Preparation method of humic acid type anti-diarrhea medicine

Abstract

The application discloses a preparation method of humic acid anti-diarrhea medicine, and belongs to the technical field of biological medicine, which can greatly improve the anti-diarrhea activity intensity of humic acid substances by homogenizing and grading the humic acid substances, and the activity intensity of the prepared anti-diarrhea raw medicine can reach three times of that before grading; in addition, the original inactive samples can also show anti-diarrhea activity, so that the invalid humic acid resources obtain medicinal value. The method systematically realizes the efficient preparation of humic acid anti-diarrhea raw medicine, and has important practical significance and application value for enhancing the stability of the quality of the medicine raw material, expanding the selection range of the medicine raw material, improving the safety of the medicine raw material and solving the bottleneck problem in the actual production of pharmaceutical enterprises. Meanwhile, the system and operability of the whole method are strong, the applicability is high, and the method is easy to be applied in industrialization.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a humic acid-based antidiarrheal drug. Background Technology

[0002] As an important component of humic substances, humic acids are a class of large-molecule phenolic acid mixtures formed from plant residues in nature through microbial decomposition and transformation, as well as a series of geochemical processes. They are widely distributed in natural environments such as soil and water bodies, with the highest content found in low-rank coals such as lignite, peat, and weathered coal, which are the main raw material sources for the industrial application of humic acids. Based on molecular weight and solubility, humic acids can be further divided into three types: fulvic acid, brown humic acid, and black humic acid. Although they possess numerous pharmacological activities, humic acids exhibit the most definite and prominent antidiarrheal activity, demonstrating good efficacy and advantages in treating diarrhea in humans and animals, and have been used clinically and in animal husbandry for a considerable period. Sodium humate tablets (granules), a single-ingredient mineral drug currently available in China for treating diarrhea, has received widespread acclaim in the treatment of childhood diarrhea. Currently, not only are the requirements for new drug development and approval becoming increasingly stringent, but existing drugs are also facing the challenges of re-evaluation and re-registration. Most of the companies that previously obtained drug approval numbers for sodium humate have gradually ceased production. The only sodium humate tablet currently on the market is also being produced intermittently and is on the verge of being withdrawn from the market. The main reasons for this are the substandard or unstable activity of the humic acid raw materials and their high ash content. On the one hand, high-quality humic acid production raw materials such as young lignite are scarce; on the other hand, existing humic acid preparation technologies cannot solve the problem of substandard antidiarrheal activity.

[0003] Humic acids are complex natural macromolecular mixtures whose pharmacological activity is easily affected by the source of the raw materials. Regarding their application in treating diarrhea, many studies and practical applications have confirmed that the antidiarrheal activity of humic acids extracted from different coal mines can vary significantly, exhibiting inconsistent activity or even no activity. Some humic acid production enterprises are limited to specific coal mines as raw materials; once high-quality resources are depleted, they face the predicament of unsustainable production. Humic acids from other lignite mines are considered to have no pharmaceutical development value due to their low activity. The inventor's team has long been engaged in basic and applied research related to humic acids. Previous research results show that the antidiarrheal activity of humic acids is often closely related to their inherent physicochemical properties such as solubility, acidity / alkalinity, molecular weight distribution, polarity, and adsorption capacity. The specific correlation varies depending on the sample. For example, the activity intensity of humic acid from a certain origin may be closely related to its acidity / alkalinity, while the activity of samples from another origin may be determined by its molecular weight distribution. Based on the latest research findings, this invention establishes a method for preparing humic acid-based antidiarrheal drugs by examining the correlation between the antidiarrheal activity of samples and their inherent physicochemical properties. This method, based on homogenization grading and biopotency detection, can significantly enhance the antidiarrheal activity of humic acid raw materials, and even enable previously inactive samples to exhibit antidiarrheal activity. Applying this method to actual production is of significant practical importance for properly addressing the raw material problems faced in the production of humic acid antidiarrheal drugs and helping enterprises resume continuous production. Summary of the Invention

