Quality detection method of health care wine containing Chinese yam in raw materials

Abstract

The invention relates to a method for detecting the quality of health-care wine containing Chinese yam in raw materials, which comprises the following steps of: concentrating a wine body, separating impurities, performing chromatographic separation on a sample under a specific wavelength by adopting a liquid chromatography technology, and judging whether the Chinese yam adopted in the health-care wine body is peeled Chinese yam or not through a chromatographic peak. According to the method, whether the used medicinal raw materials meet the pharmacopoeia standard or not can be judged by measuring the health-care wine, whether the quality of the health-care wine raw materials meets the requirement or not can be effectively judged, unqualified Chinese yams are prevented from being mistakenly used in production of the health-care wine, and the quality safety of the health-care wine product is ensured.

Description

Technical Field

[0001] This invention relates to the field of health wine quality testing technology, specifically to a method for testing the quality of health wine containing yam as a raw material. Background Technology

[0002] Yam is the dried rhizome of the Dioscorea opposita plant, belonging to the Dioscoreaceae family. The processing methods for yam all involve peeling, which helps maintain the pure white color of the yam flesh. However, the special saponins in the yam peel are irritating and can cause local itching and swelling upon skin contact. Therefore, the pharmacopoeia specifies that yam processing includes peeling, but there is no corresponding testing method to confirm whether the peel has been removed. Meanwhile, some studies have shown that yam peel contains various active ingredients and applications. For example, Chinese patent application CN115671195A discloses that yam peel contains various functional components such as total saponins, total polysaccharides, total flavonoids, and total polyphenols. Chinese patent application CN117106324A discloses a method for preparing pigments using yam peel. Because yam peel contains certain active ingredients and has color-adjusting properties, and because the peeling process for yam is more complex than the drying process for unpeeled yam, there is a possibility that some health wines containing yam may contain products made from yams with the peel on. Once the medicinal material is extracted and incorporated into the wine, it is difficult to detect whether the yam used has been peeled based solely on the appearance of the wine or conventional testing. Given this potential quality risk, it is essential to establish a targeted quality testing method for health wines. This method would determine whether the yam raw materials used in health wines containing yam meet the pharmacopoeia processing standards, thereby ensuring the safety of the product and the production process.

[0003] Yam contains polar compounds such as allantoin, as well as weakly polar compounds adenosine and diosgenin I. These three compounds coexist in yam, with varying concentrations in the peel and the yam itself. After the health wine is prepared, due to the complex composition of various medicinal ingredients, the detection process requires impurity removal and concentration to avoid interference before determining the content of trace components. The usual impurity removal process does not simultaneously collect and measure polar and weakly polar compounds, missing crucial information about the medicinal materials used in the health wine. High-performance liquid chromatography (HPLC) for the determination of compounds in health wine typically uses single-wavelength measurement, making it difficult to simultaneously measure some multiple compounds at a single wavelength. Based on the above, there is currently no existing method capable of simultaneously determining allantoin, adenosine, and diosgenin I in health wine for determining whether the yam in health wine has been peeled, severely impacting the ability to assess the quality of yam-containing health wine products. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for quality testing of health wines containing yam as a raw material, determining whether the medicinal materials used in the health wine meet the processing requirements of the pharmacopoeia standards. This method effectively determines whether the quality of health wines containing yam meets the requirements, avoiding the misuse of substandard yam in health wine production and the difficulty in judging the quality of raw materials from the appearance of the wine. It provides a method for tracing the compliance of raw materials from the product, ensuring the quality and safety of the health wine.

