Spectrophotometric method for identifying soapberry from soapberry peel and application of spectrophotometric method

Abstract

The invention relates to a method for identifying soapberry from soapberry peel based on ultraviolet-visible spectrophotometry. The method is characterized by comprising the following steps: preparing a saponin solution, adding a 5% vanillin-glacial acetic acid solution and perchloric acid, uniformly shaking, heating in a constant-temperature water bath kettle at 70 DEG C for 15 minutes, cooling in an ice-water bath for 3 minutes, adding 5mL glacial acetic acid as a solvent, and measuring the characteristic absorption spectra of the vanillin and the glacial acetic acid by adopting an ultraviolet and visible spectrophotometer. And by utilizing the obvious difference of the number, the peak pattern and the position of the characteristic peaks in the absorption spectrum, the simple and rapid identification of the two is realized. The method has the advantages that the mass of required raw materials is small, the operation is simple, the process flow is short, and the experimental conditions are easy to realize. The technology is a rapid and efficient method for identifying and identifying the pericarp, pericarp powder and pericarp extract of the Sapindus maxima and the Sapindus mukorossi.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material identification and authentication, and specifically relates to a method and application of identifying Sichuan and Yunnan soapberry and soapberry peel by spectrophotometry. Technical Background

[0002] Plants of the genus Sapindus are widely distributed in the warm temperate and tropical zones of the world, with the center of distribution located in tropical America. There are 4 species and 1 variant of the genus Sapindus in my country, including Sapindus mukorossi, Sapindus delavayi, Sapindus rarak, and Sapindus tomentosus. Sapindus is widely distributed in tropical and subtropical areas south of the Huaihe River in my country. Sapindus delavayi is a unique species of the genus Sapindus in my country, concentrated in the Yunnan-Guizhou Plateau and Sichuan Basin in my country. At present, many southern provinces have successively cultivated tens of thousands of hectares of artificial raw material forests of Sapindus mukorossi. It is an economic tree species with extremely high comprehensive development value, integrating biomass energy, medicine, natural detergents, pesticides, daily chemicals, industry, soil and water conservation and other uses. It has attracted attention due to its versatility, high development value and broad application prospects, and the peel has attracted more attention as one of the main ingredients of medicine, natural detergents, pesticides and daily chemicals.

[0003] At present, the most research is on saponins, and inter-species identification is mainly concentrated on germplasm and tree characteristics, while there is currently no identification method for Sichuan and Yunnan soapberry and soapberry. Tang Qingtao et al. published a method for determining the content of total saponins in soapberry peel by ultraviolet spectrometry, wherein the content of total saponins was determined by extraction and ultraviolet-visible spectrometry at 541 nm; a rapid detection kit and method for total saponins in soapberry peel (CN 107228833B) for detecting total saponins in soapberry peel, soapberry peel extract or daily chemical products containing soapberry saponins; a total saponins buffer solution and its application (CN116355037A), comprising total saponins in a mass volume concentration of 1 to 200 g / L, inorganic salts in a mass volume concentration of 5 to 9 g / L, a metal ion ligand in a mass volume concentration of 0.05 to 2 mmol / L and a buffer reagent in a mass volume concentration of 0.05 to 0.2 mol / L, and the remainder being water; the pH value of the total saponins buffer solution is 7.0 to 7.6.

[0004] With the development of modernization, the source of raw materials for production has changed from previous raw materials (such as soapberry fruit) to peels, peel powder, and peel extracts. In addition, the peels of Sichuan-Yunnan soapberry and soapberry are used in medicines, detergents, pesticides, and daily chemicals. However, for standardized production, the source and stability of raw materials are very important. Varieties from different sources will affect the production process, waste production costs and product quality stability. How to ensure product stability and save costs, source identification before production is the most important.

