Thin layer chromatography identification method for black sesame and roasted black sesame formula granules

Abstract

The present invention provides a thin layer identification method for black sesame and fried black sesame formula granules, including: taking a sample to be tested, dissolving with a solvent to obtain a test solution; taking sesamin and β-sitosterol reference substances, dissolving with a solvent to obtain a reference solution; taking a black sesame reference medicinal material, dissolving with a solvent to obtain a reference medicinal material solution; subjecting the test solution, the reference solution and the reference medicinal material solution to thin layer chromatography, the thin layer plate being a silica gel G thin layer plate; the developing agent being petroleum ether-toluene-chloroform; and inspecting under ultraviolet light, if there is a blue fluorescent spot at an Rf value of 0.561 in the test sample chromatogram, it is a black sesame formula granule; if there is no blue fluorescent spot at an Rf value of 0.582±10%, it is a fried black sesame formula granule. The method of the present invention is to observe whether there is a fluorescent spot in the thin layer chromatogram within a specified ratio shift value range as a distinguishing point for quickly and effectively identifying black sesame and fried black sesame formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a thin layer identification method for black sesame and fried black sesame formula particles. Background Art

[0002] Black sesame is the dried mature seed of Sesamumindicum L., a plant of the Sesamaceae family. It has the effects of nourishing the liver and kidneys, replenishing essence and blood, and moistening the intestines. The stir-fried product focuses on treating acute wind syndrome with a short course of illness and seasonal qi, so its processing purpose is to alleviate the bias and increase the extraction rate of effective substances [1]. There are differences in the physical and chemical properties and laxative effects of black sesame before and after processing. The stir-fried black sesame has better moisturizing and nourishing effects [2]. This shows that the chemical composition of black sesame and stir-fried black sesame has changed during the processing process.

[0003] The chemical components of black sesame mainly include oils, sesamin, amino acids, etc. At present, the relevant research on black sesame is concentrated on the content determination and pharmacology of different processed products. For example, Zhang Lili [3] conducted a comparative study on the changes in chemical composition and lipid-lowering pharmacological effects of different processed black sesame products, and Liu Jun [4] conducted a study on the in vitro antioxidant effects of different processed traditional Chinese medicine black sesame products. However, no effective method for identifying black sesame and fried black sesame extracts has been reported.

[0004] Since fried black sesame extracts and formula granules are made from black sesame medicinal materials through a series of processes, some of which will destroy the original properties and characteristics, the property identification method is not applicable to the identification of extracts and preparations. There is currently no effective method to distinguish between black sesame and fried black sesame in preparations.

[0005] Therefore, it is very necessary to establish a method that can distinguish black sesame, roasted black sesame extract, and formula granules. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a thin layer identification method for black sesame and roasted black sesame formula granules.

[0007] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0008] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0009] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0010] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0011] The present invention provides a thin layer identification method for black sesame and roasted black sesame formula granules, comprising:

[0012] A) Take the sample to be tested and dissolve it in solvent to obtain a test solution;

[0013] B) taking sesamin and β-sitosterol reference substances, dissolving them in a solvent to obtain reference substance solutions;

[0014] C) taking black sesame as a control medicinal material and dissolving it in a solvent to obtain a control medicinal material solution;

[0015] D) The test solution, reference solution, and reference medicinal material solution were subjected to thin layer chromatography using a silica gel G thin layer plate and a developing solvent of petroleum ether-toluene-chloroform;

[0016] E) Observe under ultraviolet light. If a blue fluorescent spot is observed at an Rf value of 0.561 in the chromatogram of the test sample, the sample is a black sesame formula granule. If no blue fluorescent spot is observed at an Rf value of 0.582 ± 10%, the sample is a roasted black sesame formula granule.

[0017] The thin layer chromatography identification method for black sesame and fried black sesame formula particles provided by the present invention first takes a sample to be tested, dissolves it with a solvent, and obtains a test solution.

[0018] The step A) of the present invention specifically comprises: dissolving the sample to be tested in water, then extracting it with ethyl acetate for 2 to 3 times; then evaporating it to dryness, and dissolving the residue in methanol to obtain a test solution.

[0019] Specifically, the mass volume ratio of the sample to be tested, water, ethyl acetate and methanol is: 0.5-1 g: 15-25 mL: 50-70 mL: 0.5-2 mL; more preferably 0.5 g: 20 mL: 60 mL: 1 mL.