[0005] The technical solution provided by this invention is as follows: A method for quality testing of health wine containing yam as a raw material, comprising the following steps: Step 1: Take the health wine containing yam and use a water bath to evaporate it dry to remove low-boiling-point substances. Step 2: Dissolve the evaporation residue from Step 1 in water, transfer it to a solid-phase extraction column for elution and separation, collect the filtrate, and then elute with water. Combine the resulting water eluent with the filtrate to obtain a mixed solution; then elute with 30% methanol solution, discard the eluent, and continue eluting with methanol, collecting the methanol eluent. Step 3: Evaporate the mixed solution and methanol eluent to dryness separately. Dissolve the residue in 90% methanol solution and bring the volume to a certain level to obtain eluent I and eluent II. Mix eluent I and eluent II in a 1:1 volume ratio, filter, and obtain the test solution. Step 4: Accurately weigh a certain amount of allantoin, adenosine, and yam extract I as standard substances, dissolve them in solvent and make up to a certain volume, shake well, filter, and take the filtrate as the standard substance solution. Step 5: Determine the test solution and standard substance solution using high performance liquid chromatography; Step Six: Determine if the raw material in the health wine is peeled yam: If the chromatogram of the test sample solution shows chromatographic peaks at the same retention time in the solutions of allantoin, adenosine, and yam extract I, then the yam raw material in the health wine prepared from the test sample solution is peeled yam; if the chromatogram of the test sample solution shows chromatographic peaks at the same retention time in the solutions of allantoin and adenosine, but no corresponding chromatographic peak at the same retention time in the solution of yam extract I, then the yam raw material in the health wine prepared from the test sample solution is peeled yam.

[0006] Preferably, in step one, 10 mL of the health wine containing yam should be precisely measured; the water bath evaporation temperature is 90°C.

[0007] Preferably, in step two, the residue is dissolved and evaporated in 5 mL of water; the solid phase extraction column used for solid phase extraction is a hydrophilic-lipophilic balanced (HLB) column, which needs to be activated before use.

[0008] Preferably, the elution solvent volumes in step two are: 20 mL of water, 5 mL of 30% methanol, and 10 mL of methanol.

[0009] Preferably, in steps three and four, the volume of the final volume is 1 mL to 25 mL, including 1 mL, 2 mL, 5 mL, 10 mL and 25 mL.

[0010] Preferably, in step four, the solvent used for dissolving and adjusting the volume of the three standard substances is 90% methanol, and the concentration of the three standard substances prepared is in the range of 50-400 mg / L.

[0011] Preferably, in step five, the liquid chromatography conditions are as follows: octadecylsilane-bonded silica gel column; column temperature 30℃; mobile phase: methanol / 0.05% phosphoric acid / water gradient elution; flow rate 1.0 mL / min; detection wavelength 230 nm for 0–7 min and 258 nm for 7–65 min; injection volume of both the test solution and standard solution is 10 μL; gradient elution conditions are as follows:

[0012] Preferably, the chromatographic column has a specification of 4.6×250mm and 5μm.

[0013] Compared with the prior art, the present invention has the following effects: This invention provides a method for quality testing of health wine containing yam as a raw material. The method uses chromatographic peaks to determine whether the yam used in the health wine is peeled. This method effectively traces the quality compliance of the raw materials used in health wine production, preventing the misuse of substandard yam and the resulting substandard health wine from entering the market, thus ensuring the safety of the health wine. Attached Figure Description

[0014] Figure 1 Chromatogram of a sample of health wine brewed from yam with skin; Figure 2 Chromatogram of a health wine sample made from peeled yam; Figure 3 Chromatogram of allantoin standard solution; Figure 4 Chromatogram of adenosine standard solution; Figure 5 Chromatogram of yam extract I standard solution; Figure 6 Comparative chromatograms of test solutions after different elution programs on solid-phase extraction columns in Example 1 and Comparative Example 1; Figure 7 The chromatograms of the test sample solutions under different single-wavelength conditions in Example 1 and Comparative Example 1 are shown. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 1. Quality control of health wines containing yam as a medicinal ingredient 1.1 Instrument Conditions High-performance liquid chromatograph (HPLC): equipped with an ultraviolet detector or a diode array detector, with a detector wavelength coverage range of 220–270 nm.

[0017] Electronic balance: Electronic balance with a measurement accuracy of one ten-thousandth.