[0004] To address the problems existing in humic acid-based antidiarrheal raw materials, this invention establishes a method for preparing humic acid-based antidiarrheal drugs, comprising the following steps: (1) The inherent physicochemical properties of humic acid substances are used as the basis for classification. The inherent physicochemical properties include solubility, acidity and alkalinity, molecular weight, polarity and adsorption capacity. Humic acid substances are classified by using one of the classification bases to achieve homogenization and classification, resulting in a series of fractions X1, X2, ..., Xn. (2) Use an antidiarrheal pharmacological model to detect the antidiarrheal index of humic acid substances and their fractions in step (1), and calculate the antidiarrheal biopotency, expressing the relative magnitude of the antidiarrheal activity intensity by biopotency; Among X1, X2, ..., Xn, if the graded fraction has a higher antidiarrheal activity than the ungraded humic acid, and the difference between the two is highly significant ( P If the fraction is <0.001, then that fraction is selected as the antidiarrheal drug; Among X1, X2, ..., Xn, if the graded substances have higher antidiarrheal activity than ungraded humic acid substances, the difference between the two is statistically significant. P<0.05), but none of them reached extremely significant ( P If the value is greater than 0.001, then the same grading technique is used to perform a second grading of that fraction, and this process is repeated until an antidiarrheal drug is selected. (3) If among X1, X2, ..., Xn, the antidiarrheal activity of ungraded humic acid is higher than that of ungraded humic acid, or although it is graded, the difference between the two is not significant ( P If the value is greater than 0.05, then another grading basis and matching grading technology are needed to homogenize the humic acid substances and repeat steps (1)-(3).

[0005] The humic substances refer to humic acid (total extract), fulvic acid, brown humic acid, black humic acid, and their organic salts or mixtures thereof.

[0006] The homogenization classification refers to classifying or segmenting humic acid substances according to a certain inherent physicochemical property, with compounds in the same fraction tending to be consistent in that property.

[0007] The aforementioned fractionation techniques refer to conventional methods for grading natural products, such as liquid-liquid extraction, pH fractionation, membrane fractionation, ultrafiltration, silica gel column chromatography, and macroporous adsorption resin column chromatography.

[0008] The evaluation method for antidiarrheal activity should select a classic, easy-to-operate, and low-cost pharmacological model, and its evaluation indicators should be clearly defined and quantifiable. For example, the following is a commonly used standardized pharmacological model in this field: The experimental animals were SPF-grade male mice, weighing 18-22g. They were fasted for 12 hours before the last pre-administration, but water was allowed. Mice were randomly divided into groups of 10 each. A blank control group was given pure water, a model group was given pure water, a positive control group was given commercially available humic acid, and the treatment groups were given humic acid substances and their fractions, administered twice daily for three days. One hour after the last administration, except for the blank control group which continued to receive pure water, all other experimental groups were given castor oil to induce diarrhea. Each mouse was placed in a separate cage with white filter paper at the bottom. The filter paper was changed hourly, and the morphology and quantity of feces were recorded over 6 hours. The defecation index (EI) for each group was calculated using the following formula: EI = 3 × (number of watery stools) + 2 × (number of semi-solid stools) + 1 × (number of normal stools). When the EI of the experimental group was higher than that of the control group, and this was statistically significant (…), the group was considered to have a higher EI than the control group. P <0.05) indicates that the animals in the experimental group experienced diarrhea or that the sample had a laxative effect; when the value was lower than that of the control group and was statistically significant ( PIf the result is <0.05, the sample is considered to have an antidiarrheal effect; otherwise, the sample is considered to have no significant effect on the defecation function of mice. The antidiarrheal biopotency of the experimental group was calculated using the following formula: Antidiarrheal biopotency = (EIc - EIt) / (EIc - EIp) × 100; EIc is the defecation index of the model control group, EIp is the defecation index of the positive drug experimental group, and EIt is the defecation index of the humic acid experimental group.

[0009] The antidiarrheal activity evaluation methods listed above are merely examples and do not constitute a limitation on the choice of evaluation methods.

[0010] The main purpose of this invention in establishing a new method for preparing humic acid-based antidiarrheal drugs is to significantly improve the antidiarrheal activity and stability of the active pharmaceutical ingredient. This effect is a comprehensive result of the above method as a whole.

