Longan leaf granules and quality control method thereof

Abstract

The invention discloses a longan leaf granule which is prepared by the following steps: adding water into longan leaves, heating, extracting, filtering, concentrating, drying, crushing to obtain dry paste powder, adding a diluent into the dry paste powder, uniformly mixing, adding a wetting agent, granulating, and drying to obtain the longan leaf granule. The forming rate, the dissolution rate, the moisture absorption rate, the fluidity and the like of the granules all meet the requirements, and the granules are good in stability, suitable for industrial batch production, capable of treating diabetes, capable of meeting the requirements of doctors for treatment based on syndrome differentiation and increase and decrease along with symptoms and convenient to carry and take by patients. Thin-layer chromatography is adopted for qualitative identification of the longan leaf granules, high performance liquid chromatography is adopted for content determination of quercetin, quercitrin and kaempferol in the longan leaf granules, operation is easy, convenient and accurate, repeatability is good, and a scientific basis is provided for quality evaluation and control of the longan leaf granules.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and particularly relates to a longan leaf granule and a quality control method therefor. Background Art

[0002] Diabetes has become the "third killer" threatening humanity after cardiovascular diseases and tumors, and the prevention and treatment of diabetes have become global issues. At present, there are many drugs with good curative effects clinically used to treat diabetes, but due to their large side effects on the human body and the fact that some drugs cannot effectively control the occurrence of various complications, they cannot be used as the first choice for long-term application. Traditional Chinese medicine has the advantages of significantly improving clinical symptoms and signs, and having few and mild adverse reactions when treating diabetes. Therefore, the treatment of diabetes with traditional Chinese medicine has attracted more and more attention.

[0003] Longan leaf is the leaf or tender bud of Dimocarpus longan Lour. of the genus Dimocarpus in the family Sapindaceae, and has the effects of inducing sweating and clearing heat, promoting diuresis and detoxifying, and is mainly used to treat cold and fever, malaria, furuncle, and eczema. The resources of longan leaves are rich in southern China. After longans are picked every year, fruit farmers cut the branches of the trees, and a large amount of leaves are discarded or burned, causing waste of resources and environmental pollution. The main chemical components of longan leaves include flavonoids, phenols, volatile oils, etc. Among them, flavonoid compounds are the main components of longan leaves. A large number of studies have shown that longan leaves have obvious hypoglycemic effects, and the flavonoid components of longan leaves can effectively prevent and treat type II diabetes.

[0004] Traditional Chinese medicine granules act quickly and are very convenient to carry and transport. Compared with traditional Chinese medicine decoction pieces, traditional Chinese medicine granules can better meet the needs of doctors for syndrome differentiation and treatment and symptom-based addition and subtraction. Longan leaves are often used alone, and the traditional taking method is the decoction. Decoction is troublesome, and the chemical properties of the decoction are unstable and easy to mildew. The literature reports on the extraction of longan leaves by traditional Chinese medicine mostly focus on using different extraction solvents or advanced technologies to improve the extraction rate of active ingredients of medicinal materials. For example, Zheng Wei et al. used ethanol as a solvent and optimized the process of extracting total flavonoids from longan leaves by ultrasonic-assisted extraction; Yu Qiuju et al. optimized the process and activity of extracting total flavonoids from Rubus idaeus by microwave-assisted deep eutectic solvent extraction method; Zhang Q et al. optimized the extraction process of two bioactive flavonoids, luteolin and apigenin, in celery by ultrasonic-assisted enzymatic hydrolysis technology, and determined the yields of luteolin and apigenin by high performance liquid chromatography. However, these methods are quite different from the water extraction method of traditional decoction, which is likely to cause unknown adverse reactions and is not suitable for the extraction process of granules. Therefore, in this study, the hydrothermal method was selected as the extraction method for longan leaf granules. In order to better exert the curative effect of longan leaves and facilitate taking, the present invention intends to develop the discarded or burned longan leaves into a granule preparation for clinical use, and has formulated a qualitative and quantitative quality control method. At present, there are no relevant reports.

[0005] The disclosure of the above background art content is only for assisting in understanding the inventive concept and technical solution of the present invention, and it does not necessarily belong to the prior art of this patent application. Without clear evidence indicating that the above content was publicly available on the filing date of this patent application, the above background art should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0006] In view of the fact that the drugs for treating diabetes in the prior art have relatively large side effects on the human body and some drugs cannot effectively control the occurrence of various complications, the present invention provides a longan leaf granule and a quality control method therefor.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] A longan leaf granule, wherein the granule is obtained by adding water to longan leaves for heating extraction, filtration, concentration, drying, and pulverization to obtain dry extract powder, adding a diluent to the dry extract powder, mixing evenly, adding a wetting agent, granulating, and drying to obtain the longan leaf granule; in the step of adding water for heating extraction: the amount of water added is preferably 18 times the weight of the longan leaves, the soaking time is preferably 30 minutes, the number of times of heating extraction is preferably 3 times, and each time is 1 hour; in the concentration step: it is preferably concentrated into an extract with a relative density of 1.25 - 1.35 at 60°C; the diluent is dextrin and mannitol, and the weight ratio of dextrin to mannitol is preferably 1:1 - 1:1.5; the best weight ratio of dextrin to mannitol is preferably 1:1.5; the wetting agent is preferably an 80% ethanol solution. The "heating extraction" of the longan leaves in the present invention is all "heating and boiling extraction".

[0009] The preparation method of the above-mentioned longan leaf granule includes the following steps: R1. Take longan leaves, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, each time for 1 hour, filter, and combine to obtain a filtrate; R2. Take the filtrate, concentrate it into an extract with a relative density of 1.25 - 1.35 at 60°C, dry it, and pulverize it to obtain dry extract powder; R3. Add the diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; the weight ratio of dextrin to mannitol is preferably 1:1 - 1:1.5; R4. Add a wetting agent to the mixture, granulate, and dry to obtain the longan leaf granule.

[0010] In the preparation method of the above-mentioned longan leaf granule, the concentration step in R2 is preferably vacuum concentration at 65 - 75°C, and the drying step is preferably vacuum drying at 60 - 80°C.

[0011] In the preparation method of the above-mentioned longan leaf granule, the concentration step in R2 is most preferably vacuum concentration at 65°C, and the drying step is most preferably vacuum drying at 60°C.

[0012] For the above-mentioned method for preparing longan leaf granules, the optimal weight ratio of dextrin to mannitol in step R3 is preferably 1:1.5.

[0013] For the above-mentioned method for preparing longan leaf granules, the wetting agent in step R4 is preferably an 80% ethanol solution.