[0020] Sesamin and β-sitosterol reference substances are dissolved in a solvent to obtain a reference substance solution. The solvent is methanol; the mass concentration of the sesamin reference substance is 0.5 to 5 mg / mL; the mass concentration of the β-sitosterol reference substance is 0.5 to 5 mg / mL.

[0021] Take black sesame as a reference medicinal material, dissolve it in a solvent, and obtain a reference medicinal material solution.

[0022] The present invention is preferably specifically:

[0023] Take black sesame as a reference medicinal material, add water to boil, then add ethyl acetate to extract, evaporate to dryness, and dissolve the residue in methanol to obtain the product.

[0024] Specifically, the mass volume ratio of the black sesame reference medicinal material, water, ethyl acetate and methanol is preferably: 0.5-1 g: 15-25 mL: 50-70 mL: 0.5-2 mL; more preferably 0.5 g: 20 mL: 60 mL: 1 mL.

[0025] The test solution, reference solution and reference medicinal material solution were subjected to thin layer chromatography.

[0026] Preferably, the test solution, reference solution, and reference medicinal material solution are aspirated and spotted on the same silica gel G thin layer plate, developed with a developing agent, taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots are clearly colored, and inspected under ultraviolet light (365nm).

[0027] The thin layer plate of the present invention is a silica gel G thin layer plate, which can be selected from the tailorable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd., Merck, and the prefabricated silica gel G plate of Qingdao Ocean Chemical Plant Branch.

[0028] The results showed that the blue fluorescent spot ratio shift values ​​(Rf) at 8.3 to 10.9 cm from the origin in the three thin layer plates were 0.613, 0.623, and 0.504, respectively, all of which could meet the identification requirements.

[0029] Check under ultraviolet light. If there is a blue fluorescent spot at the Rf value of 0.582±10% in the chromatogram of the test sample, it is a black sesame formula granule; if there is no blue fluorescent spot at the Rf value of 0.582±10%, it is a fried black sesame formula granule.

[0030] The Rf value is 0.582±10%; specifically, it can be 0.504, 0.534, 0.541, 0.561, 0.579, 0.584, 0.599, 0.613, or 0.623.

[0031] The sample spotting volume of the thin layer chromatography of the present invention is 5 to 15 μL.

[0032] In some preferred embodiments of the present invention, the mass ratio of petroleum ether-toluene-chloroform is 5:4:2; wherein the boiling point of the petroleum ether is 60-90°C.

[0033] The inspection temperature of the present invention is 4° C. to 35° C. The method of the present invention has good adaptability to different temperatures, and the black sesame formula particles show blue fluorescent spots at the ratio shift values ​​of 0.541, 0.613, and 0.534, while the roasted black sesame formula particles have no spots.

[0034] The inspection humidity of the present invention is 35% rh to 75% rh. The method of the present invention has good adaptability to different humidity, and the black sesame formula particles show blue fluorescent spots at the ratio shift values ​​of 0.584, 0.613, and 0.599, while the roasted black sesame formula particles have no spots.

[0035] The samples to be tested in the present invention are black sesame and fried black sesame formula granules.

[0036] The present invention provides a thin layer chromatography identification method for black sesame and roasted black sesame formula granules, comprising: A) taking a sample to be tested and dissolving it in a solvent to obtain a test solution; B) taking sesamin and β-sitosterol reference substances and dissolving them in a solvent to obtain a reference solution; C) taking a black sesame reference medicinal material and dissolving them in a solvent to obtain a reference medicinal material solution; D) subjecting the test solution, the reference solution, and the reference medicinal material solution to thin layer chromatography (TLC) using a silica gel G TLC plate and a developing solvent of petroleum ether-toluene-chloroform; E) examining the sample under ultraviolet light; if a blue fluorescent spot is present at an Rf value of 0.561 in the chromatogram of the sample, the sample is a black sesame formula granule; if no blue fluorescent spot is present at an Rf value of 0.582±10%, the sample is a roasted black sesame formula granule. The TLC identification method for black sesame and roasted black sesame formula granules established by the present invention uses the presence of fluorescent spots within a specified ratio shift range as a distinguishing point to quickly and effectively identify black sesame and roasted black sesame formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 To investigate the amount of samples;

[0038] Figure 2 For exclusive inspection;

[0039] Figure 3 Investigation of different thin layer plates - Tianjin Silida.