[0011] Advantages and technical effects of the present invention: (1) A method for preparing humic acid-based antidiarrheal drugs was successfully established for the first time. This method homogenizes and grades humic acid raw materials, increasing the antidiarrheal activity of the samples by 2.0-3.0 times compared to before grading, reaching 2.2-2.9 times the activity of commercially available humic acid drugs. It also enables samples that previously lacked antidiarrheal activity to exhibit activity, thus allowing humic acid resources with significantly lower activity than positive control drugs to acquire medicinal value. This method systematically achieves efficient preparation of humic acid-based antidiarrheal raw materials, which has significant practical and application value for improving the medicinal value of humic acid substances, enhancing the stability of drug raw material quality, removing restrictions on raw coal sources, expanding the range of drug raw material selection, improving drug raw material safety, and completely solving bottleneck problems in actual pharmaceutical production. (2) Humic acid, as a natural macromolecular mixture, has a very complex composition and diverse structure. Some substances in the mixture may not contribute to its antidiarrheal function, or even have antagonistic effects or toxic effects on the human body. Through homogenization and fractionation, not only can the compounds in the same fraction tend to be consistent in a certain structural characteristic (corresponding to the fractionation principle), reducing the proportion of ineffective components and thus greatly improving the activity efficiency of the sample, but also antagonistic components, ash, heavy metals and other harmful components can be removed, improving the safety and activity stability of the sample. The obtained antidiarrheal drugs are representative in terms of activity, chemical composition and structure, and can provide excellent research objects for other basic or applied research on humic acid; (3) The comprehensive method system provided by this invention detects the antidiarrheal biopotency of the drug during the preparation process. While screening out the fraction with the strongest activity, it also optimizes the corresponding grading technology. It allows for horizontal comparison of the antidiarrheal activity intensity between drugs from different coal mines, different manufacturers, and different production batches, greatly facilitating practical production applications. The entire method is systematic and highly operable, with high applicability and easy industrial application. The achieved technical effects effectively respond to the urgent needs of manufacturing enterprises and remove a major obstacle to the sustainable production of humic acid antidiarrheal drugs. Attached Figure Description

[0012] Figure 1 The defecation index of different experimental groups of sodium humate from Eshan and its fractions is shown in the figure; *** represents the difference between the control group and the control group. P <0.001; ▲ To compare with the model group, P <0.05; ▲▲▲ To compare with the model group, P <0.001; ★★★ To compare with the ungraded humic acid group, P <0.001; Figure 2 The defecation index of different experimental groups of Xundian humic acid and its primary grade is shown in the figure; *** represents the difference between the control group and the control group. P <0.01; ▲ To compare with the model group, P <0.05; ▲▲ To compare with the model group, P <0.01; ▲▲▲ To compare with the model group, P <0.001; ★ To compare with the ungraded humic acid group, P <0.05; Figure 3 The defecation index of different experimental groups of Xundian fulvic acid and its secondary grading is shown in the figure; *** represents the difference between the control group and the control group. P <0.001; ▲ To compare with the model group, P <0.05; ▲▲ To compare with the model group, P <0.01; ▲▲▲ To compare with the model group, P <0.001; ★★★ To compare with the ungraded humic acid group, P <0.001; Figure 4 The defecation index of different experimental groups for fulvic acid and its ultrafiltration grading in Milei is shown; *** in the figure represents the difference compared to the blank group.P <0.001; ▲ To compare with the model group, P <0.05; Figure 5 The defecation index of different test groups for fulvic acid and its macroporous resin in the figure is used for classification; *** in the figure represents the difference between the control group and the control group. P <0.001; ▲ To compare with the model group, P <0.05; ▲▲▲ To compare with the model group, P <0.001; ★★ To compare with the ungraded humic acid group, P <0.05; ★★★ To compare with the ungraded humic acid group, P <0.001. Detailed Implementation

[0013] The following examples further illustrate the content of the present invention, but these examples do not limit the scope of protection of the present invention. Unless otherwise specified, the methods in the examples are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods.

