Method for detecting aloe polysaccharide effective component in aloe raw material

Abstract

This application relates to the field of chemical analysis technology, specifically to a method for detecting the effective components of aloe polysaccharides in aloe vera raw materials. The method includes: obtaining a chromatogram of an aloe vera sample hydrolysate; determining the chromatographic peak corresponding to acetic acid from multiple chromatographic peaks based on the retention time and peak shape characteristics of each chromatographic peak; determining the amount of acetic acid in the aloe vera sample hydrolysate based on the hydrolysis completeness at a current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at that current moment, wherein the hydrolysis completeness at a given moment is used to characterize the degree of hydrolysis of the aloe vera sample at that moment; and determining the content of the effective polysaccharide components in the aloe vera sample based on the amount of acetic acid. This method can obtain an accurate amount of acetic acid, and based on this accurate amount of acetic acid, the content of the effective polysaccharide components in the aloe vera sample can be accurately detected.

Description

Technical Field

[0001] This application relates to the field of chemical analysis technology, specifically to a method for detecting the effective components of aloe polysaccharides in aloe raw materials. Background Technology

[0002] In related technologies, the content of acetic acid can be determined by traditional polysaccharide content determination methods, such as the method of measuring reducing sugar after hydrolysis, based on the chromatographic detection results obtained from hydrolysis, and then the effective components of aloe polysaccharides can be evaluated based on the acetic acid content.

[0003] However, in the above method, the acetic acid content obtained from chromatographic detection is actually affected by other organic acids, which leads to inaccurate determination of the acetic acid content and reduces the reliability of the detection of effective components of aloe polysaccharides. Summary of the Invention

[0004] To address the technical problem of inaccurate acetic acid content determination, which reduces the reliability of aloe polysaccharide detection, this application aims to provide a method for detecting aloe polysaccharide in aloe raw materials. The specific technical solution adopted is as follows:

[0005] This application provides a method for detecting the effective components of aloe polysaccharides in aloe raw materials, including:

[0006] A chromatogram of the hydrolysate of an aloe vera sample is obtained, which includes multiple chromatographic peaks. Based on the retention time and peak shape characteristics of each chromatographic peak, the chromatographic peak corresponding to acetic acid is identified from these multiple chromatographic peaks. Based on the hydrolysis completeness at the current time and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current time, the amount of acetic acid in the hydrolysate of the aloe vera sample is determined. The hydrolysis completeness at a certain time is used to characterize the degree of hydrolysis of the aloe vera sample at that time. Based on the amount of acetic acid, the content of polysaccharide active ingredients in the aloe vera sample is determined.

[0007] Optionally, the above method of determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak retention time and peak shape characteristics of each chromatographic peak includes: obtaining the standard peak retention time and standard peak shape characteristics of acetic acid; and determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak retention time and peak shape characteristics of each chromatographic peak, the standard peak retention time, and the standard peak shape characteristics.

[0008] Optionally, obtaining the standard peak retention time and standard peak shape characteristics of acetic acid as described above includes: obtaining an acetic acid chromatogram, which is a chromatogram of an acetic acid standard solution under the same conditions; and determining the standard peak retention time and standard peak shape characteristics based on the acetic acid chromatogram.

[0009] Optionally, the above-mentioned determination of the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the retention time and peak shape characteristics of each chromatographic peak, the retention time of the standard peak, and the standard peak shape characteristics includes: determining the retention time drift of each chromatographic peak based on the retention time of each chromatographic peak and the retention time of the standard peak, wherein the retention time drift of a chromatographic peak is used to characterize the difference between the retention time of a chromatographic peak and the retention time of the standard peak; determining the peak shape regularity of each chromatographic peak based on the peak shape characteristics of each chromatographic peak and the standard peak shape characteristics, wherein the peak shape regularity of a chromatographic peak is used to characterize the similarity between the peak shape characteristics of a chromatographic peak and the standard peak shape characteristics; and determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the retention time drift and the peak shape regularity of each chromatographic peak.

[0010] Optionally, the above-mentioned determination of the acetic acid peak from the plurality of chromatographic peaks based on the retention time drift and peak shape regularity of each chromatographic peak includes: determining the ratio of the retention time drift of each chromatographic peak to its respective peak shape regularity as the acetic acid peak verification score of each chromatographic peak, wherein the acetic acid peak verification score of a chromatographic peak is used to characterize the similarity between a chromatographic peak and a real acetic acid peak; and determining the acetic acid peak from the plurality of chromatographic peaks based on the acetic acid peak verification score of each chromatographic peak.

[0011] Optionally, the above-mentioned determination of the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the acetic acid peak verification score of each chromatographic peak includes: determining the peak area increase of each chromatographic peak in response to the addition of standard acetic acid solution to the hydrolysate of the aloe vera sample; and determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak area increase of each chromatographic peak and the acetic acid verification score of each chromatographic peak.

[0012] Optionally, the above-mentioned determination of the chromatographic peak corresponding to acetic acid from the multiple chromatographic peaks based on the peak area increase of each chromatographic peak and the acetic acid verification score of each chromatographic peak includes: obtaining the theoretical increase value of the peak area of ​​the standard acetic acid solution; determining the acetic acid peak probability of each chromatographic peak based on the peak area increase of each chromatographic peak, the theoretical increase value of the peak area of ​​the standard acetic acid solution, and the acetic acid verification score of each chromatographic peak; and determining the chromatographic peak with the highest probability of acetic acid as the chromatographic peak corresponding to acetic acid.