[0014] The identification method for the above-mentioned longan leaf granules includes the following steps: S1: Preparation of the test solution: Take longan leaf granules, grind them finely, weigh 1.0 g, add 10 mL of a methanol-hydrochloric acid solution with a volume ratio of 40:1, ultrasonically extract for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water, mix well, extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 mL of methanol to obtain the test solution; S2: Preparation of the reference solution: Weigh quercetin, quercitrin, and kaempferol reference substances respectively, add methanol to make up the volume to obtain reference solutions with concentrations of 0.25 mg / mL, 0.15 mg / mL, and 0.5 mg / mL; S3: Preparation of the control medicinal material solution: Weigh 1.0 g of longan leaf control medicinal material and prepare the control medicinal material solution in the same way as in S1; S4: Development and inspection: Pipette 10 μL of each of the above 5 solutions and spot them on the same silica gel G thin-layer plate, develop in two steps. The volume ratio of the first developing agent "ethyl acetate-formic acid-water" is 9:0.5:0.5, and the volume ratio of the second developing agent "xylene-ethyl acetate-formic acid" is 8:5:1. Take out, dry in air, spray with 3% aluminum trichloride-ethanol solution for color development, bake at 105 °C for 2 minutes, and examine under a 365 nm ultraviolet lamp.

[0015] The content determination method for the above-mentioned longan leaf granules includes the following steps: T1. Preparation of the test solution: Take 1.0 g of longan leaf granules, grind them finely, accurately weigh, add 10 mL of a methanol-hydrochloric acid solution with a volume ratio of 40:1, ultrasonically extract for 30 minutes, filter, and take the subsequent filtrate as the test solution; T2. Preparation of the reference solution: Accurately weigh quercetin, quercitrin, and kaempferol reference substances respectively, add methanol, ultrasonically treat to dissolve, shake well, and prepare a mixed reference solution with concentrations of 0.1 mg / mL, 0.2 mg / mL, and 0.02 mg / mL; T3. Chromatographic conditions: The chromatographic column is packed with octadecylsilane-bonded silica gel; the detection wavelength is 360 nm; methanol is used as mobile phase A, and 0.2% phosphoric acid is used as mobile phase B, with gradient elution (0 - 22 min, 45% A - 49% A; 22 - 35 min, 49% A - 75% A); flow rate: 1 mL / min; column temperature: 30 °C; T4. Determination: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the high-performance liquid chromatograph, and determine according to the method in step T3.

[0016] The above-mentioned application of longan leaf granules, the application of longan leaf granules in the treatment of diabetes drugs; in particular, the application in the treatment of type II diabetes drugs.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1. Aiming at the defects of traditional decoctions, such as troublesome decocting and inconvenient carrying of decoctions, the present invention adds excipients such as dextrin and mannitol to develop longan leaf into a single-component granule. Its forming rate, dissolution rate, moisture absorption rate, fluidity, etc. all meet the requirements of the Chinese Pharmacopoeia. The accelerated test results show that the product has good stability and is suitable for industrial mass production; it can exert the curative effect of longan leaf in the treatment of diabetes, especially in the treatment of type II diabetes; it can also meet the needs of doctors for syndrome differentiation and treatment, and addition and subtraction according to symptoms, and is convenient for patients to carry and take; it has a broad market demand prospect.

[0019] 2. The present invention uses thin-layer chromatography for qualitative identification of longan leaf granules. On the same silica gel G thin-layer plate, three active ingredients with large differences in polarity are simultaneously extracted and separated. After development with the developing agent, heating and color development, and examination under an ultraviolet lamp (365 nm), at the corresponding positions of quercetin, quercitrin, kaempferol reference substances and longan leaf reference medicinal materials, spots of the same color are shown. This method is simple, accurate and has good repeatability, and can be used for the quality control of longan leaf granules.

[0020] 3. The present invention also uses high-performance liquid chromatography to quantitatively determine quercetin, quercitrin and kaempferol in longan leaf granules. Flavonoid compounds in longan leaf are the main active ingredients of longan leaf, and studies have shown that they have a therapeutic effect on type II diabetes; the inventor uses UHPLC-Q-Orbitrap MS technology to study the metabolites of longan leaf in rats and finds that the pharmacodynamic material basis for longan leaf to treat T2DM may be quercetin, quercitrin and kaempferol. The present invention takes quercetin, quercitrin and kaempferol as the content determination indexes, which can more scientifically, accurately and comprehensively reflect the product quality of longan leaf granules. The test results show that the quality analysis method established by the present invention is simple, accurate and has good repeatability, providing a scientific basis for the quality evaluation and control of longan leaf granules.

[0021] Description of the drawings

[0022] Figure 1 Line chart of the change in the total flavonoid content and extract yield with different soaking times

[0023] Figure 2 Line chart of the change in the total flavonoid content and extract yield with different solvent multiples

[0024] Figure 3 Line chart of the change in the total flavonoid content and extract yield with different extraction times

[0025] Figure 4 Line chart of the total flavonoid content and extract yield obtained with different extraction times

[0026] Figure 5 Thin layer chromatography identification diagram of the longan leaf granules in Examples 1-3 of the present invention

[0027] 1. Negative control solution, 2. Quercetin, 3. Quercitrin, 4. Kaempferol, 5. Example 1, 6. Example 2, 7. Example 3, 8. Solution of the reference crude drug of longan leaf

[0028] Figure 6 HPLC diagram of the mixed reference substance solution

[0029] 1. Quercitrin, 2. Quercetin, 3. Kaempferol

[0030] Figure 7 HPLC diagram of the test solution of the longan leaf granules in Example 3 of the present invention

[0031] 1. Quercitrin 2. Quercetin 3. Kaempferol

[0032] Figure 8 HPLC diagram of the solution of the reference crude drug of longan leaf

[0033] 1. Quercitrin 2. Quercetin 3. Kaempferol

[0034] Figure 9 HPLC diagram of the negative control solution Detailed implementation manners

[0035] The present invention will be further described below in conjunction with specific embodiments, but the protection scope and application scope of the present invention are not limited.

[0036] Drugs and reagents: Commercially available longan leaf crude drugs from Wuzhou City, Guangxi; rutin reference substance (Chengdu Maidesheng Technology Co., Ltd., batch number RP210601); reference crude drug of longan leaf, identified by Professor Teng Jianbei of Guangxi University of Chinese Medicine as the leaves of Dimocarpus longan Lour of the genus Dimocarpus in the family Sapindaceae; quercetin reference substance (Chengdu Maidesheng Technology Co., Ltd., batch number RP200602); quercitrin reference substance (Chengdu Maidesheng Technology Co., Ltd., batch number RP230301); kaempferol reference substance (Chengdu Maidesheng Technology Co., Ltd., batch number RP210502); silica gel G thin layer plate; silica gel H thin layer plate; silica gel GF 254 Thin layer plate; polyamide film; acetonitrile and methanol are chromatographically pure, water is ultrapure water, and sodium nitrite, aluminum nitrate, sodium hydroxide, aluminum trichloride, and ethanol are analytical pure reagents.

[0037] Main instruments: ultraviolet-visible spectrophotometer; electronic analytical balance; high-speed multi-functional pulverizer; electrothermal constant temperature forced air drying oven; bench-top low-speed centrifuge; ultrasonic cleaner; thin-layer chromatography imaging system; semi-automatic sample applicator; high performance liquid chromatograph (Agilent Technologies, Inc. (USA), model Agilent 1100); Inert Sustain C18 chromatographic column (4.6 mm × 250 mm, 5 μm), Shimadzu Techno-Research (Shanghai) Co., Ltd.).