[0014] Example 1: In this example, lignite from Eshan, Yunnan Province, was used as raw material. Sodium humate was prepared according to the method in application number 200810233669.X, "Method for producing humic acid and its salts by oxidative degradation of lignite," and named Eshan sodium humate (HAE). Using this as raw material, the antidiarrheal drug of Eshan sodium humate was prepared using the comprehensive method system established in this invention. The specific steps are as follows: (1) Based on molecular weight, ultrafiltration technology was used to homogenize and classify sodium humate from Eshan, resulting in five fractions with apparent molecular weight ranges, which were named HAE1 (<3k), HAE2 (3k-10k), HAE3 (10k-30k), HAE4 (30k-50k), and HAE5 (>50k) in sequence. (2) The defecation index of sodium humate and its fractions in step (1) was detected using an antidiarrheal pharmacological model, and the biopotency was calculated. The specific method is as follows: The antidiarrheal activity was assessed using a pharmacological model: SPF-grade male mice, 18-22g, were used as experimental animals. They were fasted for 12 hours before the last pre-administration, but water was allowed. Mice were randomly divided into groups of 10 each. A blank control group received pure water (20mL / kg, by gavage), a model group received pure water (20mL / kg, by gavage), a positive control group received commercially available humic acid (200mg / kg, by gavage), and the treatment groups received sodium humate (HAE) and its fractions HAE1-HAE5 (200mg / kg, by gavage), respectively, twice daily for three days. The positive control group used sodium humate tablets with a national drug approval number produced by a pharmaceutical company; the dosage was calculated based on the actual sodium humate content in the tablets. One hour after the last administration, except for the blank control group which continued to receive pure water, all other experimental groups received castor oil (0.4mL / mouse, by gavage) to induce diarrhea. Each mouse was placed in a separate cage with a white filter paper at the bottom. The filter paper was changed every hour, and the form and quantity of mouse feces were recorded over 6 hours. The defecation index (EI) for each group was calculated using the following formula: EI = 3 × (number of watery stools) + 2 × (number of semi-solid stools) + 1 × (number of normal stools). When the EI of the experimental group was higher than that of the control group, and this was statistically significant ( P <0.05) indicates that the animals in the experimental group experienced diarrhea or that the sample had a laxative effect; when the value was lower than that of the control group and was statistically significant ( P <0.05) indicates the sample has an antidiarrheal effect; other results indicate no significant effect on defecation function in mice. The antidiarrheal biopotency of the experimental groups was calculated using the following formula: Antidiarrheal biopotency = (EIc - EIt) / (EIc - EIp) × 100; EIc is the defecation index of the model control group, EIp is the defecation index of the positive control group, and EIt is the defecation index of the humic acid group. The test results are as follows: Figure 1 As shown in Table 1.

[0015] Table 1

[0016] (3) Screening of antidiarrheal drugs: from Figure 1 It can be seen that the antidiarrheal activity of sodium humate HAE1-HAE5 varies with increasing molecular weight, showing a trend of first increasing and then decreasing; when the molecular weight distribution is 3k-10k, the difference in activity intensity changes from significant before fractionation (compared to the model control group), P <0.05) becomes highly significant ( P <0.001). The antidiarrheal activity of fraction HAE2 (3k-10k) was not only higher than that of unfractionated Eshan humate sodium, but the difference between the two was also highly significant. P<0.001). This indicates a strong correlation between the antidiarrheal activity of sodium humate from Eshan and its molecular weight distribution. Ultrafiltration fractionation can extract the antidiarrheal drug with a relatively concentrated active ingredient from the sample, namely fraction HAE2. Table 1 shows that after ultrafiltration fractionation, the biopotency of fraction HAE2 is 2.7 times that of sodium humate from Eshan; compared with commercially available humic acid drugs, the biopotency of fraction HAE2 is 2.9 times that of the positive control, and significantly higher than the 1.1 times that of sodium humate from Eshan; indicating that the preparation method of this invention can significantly improve the antidiarrheal activity of humic acid substances.

[0017] Example 2: In this example, lignite from Xundian, Yunnan Province, was used as raw material. Fulvic acid was prepared according to the method described in application number 200810233669.X, "Method for producing humic acid and its salts by oxidative degradation of lignite," and named Xundian fulvic acid (FAX). Using this as raw material, the comprehensive method system established in this invention was used to prepare an antidiarrheal drug from Xundian fulvic acid. The specific steps are as follows: (1) Based on molecular weight, the Xundian humic acid was homogenized and classified using ultrafiltration technology to obtain four grades with apparent molecular weight ranges, which were named FAX1 (<1k), FAX2 (1k-10k), FAX3 (10k-30k), and FAX4 (>30k) respectively.

[0018] (2) The defecation index of Xundian fulvic acid and its fractions in step (1) was detected using the same antidiarrheal pharmacological model as in Example 1, and the biopotency was calculated. The results are as follows: Figure 2 As shown in Table 2.