[0013] Optionally, the above method further includes: obtaining the peak area change of the chromatographic peak corresponding to the acetic acid during the hydrolysis process; determining the maximum peak area during the hydrolysis process based on the peak area change; and determining the completeness of hydrolysis at the current moment based on the peak area at the current moment, the maximum peak area, and the slope of the change between the peak area at the current moment and the peak area at the previous moment.

[0014] Optionally, determining the amount of acetic acid in the aloe vera sample hydrolysate based on the hydrolysis completeness at the current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment includes: obtaining the expected peak area of ​​the internal standard and the measured peak area of ​​the internal standard at the current moment; determining the ratio of the expected peak area of ​​the internal standard to the measured peak area of ​​the internal standard as the hydrolysis efficiency at the current moment; determining the optimized acetic acid peak area based on the hydrolysis efficiency at the current moment, the hydrolysis completeness at the current moment, and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment; and determining the amount of acetic acid in the aloe vera sample hydrolysate based on the optimized acetic acid peak area and the acetic acid standard curve, wherein the acetic acid standard curve is used to characterize the linear relationship between peak area and concentration.

[0015] Optionally, the above-mentioned active ingredient includes acetyl groups, and the determination of the polysaccharide active ingredient content in the aloe vera sample based on the amount of acetic acid includes: determining the acetyl content of the aloe vera sample based on the amount of acetic acid.

[0016] This application has the following beneficial effects:

[0017] Due to differences in molecular structure and polarity, different substances have different retention times for their chromatographic peaks. Furthermore, the chromatographic peak corresponding to acetic acid typically exhibits a regular and symmetrical waveform in chromatographic detection results. Therefore, the chromatographic peak corresponding to acetic acid can be determined based on the retention time and peak shape characteristics of each chromatographic peak. Since the degree of hydrolysis can affect the peak area change, the peak area of ​​acetic acid at the current moment can be corrected based on the completeness of hydrolysis to obtain a more accurate amount of acetic acid. Based on this accurate amount of acetic acid, the content of polysaccharide active ingredients in aloe vera samples can be accurately detected. Attached Figure Description

[0018] To more clearly illustrate the technical solutions and advantages in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A flowchart illustrating a method for detecting the effective components of aloe polysaccharides in aloe raw materials, provided as an embodiment of this application;

[0020] Figure 2 This is a schematic diagram of a peak area changing over time, provided as an embodiment of this application.

[0021] Figure 3 This is a flowchart illustrating another method for detecting the effective components of aloe polysaccharides in aloe raw materials, provided as an embodiment of this application. Detailed Implementation

[0022] To further illustrate the technical means and effects adopted by this application to achieve its intended purpose, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation, structure, features, and effects of a method for detecting the effective components of aloe polysaccharides in aloe raw materials according to this application. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] As a perennial herb with medicinal, health-promoting, and cosmetic value, aloe vera has become a hot topic in the pharmaceutical, cosmetic, and food industries for the research and application of its active ingredients. Among them, aloe polysaccharides are the core functional components of aloe vera gel, possessing various biological activities such as anti-oxidation, immune regulation, wound healing promotion, and moisturizing, directly determining the quality and efficacy of aloe vera raw materials and end products.

[0025] With the rapid development of the aloe vera industry, the quality of aloe vera raw materials and products on the market (such as aloe vera gel, aloe vera beverages, health products, etc.) varies greatly. Some products have problems such as insufficient polysaccharide content and adulteration with other sugars, which not only affects consumer rights but also restricts the standardized development of the industry. Therefore, establishing accurate and efficient aloe vera polysaccharide testing methods has become a key link in ensuring the quality of raw materials, standardizing production processes, and promoting industrial upgrading.

[0026] In existing technologies, traditional methods for determining polysaccharide content, such as the method of measuring reducing sugars after hydrolysis, can only provide a vague "content" value based on the total amount of monosaccharide residues due to their inherent destructive nature. They are completely unable to obtain the core structural information that determines the biological activity of polysaccharides (especially molecular weight, glycosidic bond type, and key substituents such as acetyl groups). Furthermore, in the process of inferring acetyl content from acetic acid content, the identification of the target peak of acetic acid content in the chromatographic detection results is interfered with by other organic acids. Moreover, incomplete or excessive hydrolysis during the hydrolysis process can also affect the actual acetyl content in aloe vera samples, thus reducing the reliability of the detection of effective components of aloe vera polysaccharides.

[0027] The following describes in detail, with reference to the accompanying drawings, a specific scheme for the detection method of aloe polysaccharide effective components in aloe raw materials provided in this application.

[0028] Please see Figure 1 The document illustrates a flowchart of a method for detecting the effective components of aloe polysaccharides in aloe raw materials according to an embodiment of this application.

[0029] like Figure 1 As shown, the detection methods for the effective components of aloe polysaccharides in the aloe raw material include S101-S104.

[0030] S101. Obtain the chromatogram of the hydrolysate of the aloe vera sample.