[0038] I. Process research

[0039] 1. Extraction process research

[0040] Relevant parameters of the extraction process were investigated through single-factor experiments: 50 g of crude longan leaf powder was weighed, and the solvent multiple was selected as 8, 10, 12, 14, 16, 18, 20 times; the extraction time was selected as 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h; the extraction times were selected as 1, 2, 3 times to investigate their effects on the total flavonoid extraction amount and the extract yield, providing references for selecting factors and levels in the orthogonal experiment.

[0041] Determination method of extract yield: 50 mL of the extraction solution was precisely measured and placed in an evaporating dish dried to constant weight. It was evaporated to dryness in a water bath and dried in an oven at 105 °C. After taking it out, it was cooled to room temperature in a desiccator and precisely weighed. Then it was dried at 105 °C for another 0.5 h and the mass was precisely weighed to calculate the extract yield.

[0042]

[0043] Determination method of total flavonoid content: ① Preparation of reference solution: A precisely weighed rutin reference substance dried to constant weight was placed in a volumetric flask, and 70% ethanol solution was added to volume to the scale and shaken well to prepare a rutin reference solution of 0.4020 mg / mL.

[0044] ② Preparation of test solution: Weigh 50 g of the crude powder of Euphoria longan leaves, add 18 times the amount of water, soak for 30 min, extract 3 times, each time for 1 h, filter, combine the filtrates, concentrate and make up the volume to 250 mL. Accurately measure 10 mL of the solution and transfer it to a 50-mL volumetric flask, make up the volume to the mark, and thus obtain the test solution of Euphoria longan leaves. ③ Investigation of linear relationship: Accurately measure 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of rutin reference solution respectively and transfer them to 25-mL volumetric flasks. Add 1 mL of 5% NaNO₂ solution, shake well, and let stand for 6 min; then add 1 mL of 10% Al(NO₃)₃ solution, shake well, and let stand for 6 min; then add 10 mL of 4% NaOH solution, shake well, make up the volume to the mark, and let stand for 15 min. Use the corresponding reagent as the blank, use 506 nm as the detection wavelength, determine according to the ultraviolet-visible spectrophotometry. Take the absorbance A as the ordinate and the concentration C of the rutin reference as the abscissa, plot the standard curve, and obtain the regression equation. ④ Determination of the total flavonoid content: Weigh 3 batches of samples, prepare 1 mL of the test solution according to the method under item ②, and perform the same operation as described under item ③ starting from "add 1 mL of 5% NaNO₂ solution" to determine the absorbance, and calculate the total flavonoid content of Euphoria longan leaves.

[0045] (1) Investigation of soaking time

[0046] Weigh 50 g of the crude powder of Euphoria longan leaves, a total of 5 portions, add 10 times the amount of water for soaking respectively, and the soaking times are 0 h, 0.5 h, 1 h, 1.5 h, and 2 h respectively. Extract for 1 h, and prepare the test solution according to the method under item ② above starting from "filter, combine the filtrates, and make up the volume to 250 mL". Add the color-developing solution in sequence to determine the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 1 and Figure 1 .

[0047] Table 1 Effects of different soaking times on the extraction of Euphoria longan leaves

[0048]

[0049] The test results show that: when the soaking time increases from 0 to 0.5 h, both the total flavonoid content and the extract yield in the extract gradually increase; when it exceeds 0.5 h, the total flavonoid content begins to decrease, but the change in the extract yield is not obvious. Therefore, too long soaking time will affect the extraction efficiency of total flavonoids. Thus, the preferred soaking time in the present invention is 0.5 h, that is, 30 minutes.

[0050] (2) Investigation of the multiple of water addition

[0051] Take 7 portions of crude longan leaf powder, each portion about 50 g, accurately weigh, and add 8, 10, 12, 14, 16, 18, and 20 times the amount of water respectively. Soak for 30 min and extract for 1 h. Prepare the test solution starting from "filter, combine the filtrates, and make up to 250 mL" under item ② above. Add the chromogenic solution successively to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 2, Figure 2 。

[0052] Table 2 Effects of Different Water Addition Multiples on the Extraction of Longan Leaf

[0053]

[0054] The test results show that: with the increase of the solvent multiple, both the total flavonoid content and the extract yield gradually increase. When the solvent multiple is 18 times, the total flavonoid content reaches 52.53 mg / g and the extract yield is 14.41%. Continuing to increase the solvent multiple, the total flavonoid content and the extract yield basically remain stable. Considering energy consumption and production cost, the solvent multiple of 18 times is selected in the present invention.

[0055] (3) Investigation of Extraction Time

[0056] Weigh 50 g of crude longan leaf powder, a total of 5 portions, add 18 times the amount of water to each portion. After soaking for 30 min, extract for 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h respectively. Prepare the test solution starting from "filter, combine the filtrates, and make up to 250 mL" under item ② above. Add the chromogenic solution successively to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 3, Figure 3 。

[0057] Table 3 Effects of Different Extraction Times on the Extraction of Longan Leaf

[0058]

[0059] It can be seen from the test results that with the increase of the extraction time, the total flavonoid content first increases and then decreases. When it exceeds 1 h, the total flavonoid content begins to gradually decline; the extract yield slightly decreases but the change range is not large. With too long extraction time, other impurities in the medicinal materials will be extracted, and long-term heating extraction may also damage the active ingredients of longan leaf, thus reducing the total flavonoid content extracted from longan leaf. Therefore, 1 h is selected as the optimal extraction time.

[0060] (4) Results of Investigation of Extraction Times

[0061] Weigh 50 g of the crude powder of longan leaves, a total of 3 portions. Add 18 times the amount of water, soak for 30 min, then extract for 1 h. The extraction times are 1, 2, and 3. Prepare the test solution starting from "filter, combine the filtrates, and make up the volume to 250 mL" under item ② above. Add the color-developing solution in sequence to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 4, Figure 4 .

[0062] Table 4 Effects of different extraction times on the extraction of longan leaves

[0063]

[0064] The test results show that: as the extraction times increase, the total flavonoid content and extract yield extracted gradually increase. When extracting for the 3rd time, the total flavonoid content and extract yield reach 83.95 mg / g and 20.70%, respectively. The extraction of total flavonoids is basically complete. Considering energy consumption and production costs, the preferred extraction time of this invention is 3 times.

[0065] (5) Optimization of the best extraction process by orthogonal test method

[0066] According to the results of the single-factor experiment, select the solvent multiple, extraction time, and extraction times as the investigation factors, and use the comprehensive score of total flavonoid content and extract yield as the main investigation index, that is, comprehensive score = (extract yield / maximum extract yield × 0.4 + total flavonoid content / maximum total flavonoid content × 0.6) × 100%, optimize the best extraction conditions, and determine the best extraction process of longan leaf granules. The factors and levels of the orthogonal experiment are shown in Table 5, and the orthogonal experiment design and analysis are shown in Tables 6 - 7.