[0040] Figure 4 Investigation of different thin layer plates - Merck;

[0041] Figure 5 For different thin layer plate survey - Qingdao Ocean;

[0042] Figure 6For different temperatures -4℃;

[0043] Figure 7 For different temperatures -25℃;

[0044] Figure 8 For different temperatures -35℃;

[0045] Figure 9 For different humidity -32%;

[0046] Figure 10 For different humidity - 61%;

[0047] Figure 11 For different humidity -75%;

[0048] Figure 12 Verify the results for multiple batches;

[0049] Figure 13 The result diagram of Comparative Example 1 of the present invention;

[0050] Figure 14 Comparative Example 2 method result diagram. DETAILED DESCRIPTION

[0051] To further illustrate the present invention, a thin layer identification method for black sesame and roasted black sesame formula granules provided by the present invention is described in detail below with reference to examples.

[0052] Heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel GF254 thin layer plate (Qingdao Ocean Chemical Plant, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353).

[0053] Aluminum chloride, 95% ethanol, petroleum ether (60-90°C), chloroform, toluene, ethyl acetate, and methanol were all analytical grade, and water was ultrapure water (prepared in the laboratory).

[0054] Sesamin (China Food and Drug Inspection Institute, batch number: 110836-201706), β-sitosterol (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18010303), black sesame reference material (China Food and Drug Inspection Institute, batch number: 121339-201803), black sesame formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2208001; 2208002; 2208003), fried black sesame formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2211001; 2211002; 2211003).

[0055] Example 1

[0056] Preparation of test solution

[0057] Take 0.5g of this product, grind it into powder, add 20ml of water, heat to dissolve it, add ethyl acetate and shake to extract 3 times, 20ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0058] Preparation of reference substances and reference medicinal material solutions

[0059] Separately, prepare a reference solution containing 1 mg of each of sesamin and β-sitosterol in methanol per ml. Prepare the reference solution. Also prepare a reference solution of 0.5 g of black sesame in 20 ml of water for 30 minutes. Filter the filtrate and add ethyl acetate to the filtrate. Prepare the reference solution in the same manner as in "Preparation of Test Solution."

[0060] Determination method: According to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2010 edition), 10 μl of the test solution, 5 μl of the reference solution, and 10 μl of the reference medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-toluene-chloroform (5:4:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm).

[0061] Example 2 Methodological Investigation

[0062] 2.1 Sampling quantity inspection

[0063] Under the experimental conditions proposed above, 5 μl, 10 μl and 15 μl of sesamin, β-sitosterol reference solution, black sesame reference medicinal material solution, black sesame formula granule solution and fried black sesame formula granule solution were spotted on the same silica gel G thin layer plate. Figure 1 As shown in the figure, when 5-15 μl of the reference solution, reference herbal solution, and test solution are spotted, the spots appear clear and the separation is good, indicating a suitable spot size of 5-15 μl. Furthermore, at a shift ratio of 0.561, the black sesame formula granules exhibit a blue fluorescent spot, while the roasted black sesame formula granules exhibit no spot, which can be used to distinguish between the black sesame formula granules and the roasted black sesame formula granules. The presence of a blue fluorescent spot at a shift ratio of 0.561 can be used as a distinguishing factor. Figure 1 Note: 1-3 are sesamin reference solutions; 4-6 are β-sitosterol reference solutions; 7-9 are black sesame reference solutions; 10-12 are black sesame granule solutions; 13-15 are roasted black sesame granule solutions.

[0064] 2.2 Specificity Investigation

[0065] According to the above test sample preparation method, negative solution, sesamin reference solution, β-sitosterol reference solution, black sesame reference medicinal material solution, black sesame formula granule solution, and fried black sesame formula granule solution were prepared respectively, and thin layer identification experiments were carried out. The results are shown in the figure. Figure 2 , Figure 2 Specificity investigation; Note: 1 is negative solution; 2 is sesamin reference solution; 3 is β-sitosterol reference solution; 4 is black sesame reference medicinal material solution; 5 is black sesame formula granule solution; 6 is fried black sesame formula granule solution. Figure 2 As can be seen, the negative sample did not interfere with the black sesame and roasted black sesame granules, indicating good specificity of the method. Furthermore, at a ratio shift of 0.623, the black sesame granules exhibited blue fluorescent spots, while the roasted black sesame granules showed no spots.