[0031] The chromatogram includes multiple chromatographic peaks.

[0032] In one alternative implementation, an aloe vera sample can be obtained, and a pre-set sulfuric acid solution can be added to the aloe vera sample. After sealing, the sample can be placed in a 100°C water bath for 4 hours for hydrolysis. Then, high-performance liquid chromatography (HPLC) can be used to detect the chromatogram of the aloe vera sample hydrolysate.

[0033] It should be understood that each chromatographic peak corresponds to a substance that was separated from the hydrolyzed aloe vera sample.

[0034] Optionally, the preset sulfuric acid solution can be 5 mL of sulfuric acid solution with a concentration of 2 mol / L.

[0035] S102. Based on the retention time and peak shape characteristics of each chromatographic peak, determine the chromatographic peak corresponding to acetic acid from multiple chromatographic peaks.

[0036] It should be understood that in addition to acetic acid, the hydrolysate of aloe vera samples may also contain other organic acids (such as citric acid, malic acid, etc.) or small molecule impurities released by hydrolysis. These substances will also form chromatographic peaks in the chromatogram. Different substances have different retention times in the chromatographic column due to differences in molecular structure and polarity. Therefore, the chromatographic peak corresponding to acetic acid can be determined based on the peak retention time of each chromatographic peak.

[0037] Understandably, because acetic acid has a simple molecular structure and exists in molecular form in an acidic mobile phase, the peak shape of acetic acid in chromatographic detection results usually presents a regular and symmetrical waveform. Therefore, the chromatographic peak corresponding to acetic acid can be determined based on the peak shape characteristics of each peak. Furthermore, the higher the waveform symmetry of a chromatographic peak, the greater the likelihood that the peak corresponds to acetic acid.

[0038] S103. Based on the completeness of hydrolysis at the current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment, determine the amount of acetic acid in the hydrolysate of the aloe vera sample.

[0039] The completeness of hydrolysis at a given moment is used to characterize the degree of hydrolysis of the aloe vera sample at that moment.

[0040] It should be understood that during the hydrolysis of aloe vera samples to produce acetic acid, the degree of hydrolysis can affect the accuracy of the peak area corresponding to acetic acid in the chromatographic detection results. If hydrolysis is incomplete (e.g., insufficient acid concentration, low temperature, or too short time), some acetyl groups will not be converted into acetic acid, resulting in a smaller peak area of ​​acetic acid detected by chromatography, and the final calculated acetyl content will be lower than the actual value. Conversely, if hydrolysis is excessive (e.g., prolonged reaction under strong acid and high temperature), it may trigger the degradation of the polysaccharide backbone or hydrolysis side reactions of acetic acid, such as acetic acid volatilization, decomposition, or consumption by impurities, leading to a smaller peak area of ​​acetic acid and introducing deviation.

[0041] Therefore, based on the completeness of hydrolysis at this current moment, the peak area of ​​the chromatographic peak corresponding to acetic acid in the chromatogram can be optimized.

[0042] Optionally, the current time can be the current detection time.

[0043] Understandably, during the hydrolysis of aloe vera samples, in the initial stage, as hydrolysis conditions (such as prolonged time and increased temperature) are strengthened, the rate of ester bond breakage accelerates, and the amount of acetic acid produced gradually increases. Once most acetyl groups have been converted into acetic acid (i.e., hydrolysis is complete), further strengthening of conditions (such as extending the time) no longer significantly increases the amount of acetic acid produced, at which point hydrolysis can be considered complete. Therefore, by detecting the actual amount of acetic acid, i.e., the change in peak area in the chromatographic detection results, the completeness of hydrolysis can be reflected.

[0044] In one alternative implementation, the peak area change of the chromatographic peak corresponding to the acetic acid during the hydrolysis process can be obtained; based on the peak area change, the maximum peak area during the hydrolysis process can be determined; based on the peak area at the current moment, the maximum peak area, and the slope of the change between the peak area at the current moment and the peak area at the previous moment during the hydrolysis process, the completeness of hydrolysis at the current moment can be determined.

[0045] Optionally, the peak area of ​​the chromatographic peak corresponding to acetic acid can be plotted as a function of time with hydrolysis time as the x-axis and the peak area of ​​the acetic acid peak as the y-axis. This curve can more intuitively reflect the hydrolysis process and more intuitively observe the maximum peak area and the slope of the change between the peak area at the current moment and the peak area at the previous moment.

[0046] It should be understood that when the peak area curve shows an upward trend over time, it indicates that the aloe vera sample is not completely hydrolyzed, meaning that there are still acetyl groups that have not been hydrolyzed; when the peak area curve shows a flattening trend over time, it indicates that the acetyl groups have been basically completely hydrolyzed, and the amount of acetic acid produced has reached a stable level; when the peak area curve shows a downward trend over time, it indicates that the hydrolysis has been excessive, and the amount of acetic acid decreases due to side reactions.

[0047] It is understandable that the maximum peak area is the peak area under ideal hydrolysis conditions.

[0048] For example, Figure 2 This is a schematic diagram showing the peak area of ​​the chromatographic peak corresponding to acetic acid as a function of time.

[0049] like Figure 2 As shown, time t1 is the peak area at its maximum value, and time t2 is the current time. Before time t1, the peak area curve shows an upward trend, indicating that the aloe vera sample is undergoing hydrolysis. After time t1, the peak area curve shows a downward trend.