[0005] The present invention is through the extraction of conventional saponin crude extract, and then uses common ultraviolet-visible spectrophotometer to carry out rapid determination to extracting solution, obtains both ultraviolet-visible spectrograms, and by comparing the position of their spectrogram characteristic peaks and the difference of their absorbance ratio range, quickly realizes the identification of the peel, peel powder and peel extract of two varieties of Sichuan and Yunnan soapberry and soapberry. Compared with the current technology, the method has the advantages of simple method, low cost and high credibility. In addition, the instrument used is simple and easy to operate, and does not need professional operating technology. So adopting spectrophotometry to identify the peel, peel powder and peel extract of Sichuan and Yunnan soapberry and soapberry has certain advantages. Summary of the invention

[0006] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is: a method for identifying Sichuan and Yunnan soapberry and soapberry peel by ultraviolet-visible spectrophotometry.

[0007] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0008] 1. Pretreatment of Sichuan and Yunnan soapberry and soapberry peel samples: (1) Select Sichuan and Yunnan soapberry fruits with golden yellow and wrinkled surfaces and sound when shaken, separate the peel from the seeds using a special soapberry peel separation device and clean the equipment, select Sichuan and Yunnan soapberry peels and dry them in a 60°C oven, then grind them with a multifunctional grinder and pass them through a No. 5 sieve to obtain Sichuan and Yunnan soapberry powder; (2) Select Sichuan and Yunnan soapberry fruits with golden yellow and wrinkled surfaces and sound when shaken, separate the peel from the seeds using a special soapberry peel separation device and clean the equipment, select Sichuan and Yunnan soapberry peels and dry them in a 60°C oven, then grind them with a multifunctional grinder and pass them through a No. 5 sieve to obtain soapberry powder.

[0009] 2. Preparation of crude extracts of saponin from Sichuan and Yunnan soapberry peel: (1) Weigh 2 g of sieved saponin powder, add 40 ml of methanol at a solid-liquid ratio of 1:20, use an ultrasonic machine to sonicate at 50°C for 1 h, then heat under reflux at 80°C for 4 h, filter, and evaporate to obtain a crude extract of saponin from Sichuan and Yunnan soapberry; (2) Weigh 2 g of sieved saponin powder, add 40 ml of methanol at a solid-liquid ratio of 1:20, use an ultrasonic machine to sonicate at 50°C for 1 h, then heat under reflux at 80°C for 4 h, filter, and evaporate to obtain a crude extract of saponin from Sichuan and Yunnan soapberry.

[0010] 3. Preparation of extracts of Sichuan and Yunnan soapberry and soapberry peel: (1) Weigh an appropriate amount (0.6-1.2 g) of crude extract of Sichuan and Yunnan soapberry saponins and dissolve it in methanol in a 10 mL volumetric flask to prepare Sichuan and Yunnan soapberry saponins solution. Use a pipette to accurately measure 0.1 mL of the solution into a test tube, then add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a constant temperature water bath at 70°C for 15 min, cool in an ice water bath for 3 min, add 5 mL of glacial acetic acid, shake well, and obtain Sichuan and Yunnan soapberry. Test solution; (2) Weigh an appropriate amount (0.6-1.2 g) of crude extract of sapindus saponin, dissolve it in methanol in a 10 mL volumetric flask to prepare a sapindus saponin solution, accurately measure 0.1 mL of the solution into a test tube with a pipette, then add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70°C constant temperature water bath for 15 min, cool in an ice water bath for 3 min, add 5 mL of glacial acetic acid, shake well, and obtain the sapindus test solution; the 5% vanillin-glacial acetic acid solution must be prepared and used immediately.

[0011] 4. UV-visible spectra of the extracts of Sichuan and Yunnan soapberry and soapberry peel: The blank solution was used as a reference to detect the extracts. The test solutions of Sichuan and Yunnan soapberry and soapberry peel were scanned with a UV-6100S UV-visible spectrophotometer in the range of 400-800nm ​​to obtain the UV-visible spectra and maximum absorption wavelengths of the two.

[0012] 5. Comparative analysis of the UV-visible spectra of the extracts of Sichuan and Yunnan soapberry and soapberry peel: In the spectrum of the extract of Sichuan and Yunnan soapberry peel, in the range of 400-800nm, there are two absorption peaks of Sichuan and Yunnan soapberry, located at around 476nm and 546nm respectively; in the spectrum of the extract of soapberry peel, in the range of 400-800nm, there is one absorption peak of soapberry, located at around 471nm; By comparing the number, peak shape and position of the absorption peaks, the source of Sichuan and Yunnan soapberry or soapberry peel can be determined.