[0018] Pure water system: It can produce Class I water for laboratories, and the produced Class I water meets the requirements of GB / T 6682-2008 Specifications and test methods for water used in analytical laboratories.

[0019] 1.2 Test drugs and consumables 1.2.1 Standard substances: allantoin, adenosine, and yam extract I standard substances.

[0020] 1.2.2 Acetonitrile and phosphoric acid: chromatographic grade reagents; pure water was first-grade test water prepared by a pure water machine.

[0021] 1.2.3 Solid phase extraction column: HLB (hydrophilic-lipophilic balanced) solid phase extraction column, 200mg / 6mL, purchased from Shenzhen DouDian Biotechnology Co., Ltd.

[0022] 1.2.4 Needle filter: PVDF (polyvinylidene fluoride), 0.22μm, purchased from Shanghai Hanyao Instrument Equipment Co., Ltd.

[0023] 1.3 Preparation of the test solution: 1.3.1 Activation of solid phase extraction column: Take a solid phase extraction column, add 10 mL of methanol to elute, then elute with 20 mL of water, and set aside for use.

[0024] 1.3.2 Accurately measure 10 mL of health wine containing yam, evaporate to dryness in a 90℃ water bath, add 5 mL of water to the residue to fully dissolve it, transfer to a solid-phase extraction column, collect the filtrate, and then elute with 20 mL of water, combining it with the previously collected filtrate (A). Elute with 5 mL of 30% methanol, discarding the eluent; elute with 10 mL of methanol, collecting the eluent (B). Evaporate both A and B separately in a water bath to dryness, dissolve the residue in 90% methanol solution, and dilute to a 5 mL volumetric flask, shaking well to obtain eluent I and eluent II. Mix eluent I and eluent II in a 1:1 volume ratio, and filter through a 0.22 μm (PVDF) syringe filter to obtain the test solution.

[0025] 1.3.3 The samples of health wine with skin and health wine without skin were processed according to 1.3.1-1.3.2 to obtain test solutions of health wine with skin and health wine without skin, respectively.

[0026] 1.4 Preparation of standard substance solutions: Accurately measure appropriate amounts of allantoin, adenosine, and yam extract I standard substances, dissolve them in 90% methanol, and dilute to a volumetric flask of 25 mL. The concentrations of the three standard substances prepared are 84.8 mg / L, 156.8 mg / L, and 389.6 mg / L, respectively, which are used as standard substance solutions.

[0027] 1.5 Chromatographic conditions: The chromatographic column was an octadecylsilane-bonded silica column (4.6 × 250 mm, 5 μm); the column temperature was 30℃; the mobile phase was a gradient elution of methanol / 0.05% phosphoric acid / water (elution gradient shown in Table 1); the flow rate was 1.0 mL / min; the detection wavelength was 230 nm from 0 to 7 min and 258 nm from 7 to 65 min. The injection volume of both the test sample and the standard solution was 10 μL.

[0028] Table 1 Elution gradient conditions

[0029] 1.6 Determination of Target Peak Both the standard solution and the test solution were injected into the liquid chromatograph in a volume of 10 μL. Chromatographic separation was performed under chromatographic conditions 1.5, and spectra were collected. Chromatograms of each standard solution and the test solution were obtained. Qualitative analysis was performed based on retention time, and the peak areas of allantoin, adenosine, and diosgenin I in the test solution were recorded.

[0030] The obtained chromatogram is attached. Figure 1-5 The peak areas of allantoin, adenosine, and yamin I in the test solutions of yam health wine with and without peel are as follows: Table 2 Peak areas of adenosine and yamin I

[0031] 2. Determine whether the yam used in the wine has been peeled. In the chromatogram of the test solution of the yam health wine with peel, chromatographic peaks were observed at the same retention times in the solutions of allantoin, adenosine, and yam extract I. The peak area of ​​yam extract I was 446.9 mAu·S. This indicates that the yam raw material used in the preparation of the health wine was yam with peel, consistent with the yam raw material used in the health wine. In the chromatogram of the test solution of the health wine with peeled yam, chromatographic peaks were observed at the same retention times in the solutions of allantoin and adenosine, but no corresponding chromatographic peak was observed at the same retention time in the solution of yam extract I. This indicates that the yam raw material used in the preparation of the health wine was yam with peel, consistent with the yam raw material used in the health wine.