[0067] Table 5 Factor and level table of orthogonal experiment

[0068]

[0069] Table 6 Results table of L9(3 4 ) orthogonal experiment

[0070]

[0071] Table 7 Results of variance analysis

[0072]

[0073] From the above data analysis results, it can be seen that the order of influence of the three factors from large to small is the extraction times (C) > the solvent multiple (A) > the extraction time (B). Among them, there are significant differences in the influence of factors A and C (P < 0.05), and there is no significant difference in the influence of factor B. The values of B2 and B3 are relatively close. Considering shortening the production cycle, the extraction time is selected as 1 h. To sum up, the optimal extraction process for longan leaf granules can be determined as A3B2C3, that is, the solvent multiple is 18 times, the extraction time is 1 h, and the extraction times are 3 times.

[0074] (6) Verification of the extraction process

[0075] Three repeated experiments were carried out according to the above optimal extraction process A3B2C3. The average value of the total flavonoid content was 82.04 mg / g, and the average value of the extract yield was 21.55%. The RSD values were 0.83% and 1.51% respectively. The results show that the extraction process conditions of longan leaf granules optimized by the orthogonal experiment are stable and feasible, and both the total flavonoid content and the extract yield are relatively high.

[0076] 2. Research on the purification process

[0077] Weigh the crude powder of longan leaves and extract it according to the above optimal extraction process conditions A3B2C3. After concentrating the extract, it is fixed in a 200 mL volumetric flask. Five portions are taken from it, each portion is 25 mL. One portion is used as the control solution, and the other 4 portions are respectively added with 50%, 60%, 70%, and 80% ethanol solutions for alcohol precipitation. After standing at room temperature for 24 h, the precipitate is filtered. The filtrate is concentrated and fixed in a 250 mL volumetric flask, and the color-developing solution is added successively to measure the absorbance, and the total flavonoid content, the total flavonoid transfer rate, and the extract yield are calculated. The test results are shown in Table 8.

[0078] Table 8 Investigation results of alcohol precipitation processes with different concentrations

[0079]

[0080] The test results show that after the alcohol precipitation purification process, the changes in the total flavonoid content and the extract yield show a downward trend. In order to retain the content of the main active ingredients and the extract yield to the greatest extent, the alcohol precipitation method is not adopted.

[0081] 3. Research on the concentration process

[0082] (1) Atmospheric concentration

[0083] Perform extraction according to the above-mentioned optimal water extraction method to obtain the water extract of longan leaves. Concentrate it at different temperatures of 90 °C and 100 °C respectively, and concentrate it to about 25 mL. Weigh 5 g and dissolve and make up the volume in a 100 mL volumetric flask. Add the color-developing solution in sequence to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate and extract yield. The results are shown in Table 9. It can be seen from the results that after concentration at 90 °C and 100 °C under normal pressure, the changes in the total flavonoid content and extract yield are not obvious.

[0084] Table 9 Results of investigation on concentration under normal pressure

[0085]

[0086] (2) Vacuum concentration

[0087] Measure the water extract of longan leaves by the above-mentioned optimal water extraction method, and concentrate it under reduced pressure at 55 °C, 65 °C, 75 °C, and 85 °C, and concentrate it to about 25 mL (relative density 1.25 - 1.35). Weigh 5 g and dissolve and make up the volume in a 100 mL volumetric flask. Add the color-developing solution in sequence to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate and extract yield. The results are shown in Table 10.

[0088] Table 10 Results of investigation on concentration under reduced pressure

[0089]

[0090] It can be seen from the results in Table 9 and Table 10 that the total flavonoid content in vacuum concentration is significantly higher than that in normal pressure concentration; with the increase of the concentration temperature in vacuum concentration, the total flavonoid content first increases and then decreases, and the extract yield also decreases slightly; it shows that the vacuum concentration effect is better than that of normal pressure concentration. And vacuum concentration can improve production efficiency, save production costs, and also retain the active ingredients better. Therefore, the present invention preferably uses vacuum concentration at 65 - 75 °C, and the best preferred vacuum concentration temperature is 65 °C.

[0091] 4. Study on drying process

[0092] (1) Investigation on drying under normal pressure

[0093] Weigh the crude powder of longan leaves, perform extraction by the above-determined optimal extraction method of A3B2C3, and divide the extract into 7 equal parts after concentrating it under reduced pressure at 65 °C to obtain an extract with a relative density of 1.25 - 1.35 under the above-mentioned optimal process conditions.

[0094] Take 3 portions of the extract test samples and place them in a constant temperature drying oven at 80 °C, 90 °C, and 100 °C respectively. After drying to a constant weight, crush the dried extract to obtain dry extract powder. Precisely weigh 2 g and dissolve and make up the volume in a 50 mL volumetric flask. Sequentially add the chromogenic solution to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 11. It can be seen from Table 11 that as the drying temperature gradually increases, there are no obvious changes in both the total flavonoid content and the extract yield.

[0095] Table 11 Results of atmospheric pressure drying investigation

[0096]

[0097] (2) Vacuum drying investigation

[0098] Take the remaining 4 portions of the extract test samples and carry out vacuum drying at 60 °C, 70 °C, 80 °C, and 90 °C. After drying to a constant weight, crush the dried extract to obtain dry extract powder. Precisely weigh 2 g and dissolve and make up the volume in a 50 mL volumetric flask. Sequentially add the chromogenic solution to measure the absorbance, and calculate the total flavonoid content, total flavonoid transfer rate, and extract yield. The results are shown in Table 12.

[0099] Table 12 Results of vacuum drying investigation

[0100]

[0101] It can be seen from the results of Table 11 and Table 12 that even at the same temperature of 90 °C, the total flavonoid content in vacuum drying is significantly higher than that in atmospheric pressure drying; it shows that the drying temperature has little effect on the total flavonoid in the drying process, but the pressure has a great influence; under vacuum conditions, the influence of different temperatures on the total flavonoid and extract yield indexes is relatively small. Vacuum drying can improve production efficiency, save production costs, and also has a good retention of active ingredients. Therefore, the present invention can carry out vacuum drying at 60 - 90 °C; considering equipment operation, it is preferably 60 - 80 °C for vacuum drying; the best preference is 60 °C for vacuum drying.

[0102] 5. Research on the granulation process of longan leaf granules

[0103] Weigh 50 kg of longan leaf crude powder, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, 1 hour each time, filter, combine to obtain the filtrate; take the filtrate, concentrate it under reduced pressure at 65 °C to obtain a longan leaf extract with a relative density of 1.30 at 60 °C, and dry it under reduced pressure at 60 °C and crush it to obtain longan leaf dry extract powder.

[0104] (1) Selection of diluent

[0105] In the experiment of the present invention, soluble starch, dextrin, mannitol, and microcrystalline cellulose were selected as the diluents for longan leaf granules. By investigating the types of diluents, the ratio of diluents, and the volume fraction of the wetting agent, and measuring the values of the granulation rate, angle of repose, moisture absorption rate, and dissolution rate for each factor, the comprehensive score was calculated. Based on the high and low of the score values, the optimal preparation process for longan leaf granules was preferably selected.