[0066] 2.3 Durability Investigation

[0067] 2.3.1 Comparison of different thin layer plates

[0068] The tailorable thin layer chromatography plates of Tianjin Silida Technology Co., Ltd., the prefabricated silica gel G plates of Merck and Qingdao Ocean Chemical Plant Branch were selected and tested according to the proposed test methods. Figures 3-5 The results showed that the blue fluorescent spot ratios (Rf) of the three thin-layer plates at 8.3 to 10.9 cm from the origin were 0.613, 0.623, and 0.504, respectively, meeting the identification requirements. Furthermore, the black sesame formula granules exhibited blue fluorescent spots at Rf values ​​of 0.613, 0.623, and 0.504, while the roasted black sesame formula granules showed no spots.

[0069] The results are as follows Figure 3 , Figure 3 For different thin layer plate inspections - Tianjin Silida. Among them: 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is roasted black sesame formula granule solution.

[0070] Figure 4 Different thin layer plate inspection - Merck; 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is roasted black sesame formula granule solution.

[0071] Figure 5 For the investigation of different thin layer plates - Qingdao Ocean; 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0072] 2.3.2 Comparison of different temperatures

[0073] Take the spotted thin layer plate and develop it at low temperature of 4℃, normal temperature of 25℃ and high temperature of 35℃ respectively. Figures 6-8 It can be seen that this method has good adaptability to different temperatures, and the black sesame formula particles show blue fluorescent spots at the ratio shift values ​​of 0.541, 0.613, and 0.534, while the fried black sesame formula particles have no spots.

[0074] Figure 6 For different temperatures -4°C; among them, 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0075] Figure 7 For different temperatures -25°C, among which 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0076] Figure 8 For different temperatures -35 ° C, among which 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0077] 2.3.3 Comparison of different humidity levels

[0078] The thin layer plate after spotting was developed under humidity conditions of 32%, 61% and 75% respectively. Figures 9-11 As can be seen from the figure, this method has good adaptability to different temperatures, and the black sesame formula particles show blue fluorescent spots at the ratio shift values ​​of 0.584, 0.613, and 0.599, while the roasted black sesame formula particles have no spots.

[0079] Figure 9 For different humidity levels -32%; 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is roasted black sesame formula granule solution.

[0080] Figure 10 For different humidity levels -61%; 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0081] Figure 11 For different humidity levels -75%; 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4 is black sesame formula granule solution; 5 is fried black sesame formula granule solution.

[0082] 2.3.4 Verification

[0083] Sesamin, β-sitosterol reference solution, black sesame reference medicinal material solution, black sesame formula granule solution, and stir-fried black sesame formula granule solution were spotted on the same silica gel G thin layer plate and analyzed according to the established method. The experimental results are shown in Figure 12 The results showed that the black sesame formula granules had a blue fluorescent spot at a ratio shift value (Rf) of 0.579, while the roasted black sesame formula granules did not have this spot. The fluorescent spot can be used to distinguish the two.

[0084] Figure 12 These are the results of multiple batches of verification; among them, 1 is sesamin reference solution; 2 is β-sitosterol reference solution; 3 is black sesame reference medicinal material solution; 4-6 are black sesame formula granule solutions; 7-9 are roasted black sesame formula granule solutions.

[0085] 2.4 Determination of the identification point Rf of black sesame formula granules and roasted black sesame formula granules

[0086] The data of various ratio shift values ​​in the thin layer chromatography methodology investigation are summarized and the results are shown in Table 1. Based on the summary results, it is determined that the ratio shift value (Rf) of the identification spot of the black sesame formula granules should be within the range of 10%, and the specified value is 0.582.

[0087] Table 1 Summary of methodological investigation ratios

[0088]

[0089] Comparative Example 1

[0090] Take 4g of this product, add 10mL of chloroform, ultrasonicate for 15min, and then let it stand for 1h. Filter the filtrate to evaporate and dissolve it in 1mL of chloroform as the test solution. Take 1g of black sesame control medicinal material powder and 4g of negative control material without black sesame and prepare control medicinal material solution and negative control material solution in the same way. Take 4μL of the test solution. 2~3μL of the medicinal material control solution were spotted on the same silica gel G thin layer plate and developed with cyclohexane-ether-ethyl acetate (20:5.5:2.5) as the developing agent, took it out to dry, sprayed with 10% sulfuric acid ethanol solution and dried at 100℃ for about 10min to develop color. In the chromatogram of the test product, purple-brown spots appeared at the corresponding position of the chromatogram of the control medicinal material. There was no interference from the negative control. The results are shown in the figure. Figure 13 .