[0032] Comparative Example 1 1.1 Instrument Conditions High-performance liquid chromatograph (HPLC): equipped with an ultraviolet detector or a diode array detector, with a detector wavelength coverage range of 220–270 nm.

[0033] Electronic balance: Electronic balance with a measurement accuracy of one ten-thousandth.

[0034] Pure water system: It can produce Class I water for laboratories, and the produced Class I water meets the requirements of GB / T 6682-2008 Specifications and test methods for water used in analytical laboratories.

[0035] 1.2 Test drugs and consumables 1.2.1 Standard substances: allantoin, adenosine, and yam extract I standard substances.

[0036] 1.2.2 Acetonitrile and phosphoric acid: chromatographic grade reagents; pure water was first-grade test water prepared by a pure water machine.

[0037] 1.2.3 Solid phase extraction column: HLB (hydrophilic-lipophilic balanced) solid phase extraction column, 200mg / 6mL, purchased from Shenzhen DouDian Biotechnology Co., Ltd.

[0038] 1.2.4 Needle filter: PVDF (polyvinylidene fluoride), 0.22μm, purchased from Shanghai Hanyao Instrument Equipment Co., Ltd.

[0039] 1.3 Preparation of the test solution: 1.3.1 Activation of solid phase extraction column: Take a solid phase extraction column, add 10 mL of methanol to elute, then elute with 20 mL of water, and set aside for use.

[0040] 1.3.2 Accurately measure 10 mL of health wine containing yam with peel, evaporate to dryness in a 90℃ water bath, add 5 mL of water to the residue to fully dissolve it, transfer to a solid-phase extraction column, collect the filtrate, elute with 20 mL of water, combine with the previously collected filtrate (A), and then elute with 10 mL of methanol, collecting the eluent (B). Evaporate the collected eluents A and B to dryness in a water bath, add 90% methanol solution, and dilute to a 5 mL volumetric flask, shake well, and use as eluent I and eluent II. Mix eluent I and eluent II at a volume ratio of 1:1, and filter through a 0.22 μm (PVDF) syringe filter to obtain the test solution.

[0041] 1.4 Preparation of standard substance solutions: Accurately measure appropriate amounts of allantoin, adenosine, and yam extract I standard substances, dissolve them in 90% methanol, and dilute to a volumetric flask of 25 mL to obtain the standard substance solution.

[0042] 1.5 Chromatographic conditions: The chromatographic column was an octadecylsilane-bonded silica column (4.6×250mm, 5μm); the column temperature was 30℃; the mobile phase was acetonitrile / water / 0.05% phosphoric acid gradient elution (elution gradient is shown in Table 3); the flow rate was 1.0 mL / min; and the detection wavelengths were 230 nm and 258 nm.

[0043] Table 3 Elution gradient

[0044] 1.6 Determination of Target Peak Both the standard solution and the test solution were injected into the high-performance liquid chromatograph (HPLC) at an injection volume of 10 μL. Chromatographic separation was performed under chromatographic conditions 1.5, and spectra were acquired. The peak areas of the target peaks in the standard solution and the test solution were obtained, and qualitative analysis was performed based on the retention times.

[0045] 1) The sample was processed using a solid-phase extraction column, but elution with 30% methanol was not used. Interfering peaks were present near the adenosine peak in the sample solution, affecting the adenosine peak area. In Example 1, the addition of 30% methanol elution effectively removed this interference; Example 1 is superior to Comparative Example 1. Chromatogram comparison is provided below. Figure 6 As shown.