[0106] Comprehensive score = (25 / maximum granulation rate) × granulation rate value + (25 × minimum moisture absorption rate) / moisture absorption rate value + (25 / maximum dissolution rate value) × dissolution rate value + (25 × minimum angle of repose) / angle of repose value

[0107] ① Preliminary investigation of diluent dosage

[0108] Mix the prepared longan leaf dry extract powder with the diluent dextrin according to the following table ratio, make soft materials with 80% ethanol solution, continuously knead with fingers until the state of "kneading into a ball by hand and dispersing when pressed" is achieved. Then, extrude the soft materials through a sieve, granulate with a 16-mesh sieve, dry in a 60°C forced-air drying oven for 1 h, take out and let cool, and screen with a 16-mesh sieve to obtain the granules. Record the granulation situation and calculate the comprehensive score, and preliminarily screen out the appropriate diluent dosage according to the high and low of the score values. The results are shown in Table 13.

[0109] Table 13 Results of the investigation of diluent dosage

[0110]

[0111] Through preliminary pre-experiments, when the dry extract powder:diluent = 1:2, the comprehensive score is the highest, providing a reference for the following experiments.

[0112] ② Investigation of diluent types

[0113] Mix the longan leaf dry extract powder according to the ratio of dry extract powder:diluent = 1:2. The diluents are soluble starch, dextrin, mannitol, and microcrystalline cellulose respectively. Make soft materials with 80% ethanol solution, continuously knead with fingers until the state of "kneading into a ball by hand and dispersing when pressed" is achieved. Then, extrude the soft materials through a sieve, granulate with a 16-mesh sieve, dry in a 60°C forced-air drying oven for 1 h, take out and let cool, and screen with a 16-mesh sieve to obtain the granules. Record the granulation situation and calculate the comprehensive score, and screen out the most suitable diluent type according to the high and low of the score values. Compare the granulation property, solubility, and production cost of mannitol, dextrin, and soluble starch comprehensively. The results are shown in Table 14.

[0114] Table 14 Results of the investigation of diluent types

[0115]

[0116] The results showed that the soft materials with a single diluent were more viscous, and the obtained granules were loose with a large amount of fine powder. However, the mixing of three diluents would increase the number of tests, and the investigation of the ratio and dosage of diluents became complicated. To obtain granules with good formability and solubility, therefore, two types were selected as the mixed diluents for the granules in this experiment. Mannitol is an isomer of sorbitol, which is easily soluble in water, has a sweet taste similar to sucrose, and can also be used as a flavoring agent. Dextrin has the advantages of good formability, good solubility, and moderate price. Therefore, dextrin and mannitol were selected as the diluents for longan leaf granules in this experiment.

[0117] ③ Investigation of diluent ratio

[0118] Weigh 5 portions of the above-prepared dried extract powder of longan leaf, mix them evenly with dextrin and mannitol according to different ratios respectively, add 80% ethanol solution to make soft materials, granulate through a 16-mesh sieve, dry in an incubator at 60 °C, and size the granules. Record the granulation situation and calculate the comprehensive score to select the appropriate ratio of the mixed diluents. The results are shown in Table 15.

[0119] Table 15 Results of the investigation of diluent ratio

[0120]

[0121] Based on the results of the diluent investigation, dextrin and mannitol were selected as the mixed diluents for investigation, and the optimal ratio of the two diluents was studied. The results showed that when the proportion of dextrin was relatively large, its viscosity was low, it was easy to granulate, and the fluidity of the granules was relatively good, but the formability was low; while when the proportion of mannitol was larger, the viscosity was good, granulation was easy, and the formability was good. Considering comprehensively, when dextrin:mannitol = 1:1 - 1:1.5, the soft material was moderate, easy to granulate, and had a high comprehensive score. Therefore, this ratio was selected as the ratio of the mixed diluents for longan leaf granules.

[0122] (2) Investigation of the volume fraction of the wetting agent

[0123] Weigh 4 portions of the above-prepared dried extract powder of longan leaf, stir it evenly with dextrin:mannitol (1:1.5), and add 60%, 70%, 80%, and 90% ethanol solutions respectively to make soft materials, granulate through a 16-mesh sieve, dry in an incubator at 60 °C, and size the granules. Record the granulation situation and calculate the comprehensive score to screen the appropriate volume fraction of the wetting agent. The results are shown in Table 16.

[0124] Table 16 Results of the investigation of the volume fraction of the wetting agent

[0125]

[0126] The above test results show that the molding rate of 80% ethanol is the highest, and the particles are relatively uniform, making it easier to prepare soft materials. When the volume fraction of the wetting agent is lower than 80% ethanol, the viscosity of the soft material is relatively high, making it difficult to granulate. When the volume fraction of the wetting agent is higher than 80% ethanol, the soft material is loose and easy to granulate, but there are more fine powders in the particles. Therefore, the optimal wetting agent for longan leaf particles is 80% ethanol solution.

[0127] (4) Verification experiment of small-scale molding process

[0128] Weigh the dry extract powder of longan leaves prepared above, a total of 3 portions, and add the diluents dextrin:mannitol (1:1.5) respectively. The weight ratio of the dry extract powder to the diluent is 1:2. Add 80% ethanol solution to prepare soft materials, granulate through a 16-mesh sieve, dry in an incubator at 60°C, and screen the granules. Measure 4 evaluation indexes and calculate the comprehensive score. The experimental results are shown in Table 17.

[0129] Table 17 Verification results of small-scale molding process

[0130]

[0131] For the three batches of samples prepared according to the optimized molding process, the average molding rate is 91.49%, and the RSD value is 0.75%; the average dissolution rate is 93.03%, and the RSD value is 0.35%; the average moisture absorption rate is 3.45%, and the RSD value is 1.40%; the average α value is 23.66°, and the RSD value is 1.12%; the average comprehensive score is 99.06, and the RSD is 0.29%. There is no significant difference, and all meet the requirements under the general rules for granules in Part IV of the Chinese Pharmacopoeia (2020 Edition). This indicates that the process is stable and feasible, with good repeatability, and can be used for the preparation of longan leaf granules.

[0132] II. Preparation method of longan leaf granules

[0133] Example 1

[0134] A preparation method of longan leaf granules, comprising the following steps:

[0135] R1. Take 100 kg of longan leaves, add 18 times the amount of water, soak for 30 minutes, extract by heating 3 times, each time for 1 hour, filter, combine to obtain a filtrate;

[0136] R2. Take the filtrate, concentrate it under reduced pressure at 75°C to an extract with a relative density of 1.25 at 60°C, dry it under reduced pressure at 80°C, and pulverize to obtain dry extract powder;

[0137] R3. Add the diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; the weight ratio of the dry extract powder to the diluent is 1:1.8, and the weight ratio of the dextrin to the mannitol is 1:1;

[0138] R4. Add an 80% ethanol solution to the mixture, granulate, and dry at 60°C to obtain longan leaf granules.

[0139] Example 2

[0140] A method for preparing longan leaf granules, comprising the following steps:

[0141] R1. Take 100 kg of longan leaves, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, 1 hour each time, filter, combine to obtain a filtrate;

[0142] R2. Take the filtrate, concentrate it under reduced pressure at 70°C to an extract with a relative density of 1.35 at 60°C, dry it under reduced pressure at 70°C, and pulverize to obtain dry extract powder;

[0143] R3. Add the diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; the weight ratio of the dry extract powder to the diluent is 1:2.2, and the weight ratio of dextrin to mannitol is 1:1.3;

[0144] R4. Add an 80% ethanol solution to the mixture, granulate, and dry at 60°C to obtain longan leaf granules.