[0013] The method of the present invention utilizes the characteristic difference of different absorption spectra of Sichuan and Yunnan soapberry and soapberry peel extract in the ultraviolet-visible light region, which can achieve the purpose of quickly identifying and distinguishing Sichuan and Yunnan soapberry and soapberry peel. This method breaks through the source of the soil, not only has the advantages of small sampling volume, simple and easy operation of the instrument used, and no need for professional operation technology, but also has good stability and repeatability of the test results, and is practical and feasible. It is worth noting that the identification method of the present invention does not require the use of large-scale experimental instruments and other characteristics, reducing the required unit cost investment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a photo of the fruit of the Sichuan and Yunnan soapberry.

[0015] Figure 2 This is a photo of soapberry fruit.

[0016] Figure 3 This is the UV-visible spectrum of the soapberry peel from different sources.

[0017] Figure 4 The UV-visible spectra of soapberry peels from different sources. DETAILED DESCRIPTION

[0018] The present invention will now be further described in detail with reference to the embodiments and the accompanying drawings:

[0019] The spectrophotometry of Sichuan and Yunnan soapberry and soapberry peel designed by the invention consists of five main steps: pretreatment, preparation of saponin crude extract, preparation of extract, ultraviolet-visible spectrum measurement of the extract and comparative analysis of the ultraviolet-visible spectrum of the extract.

[0020] The spectrophotometric method for distinguishing Sichuan and Yunnan soapberry and soapberry peels in the embodiment of the present invention is: using the same solvent as a reference, using The UV-6100S ultraviolet-visible spectrophotometer measured the extract in the wavelength range of 400 to 800 nm to obtain the ultraviolet-visible spectra of the extracts. The two source varieties were identified and differentiated by utilizing the obvious differences in the number, peak shape and position of the characteristic absorption peaks of the two in the ultraviolet region.

[0021] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods. It should be noted that these descriptions are only exemplary, and the claims of the present invention are not limited by the specific embodiments.

[0022] Sources of samples and their numbers in the examples: Samples are from fruits provided by enterprises from different production areas (see Tables 1 and 2 for details).

[0023] Table 1. Fruits of Sichuan and Yunnan soapberries from different sources

[0024]

[0025] Table 2 Soapberry fruits from different sources

[0026]

[0027] Example 1

[0028] 1. Pretreatment of Sichuan and Yunnan soapberry peel samples: Select Sichuan and Yunnan soapberry with golden and wrinkled surface and sound when shaken (A1, B1, C1, D1, E1) fruit, use a special separation device for soapberry peel to separate the peel from the seeds and clean the equipment, select Sichuan and Yunnan soapberry (A1, B1, C1, The peels of the seeds were dried in an oven at 60°C, crushed with a multifunctional grinder and passed through a No. 5 sieve to obtain Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) powders.

[0029] 2. Preparation of crude saponin extract from Sichuan and Yunnan soapberry peel: Weigh 2 g of sieved Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) powder, add 40 ml of methanol at a solid-liquid ratio of 1:20, use an ultrasonic machine to sonicate at 50°C for 1 h, then heat under reflux at 80°C for 4 h, filter, and evaporate to obtain the crude saponin extract from Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1).

[0030] 3. Preparation of Sichuan and Yunnan soapberry peel extract: Weigh an appropriate amount (0.6-1.2g) of Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) crude saponin extract, dissolve it in a 10mL volumetric flask with methanol to prepare Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) saponin solution, accurately measure 0.1mL of the test solution into a test tube with a pipette, then add 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid, shake well, heat in a constant temperature water bath at 70℃ for 15min, cool in an ice water bath for 3min, add 5mL of glacial acetic acid, shake well, and obtain Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) saponin solution. D1, E1) solution to be tested.

[0031] 4. Determination of UV-visible spectrum of Sichuan and Yunnan soapberry peel extract: Taking blank solution as reference, the above Sichuan and Yunnan soapberry (A1, B1, C1, D1, E1) test solutions were measured by UV-6100S UV-visible spectrophotometer within 400-800nm ​​to obtain the UV-visible spectra and maximum absorption wavelengths of different Sichuan and Yunnan soapberries.