[0050] Alternatively, the completeness of hydrolysis at a given time satisfies the following formula:

[0051]

[0052] in, express Completeness of hydrolysis at any given time Indicating the hydrolysis process Peak area of ​​the chromatographic peak corresponding to acetic acid at time t. This represents the maximum peak area during the hydrolysis process. express The slope of the change in peak area at time step 1 compared to the peak area at the previous time step 2.

[0053] Based on the above formula, it should be understood that, express The difference between the peak area at time t and the maximum peak area under ideal hydrolysis conditions; Represents the absolute value of the slope, combined with Figure 2 As shown, regardless of whether the slope is positive or negative, it indicates that the current amount of acetic acid is less than the actual amount of acetic acid. Specifically, when... A value greater than 0 indicates incomplete hydrolysis; as hydrolysis progresses, the degree of completeness of hydrolysis increases. The value gradually increases and tends to 1; the higher the degree of hydrolysis, the closer it is to the ideal hydrolysis state. The closer the value is to 1; when A value less than 0 indicates excessive hydrolysis. At this point, acetic acid begins to be consumed. The more excessive the hydrolysis, the more acetic acid is consumed. Compared to the actual amount of acetic acid in the hydrolysate of an actual aloe vera sample, the less acetic acid is present, indicating a less complete hydrolysis. The larger the value.

[0054] In one alternative implementation, the product of the hydrolysis completeness at the current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment can be determined as the peak area optimization amount at the current moment. Then, the sum of the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment and the peak area optimization amount can be determined as the optimized acetic acid peak area. Finally, based on the optimized acetic acid peak area, the amount of acetic acid in the hydrolysate of the aloe vera sample can be determined.

[0055] Understandably, the acetic acid peak area is optimized based on the completeness of hydrolysis: when hydrolysis is incomplete, acetic acid does not flow out completely, so the current acetic acid peak area is increased; when hydrolysis is excessive, some acetic acid has volatilized or been consumed, so the current acetic acid peak area is increased. This allows for the precise quantification of the amount of acetic acid under ideal hydrolysis conditions and precise quantification of acetic acid in the hydrolysate of aloe vera samples.

[0056] It is understandable that due to the short hydrolysis time or interruption of hydrolysis, the hydrolysis process may be incomplete at the current moment. In this case, the maximum peak area may not reflect the true maximum peak area. In this case, the peak area can be optimized based on the current hydrolysis efficiency after optimization based on the current maximum peak area.

[0057] In another alternative implementation, the expected peak area of ​​the internal standard and the measured peak area of ​​the internal standard at the current moment can be obtained; the ratio of the expected peak area of ​​the internal standard to the measured peak area of ​​the internal standard can be determined as the hydrolysis efficiency at the current moment; based on the hydrolysis efficiency at the current moment, the hydrolysis completeness at the current moment, and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment, the optimized acetic acid peak area can be determined; based on the optimized acetic acid peak area and the acetic acid standard curve, the amount of acetic acid in the hydrolysate of the aloe vera sample can be determined.

[0058] The acetic acid standard curve is used to characterize the linear relationship between peak area and concentration.

[0059] Optionally, an internal standard can be added to the hydrolysate before the aloe vera sample is hydrolyzed. The internal standard will undergo the same losses as the aloe vera sample, such as volatilization, decomposition, and injection error.

[0060] It should be understood that the change in the peak area of ​​the internal standard can reflect the hydrolysis efficiency. Therefore, the peak area of ​​acetic acid can be corrected by calculating the change in the area of ​​the internal standard.

[0061] It is understandable that the larger the ratio of the expected peak area of ​​the internal standard to the measured peak area of ​​the internal standard, the greater the difference between the measured peak area and the expected peak area. In this case, the hydrolysis efficiency is worse, and the acetic acid peak area can be optimized to a larger value.

[0062] For example, the internal standard could be, for instance, deuterated acetic acid or 2-ethylbutyric acid.

[0063] Optionally, the optimized acetic acid peak area satisfies the following formula:

[0064]

[0065] in, This represents the optimized peak area of ​​acetic acid. Indicating the hydrolysis process Peak area of ​​the chromatographic peak corresponding to acetic acid at time t. express Hydrolysis efficiency at any given time express Completeness of hydrolysis at any given time.

[0066] Based on the above formulas, it should be understood that by correcting the acetic acid peak area according to the completeness and efficiency of hydrolysis to compensate for this loss, a more accurate amount of acetic acid can be obtained. By amplifying the peak area to compensate for incompletely converted acetic acid and acetic acid consumed by side reactions, the theoretical value at complete hydrolysis is restored, correcting deviations caused by incomplete or excessive hydrolysis. This allows for precise quantification of acetic acid content, providing reliable data for calculating the acetyl content and evaluating the activity of aloe polysaccharides.

[0067] Optionally, based on the optimized acetic acid peak area and the acetic acid standard curve, the actual mass of acetic acid in the current aloe vera sample hydrolysate can be calculated.