[0046] 2) In the same test solution, allantoin showed a significantly higher chromatographic peak at 230 nm than at 258 nm; adenosine and diosgenin I showed obvious absorption peaks at 258 nm, but no obvious absorption peaks at 230 nm. Using a single wavelength method requires two detections to obtain the chromatographic peaks of all three compounds, while using the chromatographic conditions in Example 1, only a single measurement is needed to simultaneously acquire the chromatograms of all three compounds. Example 1 is more efficient than Comparative Example 1. Figure 7 As shown.

[0047] In comparison, the method in Example 1 is superior to that in Comparative Example 1. This method is highly efficient, and the method in Example 1 can be used to determine whether the yam raw materials in yam-containing health wines meet the pharmacopoeia processing standards.

[0048] The above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all aspects of the invention. Any modifications and refinements made by those skilled in the art without departing from the spirit and scope of the present invention are considered to fall within the protection scope of the claims of the present invention.

Claims

1. A method for quality testing of health wine containing yam as a raw material, characterized in that, Includes the following steps: Step 1: Take the health wine containing yam and use a water bath to evaporate it dry to remove low-boiling-point substances. Step 2: Dissolve the evaporation residue from Step 1 in water, transfer it to a solid-phase extraction column for elution and separation, collect the filtrate, and then elute with water. Combine the resulting water eluent with the filtrate to obtain a mixed solution; then elute with 30% methanol solution, discard the eluent, and continue eluting with methanol, collecting the methanol eluent. Step 3: Evaporate the mixed solution and methanol eluent to dryness separately. Dissolve the residue in 90% methanol solution and bring the volume to a certain level to obtain eluent I and eluent II. Mix eluent I and eluent II in a 1:1 volume ratio, filter, and obtain the test solution. Step 4: Accurately weigh a certain amount of allantoin, adenosine, and yam extract I as standard substances, dissolve them in solvent and make up to a certain volume, shake well, filter, and take the filtrate as the standard substance solution. Step 5: Determine the test solution and standard substance solution using high performance liquid chromatography; Step Six: Determine if the raw material in the health wine is peeled yam: If the chromatogram of the test sample solution shows chromatographic peaks at the same retention time in the solutions of allantoin, adenosine, and yam extract I, then the yam raw material in the health wine prepared from the test sample solution is peeled yam; if the chromatogram of the test sample solution shows chromatographic peaks at the same retention time in the solutions of allantoin and adenosine, but no corresponding chromatographic peak at the same retention time in the solution of yam extract I, then the yam raw material in the health wine prepared from the test sample solution is peeled yam.

2. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In step one, 10 mL of the health wine containing yam should be precisely measured; the water bath evaporation temperature is 90℃.

3. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In step two, the residue is dissolved and evaporated in 5 mL of water; the solid phase extraction column used for solid phase extraction is a hydrophilic-lipophilic balanced (HLB) column, which needs to be activated before use.

4. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In step two, the elution solvent volumes are: 20 mL of water, 5 mL of 30% methanol, and 10 mL of methanol.

5. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In steps three and four, the volume of the final volume is 1 mL to 25 mL.

6. The method for quality testing of a health wine containing yam as a raw material according to claim 5, characterized in that, The final volume is 1 mL, 2 mL, 5 mL, 10 mL, or 25 mL.

7. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In step four, the solvent used for dissolving and adjusting the volume of the three standard substances is 90% methanol, and the concentrations of the three standard substances prepared are in the range of 50-400 mg / L.

8. The method for quality testing of a health wine containing yam as a raw material according to claim 1, characterized in that, In step five, the liquid chromatography conditions are as follows: the column is an octadecylsilane-bonded silica gel column; the column temperature is 30℃; the mobile phase is a gradient elution of methanol / 0.05% phosphoric acid / water; the flow rate is 1.0 mL / min; the detection wavelength is 230 nm from 0 to 7 min and 258 nm from 7 to 65 min; the injection volume of both the test sample and the standard solution is 10 μL; the gradient elution conditions are as follows: 。 9. A method for quality testing of a health wine containing yam as a raw material, as described in claim 8, characterized in that, The chromatographic column has a specification of 4.6×250mm and 5μm.

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