Application of harbarin or harbutin in identifying scrophularia honey
By detecting the content of harpagoside and harpagoside in Scrophularia honey, and using ultra-high performance liquid chromatography-high resolution mass spectrometry, the problem of identifying Scrophularia honey was solved, and efficient and accurate evaluation of honey authenticity and quality monitoring were achieved.
Patent Information
- Application Number
- CN202610675617.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-15
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies make it difficult to accurately distinguish Scrophularia honey from other dark-colored honeys or adulterated products, and there is a lack of clear chemical composition identification criteria, which affects the branding and market promotion of Scrophularia honey.
UHPLC-MS/MS was used to detect harpagoside and harpagoside in Scrophularia honey. These two compounds were used as characteristic markers, and Scrophularia honey was identified by detecting their contents of ≥400 μg/kg and ≥2000 μg/kg.
It provides a highly specific and sensitive identification method with a detection limit of 5-20 μg/kg, an accuracy of 87-108%, and a coefficient of variation of less than 15%, which can accurately identify Scrophularia honey and adulterated products.
Smart Images
Figure CN122330332A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical detection technology, and in particular to the application of harpagoside or harpagoside in the identification of Scrophularia honey. Background Technology
[0002] Scrophularia ( Scrophularia ningpoensi Scrophularia ningpoensis (Hemsl.) is a perennial herb belonging to the genus Scrophularia in the family Scrophulariaceae. Honey made from its nectar by bees is a unique type of honey (Chinese honeybee honey) that combines nutritional and medicinal value. This honey is deep amber in color and, compared to lighter-colored honeys, is richer in phenols and other bioactive substances. In traditional Chinese medicine, it is often used to relieve symptoms such as sore throat, dry mouth, red eyes, and intestinal pain, making it a typical medicinal honey.
[0003] However, due to its dark color, limited production, and high medicinal value, Scrophularia honey is easily counterfeited in the market by dark-colored honey or other inexpensive commercial honeys that look similar. Because there is currently a lack of clear and specific chemical components for identifying Scrophularia honey, traditional methods such as sensory evaluation and routine physicochemical analysis are insufficient to accurately distinguish it from other dark-colored honeys or adulterated products. This, to some extent, hinders the branding and market promotion of this unique traditional Chinese medicine honey.
[0004] Therefore, there is an urgent need to establish a precise identification method for Scrophularia honey based on characteristic compounds. Summary of the Invention
[0005] To address the aforementioned technical challenges, this invention involves in-depth research on a large number of Scrophularia honeys from various sources, as well as other common honeys (such as buckwheat honey, jujube honey, rice flower honey, vitex honey, longan honey, rapeseed honey, and acacia honey), employing ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HDMS) to analyze and compare their chemical components. The results revealed that Scrophularia honey generally contains high levels of harpagoside and harpagoside, while other honey varieties contain almost none of these two components.
[0006] Further analysis using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QQQ) revealed that harpagoside and harpagoside are stably present only in Scrophularia honey, with concentrations consistently within the ranges of ≥400 μg / kg and ≥2000 μg / kg, respectively. Therefore, these two compounds can serve as characteristic markers for Scrophularia honey, used for its identification or authenticity evaluation.
[0007] Based on this, the present invention proposes the following technical solution.
[0008] In a first aspect, the present invention provides the application of harpagoside and / or harpagoside in the identification of Scrophularia honey.
[0009] The chemical structural formula of harpagoside is as follows: Figure 1 As shown, the chemical structural formula of harbazoside is as follows: Figure 2 As shown.
[0010] In some implementation schemes, if the content of harpagoside in the honey sample to be tested is ≥400 μg / kg and / or the content of harpagoside is ≥2000 μg / kg, then the honey sample to be tested is determined to be Scrophularia honey.
[0011] Secondly, the present invention provides a method for identifying Scrophularia honey, comprising: detecting the content of harpagoside and / or harpagoside in the honey sample to be tested; if the content of harpagoside in the honey sample to be tested is ≥400 μg / kg and / or the content of harpagoside is ≥2000 μg / kg, then the honey sample to be tested is determined to be Scrophularia honey.
[0012] Since the contents of the two characteristic markers mentioned above are absolute contents in honey samples, they can be determined using chromatographic techniques, chromatography-mass spectrometry, or other methods during implementation. The specific detection methods do not constitute a limitation on the implementation of this invention.