[0068] In this embodiment, since the acetic acid peak confirmed by chromatographic detection results may contain other substances with elution times close to acetic acid, these substances may be superimposed on the corresponding chromatographic peak of acetic acid in the form of shoulder peaks or baseline elevation. Direct integration to reflect the acetic acid content may be inaccurate. Therefore, the area of ​​the acetic acid peak can be optimized based on the completeness and efficiency of hydrolysis to ensure that the integration only contains the signal of acetic acid, excluding interference regions. The final integrated value is the peak area of ​​acetic acid in the chromatographic peak.

[0069] S104. Determine the content of polysaccharide active ingredients in aloe vera samples based on acetic acid content.

[0070] It should be understood that the active ingredients in aloe vera samples include acetyl groups. The acetyl groups of aloe polysaccharides are key structural features for their activity. The presence of acetyl groups affects the water solubility of polysaccharides and their ability to bind to biological targets. Therefore, the content of acetyl groups directly reflects the structural integrity and functional activity of polysaccharides—within a certain range, the higher the content, the stronger the activity.

[0071] When evaluating the activity of active ingredients in aloe vera samples, analysis can be performed by combining acetyl content with molecular weight distribution and glycosidic bond structure: size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALS) can clearly define the molecular weight range and distribution uniformity of polysaccharides, while the type of glycosidic bond determines the spatial conformation of the polysaccharide chain (such as rigid or flexible). The synergistic effect of both acetyl and acetyl groups constitutes the basis of activity, and the synergistic effect of the three constitutes the basis of the activity of active ingredients in aloe vera polysaccharides.

[0072] Understandably, determining the acetyl content essentially reflects the bioactivity of polysaccharides indirectly by quantifying key active structural units. Since acetyl groups are linked to the hydroxyl groups of sugar rings in polysaccharides via ester bonds, exhibiting high specificity among diverse functional groups, direct detection is difficult. However, through the hydrolysis of reducing sugars, acetyl groups break off from the polysaccharide chain, converting into acetic acid. Therefore, in this embodiment, the acetyl content is inferred by measuring the amount of acetic acid.

[0073] By measuring the acetic acid content in aloe vera samples, the acetyl group content can be deduced.

[0074] In one alternative implementation, the acetyl content in the aloe vera sample can be determined based on the amount of acetic acid.

[0075] It should be understood that since there is a 1:1 molar ratio between the acetyl group (-COCH3) in aloe polysaccharide and the acetic acid (CH3COOH) generated by hydrolysis, that is, one acetyl group hydrolyzes to generate one molecule of acetic acid, the mass of the acetyl group in the polysaccharide can be deduced based on the molar mass ratio of the two.

[0076] Specifically, the concentration of acetyl groups is 43 g / mol, and the concentration of acetic acid is 60 g / mol. The mass of acetyl groups can be determined by multiplying the ratio of the concentration of acetyl groups to that of acetic acid by the mass of acetic acid. The content of acetyl groups can then be obtained by multiplying the ratio of the mass of acetyl groups to the total mass of the aloe vera sample by 100%.

[0077] Understandably, the acetyl content before hydrolysis can be accurately obtained based on the above method. This value can directly reflect the structural integrity and bioactivity of aloe polysaccharides, providing an accurate description of the activity of aloe polysaccharide samples.

[0078] In this embodiment, due to the differences in molecular structure and polarity of different substances, their chromatographic peak retention times are different. Furthermore, the peak shape of the chromatographic peak corresponding to acetic acid usually presents a regular and symmetrical waveform in the chromatographic detection results. Therefore, the chromatographic peak corresponding to acetic acid can be determined based on the peak retention time and peak shape characteristics of each chromatographic peak. Since the degree of hydrolysis can affect the peak area change, the peak area of ​​acetic acid at the current moment can be corrected based on the completeness of hydrolysis to obtain a more accurate amount of acetic acid. Based on this accurate amount of acetic acid, the content of polysaccharide effective components in aloe vera samples can be accurately detected.

[0079] Combination Figure 1 ,like Figure 3 As shown, in one implementation of this application embodiment, the determination of the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak retention time and peak shape characteristics of each chromatographic peak may specifically include S301-S302.

[0080] S301. Obtain the standard peak retention time and standard peak shape characteristics of acetic acid.

[0081] It should be understood that acetic acid in aloe vera samples may be affected by other organic acids, leading to deviations in the peak retention time and peak shape of acetic acid. Therefore, multiple chromatographic peaks can be matched based on the standard peak retention time and standard peak shape characteristics of acetic acid to determine the corresponding chromatographic peak.

[0082] In one alternative implementation, an acetic acid chromatogram can be obtained, and the retention time and peak shape characteristics of the standard peak can be determined based on the acetic acid chromatogram.

[0083] The chromatogram of acetic acid is a chromatogram of a standard acetic acid solution under the same conditions.

[0084] Specifically, the acetic acid standard solution can be injected under the same chromatographic conditions (including mobile phase, flow rate, column temperature, detection wavelength, etc.) as the aloe vera sample hydrolysate to obtain an acetic acid chromatogram. The chromatographic peak with the largest area and the highest response value in the acetic acid chromatogram can be identified, and its retention time and peak shape can be recorded. The retention time is determined as the standard peak retention time of acetic acid, and its peak shape is determined as the standard peak shape characteristic.

[0085] It should be noted that the standard peak retention time of acetic acid and the peak retention time of each chromatographic peak are within a time range.