[0145] Example 3

[0146] A method for preparing longan leaf granules, comprising the following steps:

[0147] R1. Take 100 kg of longan leaves, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, 1 hour each time, filter, combine to obtain a filtrate;

[0148] R2. Take the filtrate, concentrate it under reduced pressure at 65°C to an extract with a relative density of 1.30 at 60°C, dry it under reduced pressure at 60°C, and pulverize to obtain dry extract powder;

[0149] R3. Add the diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; the weight ratio of the dry extract powder to the diluent is 1:2.0, and the weight ratio of dextrin to mannitol is 1:1.5;

[0150] R4. Add an 80% ethanol solution to the mixture, granulate, and dry at 60°C to obtain longan leaf granules.

[0151] Example 4

[0152] A method for preparing longan leaf granules, comprising the following steps:

[0153] R1. Take 20 kg of longan leaves, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, 1 hour each time, filter, combine to obtain a filtrate;

[0154] R2. Take the filtrate, concentrate it under reduced pressure at 68 °C to obtain an extract with a relative density of 1.28 at 60 °C, dry it under reduced pressure at 90 °C, pulverize it to obtain dry extract powder;

[0155] R3. Add diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; the weight ratio of the dry extract powder to the diluent is 1:2.0, and the weight ratio of dextrin to mannitol is 1:1.4;

[0156] R4. Add 80% ethanol solution to the mixture, granulate, and dry at 70 °C to obtain longan leaf granules.

[0157] Molding process verification: Weigh the longan leaf granule samples prepared in Examples 1-4 above. Measure 4 evaluation indexes and calculate the comprehensive score. The test results show that the production process of the longan leaf granules of the present invention is stable and feasible. The results are shown in Table 18.

[0158] Table 18 Results of molding process verification of examples

[0159]

[0160] III. Quality control research on longan leaf granules

[0161] 1. Thin layer chromatography identification

[0162] (1) Screening of thin layer chromatography identification conditions

[0163] ① Investigation on the preparation methods of test solution and reference crude drug solution

[0164] Weigh the longan leaf granule sample of Example 3. Six preparation methods of test solution for longan leaf granules were mainly investigated. Four of the methods are as follows: a. Heat under reflux with methanol for extraction, filter, add hydrochloric acid to the filtrate, heat in a water bath, cool, filter, and concentrate the filtrate; b. Reflux extract with water, filter, evaporate the filtrate to dryness, and extract with ethyl acetate; c. Ultrasonic extract with absolute ethanol, filter; d. Ultrasonic extract with methanol-hydrochloric acid (40:1), filter, evaporate the filtrate to dryness, add water to mix evenly, and extract with ethyl acetate. Observe the development of the test solution, reference crude drug, and reference substance on the thin layer plate to determine the best preparation methods for the test solution and reference crude drug solution.

[0165] The test results show that: the spots of quercetin and kaempferol can be clearly seen on the thin layer chromatogram, but the spot of quercitrin cannot appear; or the spot of quercitrin can be clearly seen on the thin layer chromatogram, but the spots of quercetin and kaempferol cannot appear; when the test solution and reference crude drug solution are prepared by method d, the spots of all three components are clearly visible and the resolution is good. Therefore, the preparation method d is selected for the test solution and reference crude drug solution of the present invention. For the preparation method of the test solution, please refer to the preparation method of the test solution under the "Best thin layer chromatography identification plan".

[0166] ② Investigation on thin layer plates

[0167] The inventors respectively developed using four different types of thin-layer plates: silica gel G plate, silica gel H plate, polyamide film, and silica gel GF 254 plate. After taking them out, drying them in the air, and observing the development of the thin-layer plates. The experimental results showed that: when using silica gel H thin-layer plate and silica gel GF 254 thin-layer plate, the edges of the spots were not clear, and some spots did not appear; when using polyamide film, neither the test samples nor the reference substances could be developed, and the resolution was poor; when using silica gel G thin-layer plate, the development effects of the test samples and the reference substances were the best, the spots were clear, the resolution was good, the Rf value was appropriate, and there was no tailing. Therefore, the present invention selects silica gel G thin-layer plate to carry out subsequent experiments.

[0168] ③ Investigation of developing agent

[0169] The inventors respectively investigated different developing agents: a. First development (ethyl acetate: formic acid: methanol = 8:2:1), second development (xylene: ethyl acetate: formic acid = 8:5:1); b. First development (ethyl acetate: glacial acetic acid: formic acid: water = 10:3:5:3), second development (xylene: ethyl acetate: formic acid = 8:5:1); c. First development (ethyl acetate: formic acid: water = 9:0.5:0.5), second development (n-hexane: ethyl acetate: formic acid = 3:4:1); d. First development: ethyl acetate: formic acid: water = 9:0.5:0.5, second development: toluene: ethyl acetate: formic acid = 8:4:1; e. First development (ethyl acetate: formic acid: water = 9:0.5:0.5), second development (xylene: ethyl acetate: formic acid = 8:5:1). Observe the spots on the thin-layer plate to determine the best developing system.

[0170] It can be seen from the test results that the developing agent ratios of a, b, and c have poor developing effects on quercetin and poor resolution; the developing effects of both d and e developing agents are better, but the d developing agent uses toluene as the developing agent, and toluene is a toxic and controlled reagent. Therefore, the present invention preferably selects the e developing agent.

[0171] ④ Investigation of color development method

[0172] The inventor investigated different inspection methods: a. Inspection under sunlight; b. Inspection under an ultraviolet lamp (365 nm); c. Inspection under an ultraviolet lamp (254 nm); d. Spraying with a 3% aluminum trichloride - ethanol solution, drying at 105 °C for 2 min, and then inspecting under an ultraviolet lamp (365 nm). Observe the spots on the thin layer plate to determine the best inspection method. The test results show that: before spraying the developer, the spots under sunlight or ultraviolet light at 254 nm and 365 nm are not very clear, and some spots do not appear. After spraying with a 3% aluminum trichloride - ethanol solution and drying at 105 °C for 2 min for color development, the spots can be faintly seen under sunlight, while the spots are clearer and brighter under ultraviolet light at 365 nm, and the inspection effect is the best. Therefore, the inspection method selected in the present invention is: spraying with a 3% aluminum trichloride - ethanol solution for color development, drying at 105 °C for 2 min, and then inspecting under a 365 nm ultraviolet lamp.

[0173] (2) Optimal solution for thin layer chromatography identification

[0174] ① Preparation of test solution: Respectively take the longan leaf granules of Example 1, Example 2, and Example 3 above, grind them finely, and then weigh 1.0 g each. Add 10 mL of a methanol - hydrochloric acid solution with a volume ratio of 40:1, extract ultrasonically for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water, mix well, extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1 mL of methanol to obtain the test solutions of Example 1, Example 2, and Example 3 respectively.