[0019] Ungraded Xundian fulvic acid itself possesses significant antidiarrheal activity. After ultrafiltration fractionation, the antidiarrheal activity intensity of fractions FAX1-FAX4 also changed, showing a trend of first increasing and then decreasing; however, the increase in antidiarrheal intensity among each fraction was not significant. Among them, fraction FAX2 (1k-10k) had the highest antidiarrheal potency, but compared with ungraded Xundian fulvic acid, the difference between the two was significant ( P <0.05), but not highly significant ( P >0.001), therefore FAX2 was used as the raw material for the second classification.

[0020] Table 2

[0021] (3) Based on molecular weight, using the fraction FAX2 from step (2) as raw material, a second fractionation was performed using ultrafiltration technology to obtain three fractions with apparent molecular weight ranges, which were named FAX2-1 (1k-3k), FAX2-2 (3k-5k), and FAX2-3 (5k-10k) in sequence.

[0022] (4) The same method as in step (2) was used for evaluation, and the results are as follows: Figure 3 And as shown in Table 2. From Figure 3 As can be seen, after secondary fractionation, the antidiarrheal active ingredients in the sample were further concentrated. The activity intensity of fraction FAX2-2 (3k-5k) was increased to 2.0 times that of Xundian fulvic acid and 2.4 times that of the positive control. Fraction FAX2-2 was used as an antidiarrheal drug. This embodiment demonstrates that the comprehensive method system of multiple fractionation combined with biopotency detection provided by the present invention can significantly improve the antidiarrheal activity intensity of humic acid substances.

[0023] Example 3: In this example, lignite from Mile, Yunnan Province, was used as raw material. Fulvic acid was prepared according to the method in application number 200810233669.X, "Method for producing humic acid and its salts by oxidative degradation of lignite," and named Mile fulvic acid (FAM). Using this as raw material, an antidiarrheal drug made from Mile fulvic acid was prepared using the comprehensive method system established in this invention. The specific steps are as follows: (1) Based on molecular weight, ultrafiltration technology was used to homogenize and classify fulvic acid of Milei. Six grades with apparent molecular weight ranges were obtained and named FAM1 (<1k), FAM2 (1k-3k), FAM3 (3k-5k), FAM4 (5k-10k), FAM5 (10k-30k), and FAM6 (>30k).

[0024] (2) The defecation index of Miler fulvic acid and its fractions in step (1) was detected using the same antidiarrheal pharmacological model as in Example 1. The results are as follows: Figure 4 As shown in Table 3.

[0025] Milley fulvic acid itself did not exhibit antidiarrheal activity, with its antidiarrheal potency being only about 80% of that of the positive control. After ultrafiltration fractionation, the antidiarrheal activity intensity of fractions FAM1-FAM6 did not show a significant trend; and the improvement in antidiarrheal potency of each fraction was not significant. The only fraction exhibiting antidiarrheal activity was fraction FAM2, with an antidiarrheal potency increase of only 37%. Although the activity intensity was higher than that of unfractionated fulvic acid, the difference between the two was not statistically significant. P >0.05); a different grading technique is needed to grade the fulvic acid in Mile.

[0026] Table 3

[0027] (3) Based on adsorption, the fulvic acid of Miler was homogenized and fractionated using macroporous adsorption resin column chromatography. Miler fulvic acid adsorbed on XAD-8 resin was eluted sequentially with 0.01 mol / L HCl solution, 0.01 mol / L HCl + 20% methanol mixture, 0.01 mol / L HCl + 40% methanol mixture, 0.01 mol / L HCl + 60% methanol mixture, 100% methanol and 0.01 mol / L NaOH solution to obtain 6 fractions, which were named FAMR1, FAMR2, FAMR3, FAMR4, FAMR5 and FAMR6 respectively.

[0028] (4) The same method as in step (2) was used to evaluate the classification of Miler fulvic acid and its macroporous resins FAMR1-FAMR6. The results are as follows: Figure 5 As shown in Table 3.