[0086] Optionally, standard peak shape characteristics may include peak width, symmetry, peak front width (i.e., the distance from the peak apex to the front edge), peak back width (i.e., the distance from the peak apex to the back edge), etc.

[0087] S302. Based on the retention time and peak shape characteristics of each chromatographic peak, the retention time of the standard peak, and the standard peak shape characteristics, determine the chromatographic peak corresponding to acetic acid from multiple chromatographic peaks.

[0088] Specifically, the chromatographic peak whose retention time is most similar to that of the standard peak and whose peak shape is most similar to that of the standard peak can be identified as the chromatographic peak corresponding to acetic acid.

[0089] Optionally, all chromatographic peaks appearing within the standard retention time of the standard peak can be screened first and identified as candidate peaks. Then, the chromatographic peak corresponding to acetic acid can be identified from these candidate peaks.

[0090] In one implementation of this application, the retention time drift of each chromatographic peak can be determined based on the retention time of each chromatographic peak and the retention time of the standard peak; the peak shape regularity of each chromatographic peak can be determined based on the peak shape characteristics of each chromatographic peak and the standard peak shape characteristics; and the chromatographic peak corresponding to acetic acid can be determined from the plurality of chromatographic peaks based on the retention time drift and the peak shape regularity of each chromatographic peak.

[0091] Among them, the retention time drift of a chromatographic peak is used to characterize the difference between the retention time of a chromatographic peak and the retention time of a standard peak, and the peak shape regularity of a chromatographic peak is used to characterize the similarity between the peak shape characteristics of a chromatographic peak and the peak shape characteristics of a standard peak.

[0092] It should be understood that the greater the difference between the retention time of a candidate peak and the retention time of the standard peak, the more unstable the retention time of the candidate peak is, that is, the greater the drift of the retention time, and the less likely the candidate peak is the chromatographic peak corresponding to acetic acid.

[0093] Optionally, the retention time drift of a chromatographic peak satisfies the following formula:

[0094]

[0095] in, Indicates chromatographic peaks Retention time drift Indicates chromatographic peaks Peak retention time, Indicates the retention time of the standard peak.

[0096] Based on the above formula, it should be understood that, Indicates chromatographic peaks The intersection of the peak retention time and the standard peak retention time reflects the degree of overlap between the two. The lower the overlap, the more stable the chromatographic peak. Unlike the peak retention time of acetic acid standard solution, the greater the structural difference between the substance corresponding to this chromatographic peak and acetic acid, the greater the degree of drift. Indicates chromatographic peaks The difference between the retention time of the peak and the retention time of the standard peak may be due to the chromatographic peak... The complex molecular structure of the molecules leads to faster diffusion, reflecting the degree of deviation between the two in the retention time distribution width. The greater the deviation, the more pronounced the chromatographic peak. The greater the difference between the peak broadening behavior of the sample and that of the acetic acid standard solution, the greater the degree of drift.

[0097] It should be understood that within the normal linear range, changes in the concentration of acetic acid will not alter the peak shape characteristics (width, symmetry), but will only proportionally change the peak height and peak area. Therefore, by comparing the peak width difference between the acetic acid standard solution and each chromatographic peak, the corresponding chromatographic peak of acetic acid can be determined.

[0098] Optionally, the peak shape regularity of a chromatographic peak satisfies the following formula:

[0099]

[0100] in, Indicates chromatographic peaks Peak regularity Indicates chromatographic peaks The difference between the peak width and the peak width corresponding to the standard acetic acid solution. Indicates chromatographic peaks The width of the peak front, Indicates chromatographic peaks The width of the trailing edge of the peak.

[0101] Optionally, the parameters in the above formula can be normalized or the data can be standardized.

[0102] Based on the above formula, it should be understood that acetic acid, as a structurally simple small molecule, exhibits a fixed peak width under stable chromatographic conditions. Larger peak size indicates larger chromatographic peak size. The broadening behavior of acetic acid differs significantly from that of standard acetic acid solution; the symmetry of the chromatographic peak reflects the uniformity of the elution rate of the substance in the chromatographic column. Acetic acid molecules elute stably, and the peak shape is usually nearly symmetrical (with little difference in width between the leading and trailing edges). The larger peak size indicates asymmetrical peak shape, leading to fluctuations in the effluent rate, which does not conform to the peak shape characteristics of acetic acid.

[0103] In one alternative implementation, the sum of the retention time drift of each chromatographic peak and the regularity of its peak shape can be used to determine the acetic acid validation score for each chromatographic peak, and the chromatographic peak with the highest acetic acid validation score can be identified as the chromatographic peak corresponding to acetic acid.

[0104] In another alternative implementation, the ratio of the retention time drift of each chromatographic peak to the regularity of its peak shape can be used as the acetic acid peak verification score for each chromatographic peak, and the chromatographic peak corresponding to acetic acid can be determined from the multiple chromatographic peaks based on the acetic acid peak verification score for each chromatographic peak.

[0105] The acetic acid peak verification score of a chromatographic peak is used to characterize the similarity between a chromatographic peak and the actual acetic acid peak.

[0106] It should be understood that the greater the regularity of the peak shape and the smaller the retention time drift, the higher the acetic acid peak verification score, and the greater the likelihood that the chromatographic peak corresponds to the acetic acid peak in the chromatographic detection results of the aloe vera sample.