[0032] Common absorption peaks in the UV spectra of Sichuan and Yunnan soapberry peels from different origins

[0033]

[0034] Example 2

[0035] 1. Pretreatment of soapberry peel samples: Select soapberry (A2, B2, C2, D2, E2) fruits with golden surface, wrinkles and sound when shaken, separate the peel from the seeds with a special separation device for soapberry peel and clean the equipment, select soapberry peel and dry it in a 60℃ oven, then grind it with a multifunctional grinder and pass it through a No. 5 sieve to obtain soapberry (A2, B2, C2, D2, E2) powder.

[0036] 2. Preparation of crude saponin extract from soapberry peel: Weigh 2 g of sieved soapberry (A2, B2, C2, D2, E2) powder, add 40 ml of methanol at a solid-liquid ratio of 1:20, use an ultrasonic machine to sonicate at 50°C for 1 h, then heat under reflux at 80°C for 4 h, filter, and evaporate to obtain the crude saponin extract from soapberry (A2, B2, C2, D2, E2).

[0037] 3. Preparation of soapberry peel extract: Weigh an appropriate amount (0.6-1.2 g) of crude saponin extract of soapberry (A2, B2, C2, D2, E2) and dissolve it in a 10 mL volumetric flask with methanol to prepare a test solution of soapberry (A2, B2, C2, D2, E2) saponin. Use a pipette to accurately measure 0.1 mL of the test solution into a test tube, then add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a constant temperature water bath at 70°C for 15 min, cool in an ice water bath for 3 min, add 5 mL of glacial acetic acid, shake well, and obtain the soapberry (A2, B2, C2, D2, E2) test solution.

[0038] 4. UV-visible spectrum determination of soapberry peel extract: Taking blank solution as reference, the above soapberry (A2, B2, C2, D2, E2) test solutions were measured by UV-6100S UV-visible spectrophotometer within 400-800nm ​​to obtain the UV-visible spectra and maximum absorption wavelengths of different Sichuan and Yunnan soapberries.

[0039] Common absorption peaks in the UV spectra of soapberry peels from different origins

[0040]

[0041] Figure 1 UV-visible spectra of the extracts from Sichuan and Yunnan soapberry peels from different origins, A1 (Zhenfeng, Guizhou), B1 (Anning, Yunnan), C1 (Chenggong, Yunnan), D1 (Yuxi, Yunnan), E1 (Yilang, Yunnan) The number of characteristic absorption peaks in the ultraviolet region is 2, the peak shapes are consistent and the positions are close, among which the first absorption peak is located at 474-479nm, and the second absorption peak is located at 543-550nm.

[0042] Figure 2 The UV-visible spectra of the soapberry peel extracts from different origins, A2 (Zhenfeng, Guizhou), B2 (Suzhou, Anhui, Anning, Yunnan), C2 (Kunshan, Jiangsu), D2 (Dongyang, Zhejiang), and E2 (Wuhan, Hubei) all have 1 characteristic absorption peak in the UV region, the peak shape is consistent and the position is similar, and the absorption peak is located at 471-472nm.

[0043] Figure 1 A1 and Figure 2 A2 is from Bike Village, Yongfeng Street, Zhenfeng County, Qianxinan Prefecture, Guizhou Province, at 25.45 north latitude, 105.65 east longitude, and 1116.6m above sea level. The altitude, climate, and soil conditions are the same. Figure 1 , Figure 2 Obvious differences can be seen in the results, and the source varieties can be distinguished as Sichuan and Yunnan soapberry and soapberry; the differences between varieties from different sources have been eliminated, indicating the practicality of this method.

[0044] Figure 1 and Figure 2 The source varieties involved involved multiple provinces and regions. The differences between the same varieties were not obvious, but the differences between different varieties were large, indicating that this method has a wide range of applications for distinguishing the peel, peel powder, and peel extract of Sichuan and Yunnan soapberry and soapberry.