[0013] As a preferred implementation scheme, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used to detect the honey sample to be tested.
[0014] Preferably, the conditions for ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection include: using a reversed-phase C18 column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous formic acid solution, and the formic acid accounts for 0.08%-0.12% of the volume of mobile phase A, and mobile phase B is acetonitrile; the flow rate is 0.2-0.4 mL / min; and the column temperature is 28-32℃.
[0015] Preferably, a gradient elution program is used, wherein the sum of the volume percentages of mobile phase A and mobile phase B during the gradient elution process is 100%, and the volume percentage of mobile phase B during the gradient elution process is as follows: 0-2 min, 95% mobile phase B; 2-5 min, 95-90% mobile phase B; 5-10 min, 90-50% mobile phase B; 10-15 min, 50-15% mobile phase B; 15-20 min, 15% mobile phase B; 20-20.1 min, 15-95% mobile phase B; 20.1-25 min, 95% mobile phase B.
[0016] Preferably, electrospray ionization mass spectrometry is used for detection, with the scanning mode being negative ion mode and multiple reaction monitoring mode; wherein, The parent ion of harbazoside is m / z 363.03, and the daughter ions are m / z 201.05 and m / z 182.97; The parent ion of harpagoside is m / z 493.22, and the daughter ions are m / z 345.04 and m / z 146.99.
[0017] When the above-mentioned preferred detection method is used to detect harbazoside and harbazoside, the accuracy is between 87% and 108%, the coefficient of variation is less than 15%, and the detection limit can reach 5~20 μg / kg, which fully meets the requirements of analytical detection.
[0018] In some implementation schemes, the honey sample to be tested is pretreated before UHPLC-QQQ detection. The pretreatment includes: weighing the honey sample to be tested, dissolving it in ultrapure water, purifying and enriching it with a solid phase extraction column (such as Oasis HLB) (washing with water and methanol in sequence), collecting the eluent, concentrating it with nitrogen blowing, redissolving it (preferably with 80% methanol aqueous solution), filtering it through a membrane, and then detecting it on the instrument.
[0019] Thirdly, the present invention provides the application of the identification method for Scrophularia honey in the identification of the authenticity of Scrophularia honey.
[0020] Specifically, if the content of harpagoside in the honey sample to be tested is ≥400 μg / kg and / or the content of harpagoside is ≥2000 μg / kg, then the honey sample to be tested is determined to be Scrophularia honey; otherwise, it is other honey (non-Scrophularia honey) or adulterated Scrophularia honey.
[0021] Fourthly, the present invention provides the application of the identification method of Scrophularia honey in the quality monitoring of Scrophularia honey products.
[0022] Fifthly, the present invention provides the application of the identification method of Scrophularia honey in the quality evaluation of Scrophularia honey.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention marks the first discovery that harpagoside and harpagoside can serve as characteristic marker compounds for the identification of Scrophularia ningpoensis honey. The identification method for Scrophularia ningpoensis honey provided by this invention exhibits high specificity and sensitivity, with detection limits for the target compounds reaching 5–20 μg / kg, accuracy between 87–108%, and coefficients of variation all less than 15%. This identification method is simple to operate, highly efficient, and easy to promote and apply. It is of great significance for the identification and authenticity evaluation of Scrophularia ningpoensis honey and has broad application prospects. Attached Figure Description
[0024] Figure 1 This is the chemical structural formula of harpagoside.
[0025] Figure 2 Here is the chemical structural formula of harbazoside.
[0026] Figure 3 This is a secondary mass spectrum of harpagoside in a Scrophularia honey sample.
[0027] Figure 4 This is a secondary mass spectrum of harbazoside in a Scrophularia honey sample.
[0028] Figure 5 The qualitative ion chromatogram (m / z 345.04) of pure harpagoside is shown.
[0029] Figure 6 The qualitative ion chromatogram (m / z 146.99) of pure harpagoside is shown.
[0030] Figure 7 The chromatogram of harpagoside qualitative ions (m / z 345.04) in a Scrophularia honey sample.
[0031] Figure 8 The chromatogram of harpagoside qualitative ions (m / z 146.99) in a Scrophularia honey sample.
[0032] Figure 9 The qualitative ion chromatogram (m / z 201.05) of pure harbazoside is shown.
[0033] Figure 10 The qualitative ion chromatogram (m / z 182.97) of pure harbazoside is shown.
