the detection time is about 1 minute. Compared with the existing literature and industry standard methods that require 6-8 hours to determine "wild" ginseng by physical and chemical properties-saponin content, the operation time can be significantly reduced.

[0089] Example 2

[0090] A total of 43 samples containing ordinary garden ginseng and wild ginseng powder were tested blindly.

[0091] 1) The test results are summarized according to two different methods, as follows

[0092]

[0093] 2) Compare the consistency of the two methods using paired chi-square test

[0094]

[0095] Table 2: Paired χ 2 test

[0096]

[0097] Result judgment: kappa=0.767, P<0.05, which proves that the two methods have good consistency and can pass statistical tests.

Claims

1. A method for identifying wild ginseng and garden ginseng using terahertz spectroscopy, characterized in that The steps include: 1) A step to obtain the terahertz fingerprint of the ginseng sample to be tested: a) taking the dried root tissue of the ginseng sample to be tested, processing it through a grinder to make ginseng powder, taking 20-200 mg, and drying it; b) mixing the dried ginseng powder to be tested with the light-transmitting auxiliary material; c) grinding and mixing the mixture in step b) and pressing into flakes; d) testing the thin slice sample in step c) using terahertz spectroscopy to obtain a full-frequency-domain absorption data table of the terahertz spectrum of the sample to be tested; 2) Steps for analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum; a) Import the collected absorption data table of the full-frequency domain of the terahertz spectrum of the sample to be tested into Origin 14.1 to generate a spectral absorption diagram, with the X-axis being the terahertz frequency domain and the Y-axis being the optical absorption value; b) Take the first-order derivative of the absorption spectrum value of the Y axis corresponding to the X axis between 8.25-9.50 THz, take the 8.25-9.50 THz spectrum region as the horizontal coordinate X, and the first-order derivative of the terahertz optical absorption data of the sample to be tested in this interval as the vertical coordinate Y, and take the maximum value of the Y value in this interval as Ymax, and the minimum value as Ymin; c) Let ΔY = Ymax - Ymin If: ΔY> 0.15, it is garden ginseng; If ΔY< 0.15, it is forest ginseng.

2. The method for identifying wild ginseng and garden ginseng using terahertz spectroscopy according to claim 1, characterized in that: In obtaining the terahertz fingerprint of the sample to be tested, the sample to be tested and the light-transmitting auxiliary material are mixed, and the mass ratio of the sample to be tested and the light-transmitting auxiliary material is 1:1-1:

10.

3. The method for identifying wild ginseng and garden ginseng using terahertz spectroscopy according to claim 1, characterized in that: The light-transmitting auxiliary material is a polyethylene polymer compound.

4. The method for identifying wild ginseng and garden ginseng using terahertz spectroscopy according to claim 1, characterized in that: In a step of analyzing and processing the absorption data of the full frequency domain of the terahertz spectrum, it was found that the garden ginseng had specific absorption at 9.2 THz, while the wild ginseng in the forest had no absorption peak.

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