[0175] ② Preparation of reference solution: Respectively weigh the reference substances of quercetin, quercitrin, and kaempferol, add methanol to make up the volume, and prepare reference solutions of 0.25 mg / mL, 0.15 mg / mL, and 0.5 mg / mL respectively.

[0176] ③ Preparation of reference medicinal material solution: Weigh 1.0 g of the longan leaf reference medicinal material, and prepare the reference medicinal material solution in the same method as the "test solution".

[0177] ④ Preparation of the negative sample of longan leaf granules: Except for longan leaves, take dextrin and mannitol, and prepare the negative sample of longan leaf granules according to the preparation method of Example 3.

[0178] ⑤ Preparation of negative control solution: Weigh the negative sample of longan leaf granules and process it in the same way to prepare the negative control solution;

[0179] ⑥ Development and examination: Pipette 10 μL of each of the above 5 solutions and spot them on the same silica gel G thin-layer plate. Develop in two steps. For the first development, the volume ratio of the developing agent "ethyl acetate - formic acid - water" is 9:0.5:0.5. For the second development, the volume ratio of the developing agent "xylene - ethyl acetate - formic acid" is 8:5:1. Take out the plate, dry it in air, spray it with 3% aluminum trichloride - ethanol solution for color development, bake it at 105 °C for 2 minutes, and examine it under a 365 nm ultraviolet lamp. The experimental results are shown in Figure 5 。

[0180] The test results show that: in the thin-layer chromatograms of the test samples and the reference substance of the longan leaf granules of Examples 1, 2, and 3 of the present invention, quercetin and kaempferol both show the same blue spots, and quercitrin shows yellow spots, and the resolution is good. The inventor also conducted durability investigations on thin-layer plates from different manufacturers, temperature, humidity, etc. of the development environment, indicating that the established thin-layer chromatography identification method for longan leaf granules has good stability and durability.

[0181] 2. Content determination study by high performance liquid chromatography

[0182] (1) Preparation of the reference substance solution

[0183] Accurately weigh the reference substances of quercetin, quercitrin, and kaempferol respectively, place them in a volumetric flask, add methanol, sonicate to dissolve, cool to room temperature, and then make up the volume to the mark and shake well to prepare a mixed reference substance solution with concentrations of 0.1286 mg / mL, 0.1904 mg / mL, and 0.0234 mg / mL.

[0184] (2) Preparation of the test sample solution

[0185] Take 1.0 g of the longan leaf granules of Example 3, grind them finely, accurately weigh, add 10 mL of methanol - hydrochloric acid (40:1) solution, weigh, ultrasonically extract for 30 min, make up the weight, filter, and pass the subsequent filtrate through a 0.22 μm filter membrane to obtain the test sample solution.

[0186] (3) Preparation of the reference medicinal material solution

[0187] Take 1.0 g of the reference medicinal material of longan leaf, accurately weigh, place it in a stoppered conical flask, and prepare the reference medicinal material solution of longan leaf according to the preparation method of the test sample.

[0188] (4) Preparation of the negative control solution

[0189] Take 1.0 g of the prepared negative sample of longan leaf granules, grind it finely, accurately weigh, and prepare the negative control solution of longan leaf granules according to the preparation method of the test sample solution.

[0190] (5) Chromatographic conditions

[0191] Chromatographic column: Inert Sustain C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol (A) - 0.2% phosphoric acid (B); Gradient elution (0 - 22 min, 45% A - 49% A; 22 - 35 min, 49% A - 75% A); Detection wavelength: 360 nm; Flow rate: 1 mL / min; Column temperature: 30 °C, Injection volume: 10 μL.

[0192] (6) Methodology investigation

[0193] ① System suitability test

[0194] Precisely pipette 10 μL each of the reference substance solution, test solution, reference crude drug solution, and negative control solution, inject them into the liquid phase for analysis, and record the liquid chromatogram.

[0195] The test results showed that: the retention times of the three components to be measured in the test solution of Longan Leaf Granules were consistent with those in the reference substance solution and the reference crude drug solution, and there was no interference from the negative control. Moreover, the theoretical plate numbers of quercetin, quercitrin, and kaempferol were all greater than 5000; the peak symmetry was good, and the tailing factor was 0.96 - 1.06; the resolution between the main component and the adjacent peaks was greater than 1.5; excipients, etc. did not interfere with the determination of the main component, and the specificity was good. See Figures 6 to 9 。

[0196] ② Linear relationship investigation

[0197] Precisely pipette 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL of the above mixed reference substance solution respectively, place them in 10 mL volumetric flasks, add methanol and slowly make up to the mark, and prepare a series of solutions. Inject them according to the above chromatographic conditions. Taking the peak area as the ordinate and the concentration of the reference substance solution as the abscissa, plot the standard curve and obtain the regression equation.

[0198] The linear relationships of the reference substance concentrations of quercetin, quercitrin, and kaempferol were good in the ranges of 0.0129 - 0.1286 mg / mL, 0.0190 - 0.1904 mg / mL, and 0.0023 - 0.0234 mg / mL. The regression equations and correlation coefficients are shown in Table 19.

[0199] Table 19 Linear relationships of three chemical components in Longan Leaf Granules

[0200]

[0201] ③ Precision test

[0202] Take the above mixed reference substance solution and inject it continuously for 6 times under the above chromatographic conditions, and calculate the RSD of the peak areas of quercetin, quercitrin, and kaempferol. Test results: The RSDs of the peak areas of quercetin, quercitrin, and kaempferol are 1.01%, 1.12%, and 1.74% respectively, indicating good instrument precision. The results are shown in Table 20.

[0203] Table 20 Results of precision experiment (n = 6)

[0204]

[0205] ④ Repeatability experiment

[0206] Weigh 6 portions of the longan leaf granules sample of Example 3, 1.0 g for each portion, grind them finely, accurately weigh them, and prepare the test solution according to the above preparation method of the test solution respectively. Then, determine the test solution under the above chromatographic conditions, record the peak areas, and calculate the RSDs of the contents of quercetin, quercitrin, and kaempferol. Test results: The RSDs of the contents of quercetin, quercitrin, and kaempferol are 2.68%, 2.81%, and 2.25% respectively, indicating good repeatability of this method. The results are shown in Table 21.

[0207] Table 21 Results of repeatability experiment (n = 6)

[0208]

[0209] ⑤ Stability experiment

[0210] Weigh 1.0 g of the longan leaf granules of Example 3, grind them finely, accurately weigh them, prepare the test solution according to the above preparation method of the test solution, inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h time points under the above chromatographic conditions, and calculate the RSDs of the peak areas of quercetin, quercitrin, and kaempferol. Test results: The RSDs of the peak areas of quercetin, quercitrin, and kaempferol are 1.86%, 1.96%, and 2.60% respectively, indicating good stability of the test solution within 24 h. The results are shown in Table 22.