[0107] Optionally, the chromatographic peak with the highest acetic acid validation score can be identified as the chromatographic peak corresponding to acetic acid.

[0108] Understandably, by comparing each chromatographic peak with the chromatographic peak of a real acetic acid standard solution, the difference between the retention time of each chromatographic peak and the retention time of the standard peak can be obtained, as well as the similarity between the peak shape characteristics of each chromatographic peak and the standard peak shape characteristics. Based on this, the chromatographic peak corresponding to acetic acid can be accurately determined.

[0109] It should be understood that due to the complex matrix of aloe polysaccharide samples, which may contain structurally similar organic acids and polysaccharide degradation products, some interfering acids may be mixed into the chromatographic detection results. In this case, although the retention time of the peak is consistent with that of the standard, it is actually a mixed peak of multiple substances, not a pure acetic acid peak. Therefore, there is still a risk of misjudgment when screening the chromatographic peak corresponding to acetic acid based on the above acetic acid validation score. Therefore, each chromatographic peak can be spiked for validation, and the chromatographic peak corresponding to acetic acid can be identified based on the increase in peak area for final confirmation.

[0110] In one implementation of this application, in response to the addition of standard acetic acid solution to the hydrolysate of aloe vera sample, the increase in peak area of ​​each chromatographic peak can be determined; based on the increase in peak area of ​​each chromatographic peak and the acetic acid verification score of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks.

[0111] It should be understood that this standard acetic acid solution is an acetic acid standard with a known mass and concentration.

[0112] Specifically, add a known mass and concentration of acetic acid solution to the hydrolysate of the aloe vera sample, and then perform chromatographic analysis again. At this time, the peak area of ​​the acetic acid solution in the second chromatographic analysis is a fixed amount that can be obtained. Identify the increase in peak area of ​​each chromatographic peak in the second chromatographic analysis result. If the peak area of ​​a certain chromatographic peak increases significantly in the second chromatographic analysis result after spiked analysis, and the increase in peak area is directly proportional to the amount of standard acetic acid solution added, then the chromatographic peak is more likely to be the chromatographic peak corresponding to acetic acid.

[0113] In one alternative implementation, the theoretical increase in peak area of ​​the standard acetic acid solution can be obtained. Then, based on the increase in peak area of ​​each chromatographic peak, the theoretical increase in peak area of ​​the standard acetic acid solution, and the acetic acid verification score of each chromatographic peak, the probability of each chromatographic peak being an acetic acid peak can be determined, and the chromatographic peak with the highest probability of being an acetic acid peak can be identified as the chromatographic peak corresponding to acetic acid.

[0114] Alternatively, the probability of an acetic acid peak in a chromatographic peak satisfies the following formula:

[0115]

[0116] in, Indicates chromatographic peaks The probability of an acetic acid peak, Indicates chromatographic peaks Verification score of acetic acid peak. Indicates chromatographic peaks The increase in peak area This represents the theoretical increase in peak area of ​​the standard acetic acid solution.

[0117] Based on the above formula, it should be understood that, Indicates chromatographic peaks The rationality of the increase in peak area Indicates chromatographic peaks The smaller the absolute deviation between the actual increase in peak area and the theoretical increase, the better the chromatographic peak after spiking. The closer the area change matches the theoretical expectation, the better the chromatographic peak... The signal increment mainly comes from the added acetic acid standard; This is to prevent the denominator from being zero; combined with chromatographic peaks The acetic acid peak is validated and scored; the larger the product of the two, the stronger the chromatographic peak. The higher the probability that the chromatographic peak corresponds to acetic acid.

[0118] Alternatively, the top 5 chromatographic peaks can be screened based on the acetic acid validation score, and then the probability of the top 5 chromatographic peaks being acetic acid peaks can be calculated. This can improve detection efficiency while ensuring accuracy.

[0119] Understandably, by identifying the chromatographic peak with the highest probability of acetic acid as the acetic acid peak in the chromatographic detection results of the aloe vera sample, the position of the corresponding chromatographic peak can be accurately determined.

[0120] In summary, the method for detecting the effective components of aloe polysaccharides in aloe raw materials provided in this application first screens chromatographic peaks based on their retention time and peak shape characteristics and quantifies the acetic acid verification score. This allows for targeted identification of potential candidate peaks, significantly reducing interference from irrelevant peaks and improving the efficiency and specificity of target peak identification. Next, spiked verification combined with peak area changes confirms the chromatographic peak corresponding to acetic acid, eliminating misjudgments of structurally similar impurities and ensuring accurate peak attribution. Further consideration and optimization of the interference of the hydrolysis process on the acetic acid peak area effectively corrects deviations caused by incomplete or excessive hydrolysis, eliminating systematic errors introduced in the pretreatment stage. Finally, based on the optimized peak area, the amount of acetic acid is determined and the acetyl content is inferred, achieving precise conversion from chromatographic signal to active ingredient content and enabling effective quantitative analysis of the effective components of aloe polysaccharides.