[0091] Figure 13 Results from Comparative Example 1 of the present invention. 1 is a black sesame negative control solution; 2 is a black sesame control medicinal solution; 3 is a black sesame granule solution; and 4 is a roasted black sesame granule solution. The results show that using the TLC method described above, the black sesame and roasted black sesame granules lack obvious spots, making them indistinguishable.

[0092] Comparative Example 2

[0093] Take 0.5g of this product, crush it, add 20ml of anhydrous ethanol, ultrasonically treat it for 20 minutes, filter it, evaporate the filtrate to dryness, add 1ml of anhydrous ethanol to dissolve the residue, let it stand, and take the supernatant as the test solution. Take another 0.5g of black sesame reference medicinal material, and prepare the control medicinal material solution, sesamin reference substance, and β-sitosterol reference substance in the same way. Add anhydrous ethanol to make a solution containing 1mg per 1ml, which is used as the reference solution. According to the thin layer chromatography method (General Rule 0502), 8μl of the above test solution and control medicinal material solution and 4μl of the reference solution are respectively taken and spotted on the same silica gel G thin layer plate, and cyclohexane-ether-ethyl acetate (20:5.5:2.5) is used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat until the spots are clearly colored. In the test sample chromatogram, spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the reference product chromatogram. Figure 14 Comparative Example 2 results. Figure 1 shows a sesamin reference solution; 2 shows a β-sitosterol reference solution; 3 shows a black sesame reference solution; 4 shows a black sesame granule solution; and 5 shows a roasted black sesame granule solution. The results show that using the TLC method, no significant differences were observed between the black sesame and roasted black sesame granules, making them indistinguishable.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for identifying black sesame and roasted black sesame granules through thin layer chromatography, comprising: A) Dissolve the sample in water, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to obtain a test solution; B) taking sesamin and β-sitosterol reference substances, dissolving them in a solvent to obtain reference substance solutions; C) taking black sesame as a control medicinal material and dissolving it in a solvent to obtain a control medicinal material solution; D) The test solution, the reference solution, and the reference medicinal material solution were subjected to thin layer chromatography using a silica gel G thin layer plate; the developing solvent was petroleum ether-toluene-chloroform; the mass ratio of the petroleum ether-toluene-chloroform was 5:4:2; The boiling point of the petroleum ether is 60-90°C; E) Observe under ultraviolet light. If a blue fluorescent spot is observed at an Rf value of 0.582 ± 10% in the chromatogram of the test sample, it is the black sesame granules. If there is no blue fluorescent spot at the Rf value of 0.582±10%, it is a roasted black sesame formula granule.

2. The method according to claim 1, characterized in that The step A) is specifically as follows: The mass volume ratio of the sample to be tested, water, ethyl acetate and methanol is: 0.5-1 g: 15-25 mL: 50-70 mL: 0.5-2 mL.

3. The method according to claim 1, characterized in that Step B) The solvent is methanol; the mass concentration of the sesamin reference substance is 0.5-5 mg / mL; the mass concentration of the β-sitosterol reference substance is 0.5-5 mg / mL.

4. The method according to claim 1, wherein The step C) is specifically as follows: Take black sesame as a reference medicinal material, add water to boil, then add ethyl acetate to extract, evaporate to dryness, and dissolve the residue in methanol to obtain the product.

5. The method according to claim 1, wherein The thin layer chromatography spotting volume is 5 to 15 μL.

6. The method according to claim 1, characterized in that In step E), the wavelength of the ultraviolet lamp is 365 nm.

7. The method according to claim 1, characterized in that Step E) Check the temperature to be between 4°C and 35°C.

8. The method according to claim 7, characterized in that In step E), the humidity is checked to be 35% rh to 75% rh.

9. The method according to claim 1, characterized in that Step A) The sample to be tested is black sesame or roasted black sesame granules.

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