[0211] Table 22 Results of stability experiment

[0212]

[0213] ⑥ Spiked recovery experiment

[0214] Take 0.5 g of the longan leaf granules in Example 3, grind them finely, and weigh accurately. There are 6 portions in total. Add appropriate amounts of quercetin, quercitrin, and kaempferol reference substances respectively, and prepare 6 portions of test solution according to the above preparation method of test solution. Then, determine according to the above chromatographic conditions and calculate the recovery rate of sample addition. The test results are as follows: the average recovery rates of quercetin, quercitrin, and kaempferol are 98.17%, 99.71%, and 101.45% respectively, and the RSDs are 2.61%, 1.68%, and 2.75% respectively, indicating that the accuracy of this method is good. The results are shown in Table 23.

[0215] Table 23 Results of the experiment on recovery rate of sample addition (n = 6)

[0216]

[0217] (7) Determination of the contents of quercetin, quercitrin, and kaempferol in three batches of longan leaf granules

[0218] Respectively take the samples of the longan leaf granules in Example 1, Example 2, and Example 3 prepared above, prepare the test solutions of three batches of longan leaf granules according to the preparation method of test solution, then determine according to the above chromatographic conditions, and calculate the contents of quercetin, quercitrin, and kaempferol in the samples of the longan leaf granules in Example 1, Example 2, and Example 3. The contents of quercetin, quercitrin, and kaempferol in three batches of longan leaf granules are shown in detail in Table 24.

[0219] Table 24 Results of the determination of the contents of three batches of longan leaf granules

[0220]

[0221] III. Investigation on the stability of the product of the longan leaf granule example of the present invention

[0222] 1. Samples: Take the samples of the longan leaf granules in Example 3 of the present invention, and investigate the stability under room temperature conditions (temperature: 25°C ± 5, relative humidity: 60% ± 10%) and accelerated conditions (temperature: 40°C ± 2, relative humidity: 75% ± 5%).

[0223] 2. Investigation items: Appearance, identification, inspection (moisture content), content determination. The determination results are shown in Table 25.

[0224] Table 25 Results of the stability test of longan leaf granules

[0225]

[0226] It can be seen from the results in Table 25 that the samples of the longan leaf granules in Example 3 under investigation are placed for 6 months under the packaging for intended market launch, room temperature conditions, and accelerated conditions, and the results of each investigation and detection show no obvious changes compared with those at month 0, indicating that the quality of this product is stable.

[0227] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, they can also make several substitutions or modifications to these described embodiments, and these substitution or modification methods should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A longan leaf granule, characterized in that, The granules are prepared by adding water to longan leaves for heating extraction, filtration, concentration, drying, and pulverization to obtain dry extract powder. A diluent is added to the dry extract powder, mixed evenly, a wetting agent is added, granulated, and dried to obtain longan leaf granules; The step of adding water for heating extraction is as follows: the amount of added water is 18 times the weight of longan leaves, soaked for 30 minutes, the number of times of heating extraction is 3 times, and each time is 1 hour; The concentration step: concentrate into an extract with a relative density of 1.25 - 1.35 at 60 °C; The diluent is dextrin and mannitol, and the weight ratio of dextrin to mannitol is 1:1 - 1:1.

5.

2. The longan leaf granule according to claim 1, characterized in that, The wetting agent is an 80% ethanol solution.

3. A preparation method of the longan leaf granules as described in claim 1, characterized in that, It includes the following steps: R1. Take longan leaves, add 18 times the amount of water, soak for 30 minutes, heat and extract 3 times, each time for 1 hour, filter, combine to obtain a filtrate; R2. Take the filtrate, concentrate it into an extract with a relative density of 1.25 - 1.35 at 60 °C, dry, and pulverize to obtain dry extract powder; R3. Add diluents dextrin and mannitol to the dry extract powder, mix evenly to obtain a mixture; The weight ratio of dextrin to mannitol is 1:1 - 1:1.5; R4. Add a wetting agent to the mixture, granulate, and dry to obtain longan leaf granules.

4. The preparation method of the longan leaf granules according to claim 3, characterized in that, In the concentration step in R2, it is concentration under reduced pressure at 65 - 75 °C, and the drying step is drying under reduced pressure at 60 - 80 °C.

5. The preparation method of the longan leaf granules according to claim 3, characterized in that, In the concentration step in R2, it is concentration under reduced pressure at 65 °C, and the drying step is drying under reduced pressure at 60 °C.

6. The preparation method of the longan leaf granules according to claim 3, characterized in that, In step R3, the weight ratio of dextrin to mannitol is 1:1.

5.

7. The preparation method of the longan leaf granules according to claim 3, characterized in that, In step R4, the wetting agent is an 80% ethanol solution.

8. A method for identifying the longan leaf granules as described in claim 1, characterized in that, It includes the following steps: S1: Preparation of test solution: Take longan leaf granules, grind them finely, weigh 1.0 g, add 10 mL of methanol - hydrochloric acid solution with a volume ratio of 40:1, extract ultrasonically for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water, mix evenly, extract with ethyl acetate 2 times, each time with 20 mL, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 mL of methanol as the test solution; S2: Preparation of reference substance solution: Weigh quercetin, quercitrin, and kaempferol reference substances respectively, add methanol to make a constant volume to prepare reference substance solutions of 0.25 mg / mL, 0.15 mg / mL, and 0.5 mg / mL; S3: Preparation of control medicinal material solution: Weigh 1.0 g of longan leaf control medicinal material, and prepare a control medicinal material solution in the same method as S1; S4: Development and inspection: Pipette 10 μL of each of the above 5 solutions, respectively spot them on the same silica gel G thin layer plate, develop in 2 times. The volume ratio of the first developing agent "ethyl acetate - formic acid - water" is 9:0.5:0.5, and the volume ratio of the first developing agent "xylene - ethyl acetate - formic acid" is 8:5:

1. Take out, dry in the air, spray with 3% aluminum trichloride - ethanol solution for color development, bake at 105 °C for 2 minutes, and examine under a 365 nm ultraviolet lamp.

9. A method for determining the content of longan leaf granules as described in claim 1, characterized in that, It includes the following steps: T1. Preparation of test solution: Take 1.0 g of longan leaf granules, grind them finely, accurately weigh, add 10 mL of methanol - hydrochloric acid solution with a volume ratio of 40:1, extract ultrasonically for 30 minutes, filter, take the continuous filtrate as the test solution; Preparation of reference substance solution: Weigh accurately the reference substances of quercetin, quercitrin, and kaempferol respectively, add methanol, and ultrasonically treat to dissolve. Shake well to prepare a mixed reference substance solution with concentrations of 0.1 mg / mL, 0.2 mg / mL, and 0.02 mg / mL. T3. Chromatographic conditions: The chromatographic column is packed with octadecylsilane chemically bonded silica gel; the detection wavelength is 360 nm; methanol is used as mobile phase A, and 0.2% phosphoric acid is used as mobile phase B, with gradient elution (0 - 22 min, 45% A - 49% A; 22 - 35 min, 49% A - 75% A); flow rate: 1 mL / min; column temperature: 30 °C. T4. Determination: Accurately pipette 10 μL each of the reference substance solution and the test sample solution, inject them into the high-performance liquid chromatograph, and determine according to the method in step T3.

10. The application of the longan leaf granules according to claim 1, characterized in that, Application of the longan leaf granules in the treatment of diabetes drugs.

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