[0121] It should be noted that the order of the embodiments described above is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. The processes depicted in the accompanying drawings do not necessarily require a specific or sequential order to achieve the desired result. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0122] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

Claims

1. A method for detecting an effective component of aloe polysaccharide in an aloe raw material, characterized by, The method includes: Obtain a chromatogram of the hydrolysate of an aloe vera sample, wherein the chromatogram includes multiple chromatographic peaks; Based on the peak retention time and peak shape characteristics of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks; Obtain the peak area changes of the chromatographic peak corresponding to acetic acid during the hydrolysis process; Based on the change in peak area, the maximum peak area during the hydrolysis process is determined; The completeness of hydrolysis at the current moment is determined based on the peak area, the maximum peak area, and the slope of the change between the peak area at the current moment and the peak area at the previous moment during the hydrolysis process. The amount of acetic acid in the hydrolysate of the aloe vera sample is determined based on the completeness of hydrolysis at the current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment. The completeness of hydrolysis is used to characterize the degree of hydrolysis, and the current moment is any moment in the hydrolysis process. The content of polysaccharide active ingredients in the aloe vera sample was determined based on the amount of acetic acid.

2. The method for detecting the effective components of aloe polysaccharides in aloe vera raw materials according to claim 1, characterized in that, The step of determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak retention time and peak shape characteristics of each chromatographic peak includes: Obtain the standard peak retention time and standard peak shape characteristics of acetic acid; Based on the retention time and peak shape characteristics of each chromatographic peak, the retention time of the standard peak, and the standard peak shape characteristics, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks.

3. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 2, characterized in that, The acquisition of the standard peak retention time and standard peak shape characteristics of acetic acid includes: Obtain an acetic acid chromatogram, wherein the acetic acid chromatogram is a chromatogram of the hydrolysate of an acetic acid standard solution under the same conditions; The retention time and peak shape characteristics of the standard peak are determined based on the acetic acid chromatogram.

4. The method for detecting the effective components of aloe polysaccharides in aloe vera raw materials according to claim 2, characterized in that, The process of determining the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the peak retention time and peak shape characteristics of each chromatographic peak, the retention time of the standard peak, and the standard peak shape characteristics includes: Based on the retention time of each chromatographic peak and the retention time of the standard peak, the retention time drift of each chromatographic peak is determined. The retention time drift of a chromatographic peak is used to characterize the difference between the retention time of a chromatographic peak and the retention time of the standard peak. Based on the peak shape characteristics of each chromatographic peak and the standard peak shape characteristics, the peak shape regularity of each chromatographic peak is determined. The peak shape regularity of a chromatographic peak is used to characterize the degree of similarity between the peak shape characteristics of a chromatographic peak and the standard peak shape characteristics. Based on the retention time drift and peak shape regularity of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks.

5. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 4, characterized in that, Based on the retention time drift and peak shape regularity of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks, including: The ratio of the retention time drift of each chromatographic peak to the regularity of its peak shape is determined as the acetic acid peak verification score of each chromatographic peak. The acetic acid peak verification score of a chromatographic peak is used to characterize the similarity between a chromatographic peak and a real acetic acid peak. Based on the acetic acid peak verification score of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks.

6. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 5, characterized in that, The determination of the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the acetic acid peak verification score for each chromatographic peak includes: In response to the addition of standard acetic acid solution to the hydrolysate of aloe vera samples, the increase in peak area for each chromatographic peak was determined. Based on the increase in peak area of ​​each chromatographic peak and the acetic acid verification score of each chromatographic peak, the chromatographic peak corresponding to acetic acid is determined from the plurality of chromatographic peaks.

7. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 6, characterized in that, The determination of the chromatographic peak corresponding to acetic acid from the plurality of chromatographic peaks based on the increase in peak area of ​​each chromatographic peak and the acetic acid verification score of each chromatographic peak includes: Obtain the theoretical increase in peak area of ​​the standard acetic acid solution; Based on the increase in peak area of ​​each chromatographic peak, the theoretical increase in peak area of ​​the standard acetic acid solution, and the acetic acid verification score of each chromatographic peak, the probability of each chromatographic peak being an acetic acid peak is determined. The chromatographic peak with the highest probability of being an acetic acid peak is identified as the chromatographic peak corresponding to acetic acid.

8. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 1, characterized in that, The determination of the amount of acetic acid in the aloe vera sample hydrolysate based on the hydrolysis completeness at the current moment and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment includes: Obtain the expected peak area and the measured peak area of ​​the internal standard at the current time; The ratio of the expected peak area of ​​the internal standard to the measured peak area of ​​the internal standard is determined as the hydrolysis efficiency at the current moment. Based on the hydrolysis efficiency at the current moment, the hydrolysis completeness at the current moment, and the peak area of ​​the chromatographic peak corresponding to acetic acid at the current moment, the optimized acetic acid peak area is determined; Based on the optimized acetic acid peak area and acetic acid standard curve, the amount of acetic acid in the hydrolysate of the aloe vera sample is determined. The acetic acid standard curve is used to characterize the linear relationship between peak area and concentration.

9. The method for detecting the effective components of aloe polysaccharides in aloe raw materials according to claim 1, characterized in that, The active ingredient includes acetyl groups, and determining the content of polysaccharide active ingredients in the aloe vera sample based on the amount of acetic acid includes: The acetyl content in the aloe vera sample was determined based on the amount of acetic acid.

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