[0029] from Figure 5 It can be seen that the antidiarrheal activity of the fulvic acid fractions FAMR1-FAMR6 varies due to differences in adsorption, showing a trend of first increasing and then decreasing. The antidiarrheal activity of fraction FAMR3 is not only higher than that of the unfractionated fulvic acid, but there is also a highly significant difference between the two fractions. P <0.001). This indicates a strong correlation between the antidiarrheal activity of fulvic acid and its adsorption properties. Macroporous adsorption resin column chromatography can extract the antidiarrheal drug with a relatively concentrated active ingredient from the sample, namely fraction FAMR3. Comparative analysis of the biopotency data listed in Table 3 reveals that, after homogenization and fractionation, fulvic acid, which originally had no significant antidiarrheal activity, exhibited extremely significant activity. The activity intensity of fraction FAMR3 is 3.0 times that of fulvic acid and 2.2 times that of commercially available humic acid drugs. This demonstrates that the preparation method provided by this invention can not only significantly improve the antidiarrheal activity of humic acid substances but also enable samples that originally had no antidiarrheal activity to exhibit activity, directly endowing samples with activity significantly weaker than positive control drugs with medicinal value.

[0030] Example 4: Cross-sectional comparison of antidiarrheal activity intensity Table 4

[0031] Table 4 lists the biopotency of humic acid substances from different origins and the prepared antidiarrheal drugs in Examples 1-3. Since these data are relative values ​​of the antidiarrheal activity intensity of the same positive drug, comparative analysis is possible. Table 4 shows that sodium humate from Eshan lignite mine and fulvic acid from Xundian lignite mine have medicinal development value. After processing the raw materials using the preparation method of this invention, their antidiarrheal activity intensity is greatly enhanced. Although fulvic acid from Mile lignite mine does not have direct medicinal value, its antidiarrheal activity intensity is also greatly enhanced after processing using the preparation method of this invention. On the one hand, the preparation method of this invention may improve medicinal value, reduce costs, and increase enterprise benefits; on the other hand, the biopotency of the antidiarrheal drugs of this invention is tested during the preparation process, allowing for horizontal comparison of antidiarrheal activity intensity between drugs from different coal mines, different manufacturers, and different production batches, greatly facilitating practical production applications.

[0032] The experimental results of Examples 1-4 show that the preparation method of humic acid antidiarrheal drugs based on homogenization grading and biopotency detection provided by this invention can increase the antidiarrheal intensity of samples by up to 3.0 times before grading, and up to 2.9 times the activity intensity of commercially available humic acid drugs. It can also make samples that originally had no antidiarrheal activity exhibit activity, thus enabling humic acid resources with significantly lower activity than positive control drugs to acquire medicinal value. This method systematically realizes the efficient preparation of humic acid antidiarrheal raw materials, which has significant practical significance and application value for improving the medicinal value of humic acid substances, enhancing the stability of drug raw material quality, removing restrictions on raw coal sources, expanding the range of drug raw material selection, improving raw material safety, and completely solving the bottleneck problems in actual pharmaceutical production.

Claims

1. A method for preparing a humic acid-based antidiarrheal drug, characterized in that, Includes the following steps: (1) The inherent physicochemical properties of humic acid substances are used as the basis for classification. The inherent physicochemical properties include solubility, acidity and alkalinity, molecular weight, polarity and adsorption capacity. Humic acid substances are classified by a corresponding classification method in the classification basis to obtain a series of fractions X1, X2, ..., Xn. (2) Use an antidiarrheal pharmacological model to detect the antidiarrheal index of humic acid substances and their fractions in step (1), and calculate the antidiarrheal biopotency, expressing the relative magnitude of the antidiarrheal activity intensity by biopotency; Among X1, X2, ..., Xn, if the graded fraction has a higher antidiarrheal activity than the ungraded humic acid, and the difference between the two is highly significant. P If the value is <0.001, then this fraction should be selected as the antidiarrheal drug; Among X1, X2, ..., Xn, if the fractionated antidiarrheal activity is higher than that of the unfractionated humic acid, the difference is statistically significant. P <0.05, but none of them reached extremely significant. P If the value is greater than 0.001, the same grading technique is used to perform a second grading of the fraction, and this process is repeated until an antidiarrheal drug is selected. (3) Among X1, X2, ..., Xn, if the antidiarrheal activity of the ungraded substance is higher than that of the ungraded humic acid substance, or if the substance is graded but the difference between the two is not significant. P If the value is greater than 0.05, then change to another grading basis and a matching grading method to perform homogenization grading of humic acid substances, and repeat steps (1)-(3).

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