[0034] Figure 11 The chromatogram of harbazoside (m / z 201.05) in a Scrophularia honey sample is shown.
[0035] Figure 12 The chromatogram of harbazoside (m / z 182.97) in a Scrophularia honey sample is shown. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0037] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0038] Example 1 This embodiment provides a method for detecting harpagoside and harpagoside using UHPLC-QQQ, the steps of which are as follows: Sample pretreatment: Weigh 5 g of honey sample (accurate to 0.01 g), add 10 mL of ultrapure water, and vortex to dissolve completely. Activate the Waters Oasis HLB column (500 mg, 60 μm) sequentially with 5 mL of methanol and 5 mL of water. Slowly add 10 mL of honey solution to the activated column at a natural flow rate of 1 mL / min. Elute with 5 mL of ultrapure water to remove sugars, then elute with 8 mL of methanol. Collect the eluent, dry it under nitrogen at 50°C, redissolve it with 1 mL of 80% (v / v) methanol aqueous solution, pass it through a 0.22 μm nylon membrane, and collect the filtrate in a sample vial for analysis.
[0039] Liquid chromatography detection conditions: A BEH C18 (1.7 μm, 2.1 × 100 mm) column was used. The mobile phases were 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B). The gradient elution program was as follows: 0–2 min, 95% mobile phase B; 2–5 min, 95–90% mobile phase B; 5–10 min, 90–50% mobile phase B; 10–15 min, 50–15% mobile phase B; 15–20 min, 15% mobile phase B; 20–20.1 min, 15–95% mobile phase B; 20.1–25 min, 95% mobile phase B; the flow rate was 0.2 mL / min, the column temperature was 30 °C, and the injection volume was 2 μL.
[0040] The mass spectrometry detection conditions were as follows: electrospray ionization mass spectrometry, capillary voltage: 3.0 kV, cone voltage: 26 V, ion source temperature: 150℃, desolvation temperature: 400℃, desolvation gas flow rate: 900 L / hr, cone gas flow rate: 150 L / hr, scanning mode: negative ion mode, multiple reaction monitoring mode. The precursor ion of harpagoside was m / z 363.03, and the daughter ions were m / z 201.05 and m / z 182.97; the precursor ion of harpagoside was m / z 493.22, and the daughter ions were m / z 345.04 and m / z 146.99.
[0041] The effectiveness of the above detection methods was evaluated, and the results are as follows: The secondary mass spectrum of harpagoside in Scrophularia honey sample is as follows: Figure 3 As shown, the secondary mass spectrum of harbazoside in the Scrophularia honey sample is as follows. Figure 4 As shown, the chromatograms of the qualitative ions (m / z 345.04) and (m / z 146.99) of pure harpagoside are respectively as follows: Figure 5 and Figure 6 As shown, the chromatograms of harpagosine ions (m / z 345.04) and (m / z 146.99) in the Scrophularia honey sample are as follows: Figure 7 and Figure 8 As shown, the chromatograms of the qualitative ions (m / z 201.05) and (m / z 182.97) of pure harbazoside are respectively as follows: Figure 9 and Figure 10 As shown, the chromatograms of the qualitative ions (m / z 201.05) and (m / z 182.97) of harpain in the Scrophularia honey sample are as follows: Figure 11 and Figure 12 As shown.
[0042] Accuracy analysis: Low, medium, and high concentrations of harpagoside and harpagoside standards were added to known negative honey samples, with each concentration determined in triplicate. The results showed that the average recoveries of harpagoside and harpagoside were 87.2%–106.5% and 89.6%–107.8%, respectively, both falling within the range of 87%–108%, indicating good accuracy of the method. Specific data are shown in Table 1.
[0043] Table 1
[0044] Precision analysis: The same Scrophularia honey sample was injected six times consecutively, and the relative standard deviation (RSD) of the peak area was calculated. The RSDs of the peak areas of harpagoside and harpagoside were 4.6% and 3.8%, respectively, both less than 15%. Another sample of the same type was injected twice daily for three consecutive days. The intra-day RSDs were 5.1% and 4.2%, and the inter-day RSDs were 6.3% and 5.5%, respectively, indicating that the method has good precision.
[0045] Stability analysis: The same sample solution of Scrophularia ningpoensis and honey was analyzed at 0, 2, 4, 8, 12, and 24 hours. The peak area RSDs of harpagoside and harpagoside were 5.8% and 4.9%, respectively, both less than 15%, indicating that the test solution had good stability within 24 hours.