[0045] In the embodiment of the present invention, 10 samples from different origins are identified by the method, and the characteristics are obvious, so that soapberries or Sichuan and Yunnan soapberries of different origins, varieties and types can be distinguished.

[0046] In summary, the spectrophotometric method described in the present invention can simply and quickly identify the peels, peel powders and peel extracts of two tree species, Sichuan and Yunnan soapberry and soapberry, and the operation is simple and the process flow is short. Not only is the instrument simple and easy to operate, but also no professional operating technology is required, which is beneficial to the promotion of daily or industrial identification to a certain extent.

Claims

1. A method and application for identifying Sichuan and Yunnan soapberry and soapberry peel, characterized in that: include: Preparation of peel powder, extraction of the powder and preparation of the test solution; Add 5% vanillin-glacial acetic acid solution and perchloric acid to the saponin solution, shake well, heat in a 70°C constant temperature water bath for 15 min, cool in an ice water bath for 3 min, and add 5 mL of glacial acetic acid solution; The UV-visible spectra of the two extracts were measured by a spectrophotometer. The two peels were distinguished by the fact that they had selective absorption in the UV-visible region, showing significantly different numbers, peak shapes and positions of characteristic peaks.

2. A spectrophotometric method for identifying Sichuan and Yunnan soapberry and soapberry peel according to claim 1, which adopts the following process: Step 1: Pretreatment of Sichuan and Yunnan soapberry and soapberry peel samples: Select fruits with golden and wrinkled surfaces and sounds when shaken, separate the peel from the seeds using a special soapberry peel separation device and clean the device, select the peel and dry it in a 60°C oven, then crush it with a multifunctional grinder and pass it through a No. 5 sieve to obtain powder; Step 2: Preparation of crude extracts of saponin from Sichuan and Yunnan soapberry and soapberry peel: weigh 2 g of the sieved powder, add 40 ml of methanol at a solid-liquid ratio of 1:20, use an ultrasonic machine to sonicate at 50°C for 1 h, then heat under reflux at 80°C for 4 h, filter, and evaporate to obtain a crude extract of saponin; Step 3: Preparation of test solution extracted from Sichuan and Yunnan soapberry and soapberry peel: Weigh an appropriate amount (0.6-1.2 g) of crude saponin extract and dissolve it in a 10 mL volumetric flask with methanol to prepare a saponin solution. Use a pipette to accurately measure 0.1 mL of the solution into a test tube, then add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70°C constant temperature water bath for 15 min, cool in an ice water bath for 3 min, add 5 mL of glacial acetic acid, shake well, and obtain the test solution; Step 4: UV-visible spectra of the extracts of Sichuan and Yunnan soapberry and soapberry peel: The blank solution is used as a reference to detect the extracts. A UV-6100S UV-visible spectrophotometer is used to perform full wavelength scanning, and the Sichuan and Yunnan soapberry and soapberry peel test solutions are measured in the wavelength range of 400 to 800 nm to obtain the UV-visible spectra and maximum absorption wavelengths of the two, respectively.

3. The spectrophotometric method for distinguishing Sichuan and Yunnan soapberry and soapberry peel according to claim 2, characterized in that: Under the same extraction conditions, the spectra of the extracts of Sichuan and Yunnan soapberry and soapberry peel have obvious differences in the number, peak shape and position of characteristic absorption peaks in the ultraviolet region.

4. The 5% vanillin-glacial acetic acid solution according to step 3 of claim 2 needs to be prepared and used immediately.

5. According to step 4 of claim 2, the ultraviolet spectrum region ranges from 400 to 800 nm.

6. The method for identifying the two peels according to claim 3 is specifically that the UV-visible spectrum of the extracts of Sichuan and Yunnan soapberry and soapberry peel has 2 or 1 absorption peaks within the ultraviolet wavelength range, and the peak shape is double peak or single peak.

7. According to the absorption peaks in claim 6, the Sichuan and Yunnan soapberry peel extract has two peaks in the ultraviolet spectrum region of 400-800nm, which are bimodal and located at about 476nm and 546nm; the soapberry peel extract has one peak in the ultraviolet spectrum region of 400-800nm, which is unimodal and located at about 471nm; rapid identification and differentiation are achieved.

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