[0046] Analysis of limits of detection and limits of quantitation: The limit of detection for harpagoside was 5 μg / kg and the limit of quantitation was 10 μg / kg; the limit of detection for harpagoside was 20 μg / kg and the limit of quantitation was 40 μg / kg.
[0047] Example 2 In this embodiment, harpagoside and harpagoside are used as characteristic markers for the identification of commercially available Scrophularia honey. The steps are as follows: Sample sources: 10 samples labeled as Scrophularia honey were purchased from the market (and identified as Scrophularia honey through palynological analysis), and 14 other dark-colored or inexpensive honey samples (including 2 samples each of buckwheat honey, jujube flower honey, rice flower honey, vitex honey, longan honey, rapeseed honey, and acacia honey, and the types of honey have been identified by other methods).
[0048] The contents of harpagoside and harpagoside in each honey sample were blindly tested using the UHPLC-QQQ detection method of Example 1, and the consistency was compared with the identified honey types.
[0049] The results are shown in Table 2. Harpaginoside and harpagoside were detected in 10 honey samples, and their contents all met the judgment criteria set by this invention (harpaginoside ≥ 400 μg / kg, harpagoside ≥ 2000 μg / kg). Therefore, they were identified as Scrophularia honey. The identification results of this invention are completely consistent with the results of other methods. These 10 honey samples are all Scrophularia honey. Harpaginoside and harpagoside were not detected in 14 honey samples, and therefore they were identified as non-Scrophularia honey. The identification results of this invention are completely consistent with the results of other methods.
[0050] Table 2
[0051] Note: "-" indicates that it was not detected in the honey sample.
[0052] The results show that harpagoside and harpagoside can be used as characteristic markers for the accurate identification of Scrophularia honey. Moreover, the identification method of the present invention can accurately identify Scrophularia honey and can also identify adulterated Scrophularia honey.
[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Application of harpagoside and / or harpagoside in the identification of Scrophularia honey.
2. The application according to claim 1, characterized in that, If the content of harpagoside in the honey sample to be tested is ≥400 μg / kg and / or the content of harpagoside is ≥2000 μg / kg, then the honey sample to be tested is determined to be Scrophularia honey.
3. A method for identifying Scrophularia honey, characterized in that, include: The content of harpagoside and / or harpagoside in the honey sample to be tested is detected. If the content of harpagoside in the honey sample to be tested is ≥400 μg / kg and / or the content of harpagoside is ≥2000 μg / kg, then the honey sample to be tested is determined to be Scrophularia honey.
4. The identification method according to claim 3, characterized in that, The honey samples were analyzed using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry.
5. The identification method according to claim 4, characterized in that, The conditions for ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection include: using a reversed-phase C18 column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous formic acid solution, and the formic acid accounts for 0.08%-0.12% of the volume of mobile phase A, and mobile phase B is acetonitrile; the flow rate is 0.2-0.4 mL / min; and the column temperature is 28-32℃.
6. The identification method according to claim 5, characterized in that, A gradient elution process was used, in which the sum of the volume percentages of mobile phase A and mobile phase B was 100%. The volume percentage of mobile phase B during the gradient elution process is as follows: 0-2 min, 95% mobile phase B; 2-5 min, 95-90% mobile phase B; 5-10 min, 90-50% mobile phase B; 10-15 min, 50-15% mobile phase B; 15-20 min, 15% mobile phase B; 20-20.1 min, 15-95% mobile phase B; 20.1-25 min, 95% mobile phase B.
7. The identification method according to any one of claims 4 to 6, characterized in that, Electrospray mass spectrometry was used for detection, with scanning in negative ion mode and multiple reaction monitoring mode; among which... The parent ion of harbazoside is m / z 363.03, and the daughter ions are m / z 201.05 and m / z 182.97; The parent ion of harpagoside is m / z 493.22, and the daughter ions are m / z 345.04 and m / z 146.
99.
8. The application of the identification method of Scrophularia honey according to any one of claims 3 to 7 in the identification of the authenticity of Scrophularia honey.
9. The application of the identification method of Scrophularia honey according to any one of claims 3 to 7 in the quality monitoring of Scrophularia honey products.
10. The application of the identification method of Scrophularia honey according to any one of claims 3 to 7 in the quality evaluation of Scrophularia honey.