A method for preparing smilax glabra extract

Abstract

The invention relates to the technical field of smilax glabra extraction, and in particular to a method for preparing a smilax glabra extract. The method comprises taking smilax glabra slices, soaking them in water, and then performing water bath extraction under a temperature condition of 65-75°C to obtain a medicinal solution, filtering the medicinal solution, decocting with water, filtering, concentrating the filtrate, and drying the extract, which not only significantly reduces the formation of insoluble matter in the extract, but also has the most significant effect on improving solubility, and also significantly increases the content of astilbin.

Description

Technical Field

[0001] The invention relates to the technical field of smilax glabra extraction, and in particular to a method for preparing a smilax glabra extract. Background Art

[0002] Smilax glabra Roxb. is a dried rhizome of the lily family plant. It is sweet, light and flat in nature and flavor. It enters the liver, stomach and spleen meridians. It has the effects of detoxification, dehumidification and joint dredge. It can be used for limb cramps and tendon pain caused by syphilis and mercury poisoning; damp-heat stranguria, leucorrhea, carbuncle, scrofula, scabies and other symptoms. Smilax glabra is often used in clinical treatment of hyperuricemia and gout. Modern research shows that Smilax glabra also has many pharmacological activities such as anti-inflammatory, analgesic, anti-tumor, antioxidant, immunomodulatory, liver protection, diuretic and antibacterial.

[0003] The Chinese smilax glabra formula granules are granules made from Chinese smilax glabra slices through water extraction, separation, concentration, drying, granulation and other processes. Compared with traditional decoctions, they have the advantages of convenient adjustment, convenient production and use, accurate dosage, easy to carry, and can be added or subtracted according to symptoms. However, the research team found in the early stage that some of the Chinese smilax glabra formula granules on the market still have a large number of undissolved particles (such as Figure 1 ), and the Tufuling formula granules from three different manufacturers all had similar problems, which did not meet the quality requirements for granules specified in the 2020 edition of the "Chinese Pharmacopoeia".

[0004] The presence of a large amount of insoluble matter will reduce the patient's acceptance and compliance, resulting in a decline in the market competitiveness of the product. In order to improve the solubility of the Smilax glabra formula granules, the research team initially planned to improve the solubility by improving the excipients and production process. However, methods such as excipient screening, adding dispersants, powder modification, shortening the extraction time, and centrifuging the extract to remove sediments cannot effectively solve the problem of poor solubility of the Smilax glabra formula granules. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defect of poor solubility of the granules of the Smilax glabra extract in the prior art, thereby providing a method for preparing the Smilax glabra extract.

[0006] To this end, the present invention provides a method for preparing a smilax glabra extract, comprising taking smilax glabra slices, soaking them in water, and then performing water bath extraction at a temperature of 65-75°C to obtain a medicinal solution, filtering the medicinal solution, decocting it with water, filtering it, concentrating the filtrate, and drying it to obtain the extract.

[0007] Furthermore, the number of water bath extractions is 1-5 times, and the time of each water bath extraction is 2-4 hours; preferably, the number of water bath extractions is 2-3 times, and the time of each water bath extraction is 2.5-3.5 hours.

[0008] Furthermore, the water bath extraction was performed twice, and the time of each water bath extraction was 2 hours.

[0009] Furthermore, the temperature of the water bath extraction is 70°C.

[0010] Furthermore, before water bath extraction, 6-10 times the amount of water of the Rhizoma Smilacis Glabrae slices is added for soaking.

[0011] Further, the immersion is carried out at room temperature for 20-40 minutes.

[0012] Furthermore, the mass of water added during the decoction process is 5-8 times the mass of the Smilax glabra slices, and the decoction time is 2-4 hours.

[0013] Furthermore, the ratio of the volume of the concentrated medicinal solution to the mass of the Smilax glabra decoction pieces is 1.5-2L:2kg.

[0014] Further, it includes taking the pieces of Smilax glabra and adding 8 times the amount of water to soak for 30 minutes, directly extracting in a 70°C water bath for 2 hours, filtering through a 120-mesh filter, adding 6 times the amount of water and boiling for 2 hours, pouring out the medicinal solution and filtering through a 120-mesh filter, combining the filtrate, concentrating, and drying to obtain the product.

[0015] The present invention also provides a Smilax glabra extract obtained by any of the above methods for preparing the Smilax glabra extract.

[0016] The present invention also provides a smilax glabra formula granule, comprising the smilax glabra extract prepared by any of the above methods for preparing the smilax glabra extract.

[0017] The technical solution of the present invention has the following advantages:

[0018] The method for preparing a smilax glabra extract provided by the present invention comprises the following steps: taking smilax glabra slices, soaking them in water, and then performing water bath extraction at a temperature of 65-75 DEG C to obtain a medicinal liquid, filtering the medicinal liquid, then boiling them with water, filtering, concentrating the filtrate, and drying them. The method of soaking them in water and then performing water bath extraction at a specific temperature not only significantly improves the solubility of the smilax glabra extract, but also significantly increases the astilbin content. In addition, the decomposition rate of flavonoid compounds is also significantly reduced, and the total flavonoid content is increased. In particular, when the water bath extraction temperature is 70 DEG C, the best temperature is 70 DEG C, and the astilbin content obtained by the water bath extraction is the highest. Moreover, only water is used as a solvent, which meets the national standards for formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The dissolution of smilax glabra granules from different manufacturers in the background technology;

[0021] Figure 2 The dissolution conditions of the Smilax glabra extracts prepared in the examples and comparative examples;

[0022] Figure 3 is the proportion of insoluble matter in the Smilax glabra extracts prepared in the Examples and Comparative Examples (%);

[0023] Figure 4 It is a transmission light change curve of the Smilax glabra extract solution prepared in the examples and comparative examples; A is comparative example 1, B is comparative example 2, C is comparative example 3, D is comparative example 4, and E is example 1;

[0024] Figure 5 This is the TSI change trend chart;

[0025] Figure 6 It is a radar chart of the overall TSI value of the extract solution, the average value of the bottom and top transmitted light intensity, and the average value of the top and bottom peak thickness;

[0026] Figure 7 UPLC characteristic spectra of Smilax glabra extracts prepared by five extraction methods; Peak 1 is the astilbin peak; Peak 5 is the flavonoids glycoside peak. DETAILED DESCRIPTION

[0027] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0028] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0029] instrument:

[0030] BSA 224SA 1 / 10,000 analytical balance and BT25S 1 / 100,000 analytical balance (Sartorius, Germany); water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.); electric heating jacket (Beijing Kewei Yongxing Instrument Co., Ltd.); deionized water (Sichuan Youpu Ultrapure Technology Co., Ltd.); electric constant temperature blast drying oven (Shanghai Kailang Instrument Factory); freeze dryer (Ningbo Xinzhi Freeze Drying Equipment Co., Ltd.); Ultimate 3000UPLC high performance liquid chromatograph (Thermo Fisher Scientific); Turbiscan Lab stability analyzer (Formulaction, France).

[0031] Drug testing:

[0032] The control drug of Smilax glabra (ID: 61T3-EPRG) was purchased from the China Food and Drug Inspection Institute; astilbin (batch number: DST201015-054, purity ≥ 98%), cyperoside (batch number: DST210604-045, purity ≥ 98%), and D(+)-anhydrous glucose (batch number: DSTW000501, purity ≥ 98%) were all purchased from Lemeitian Pharmaceutical / Desit Biotechnology; starch test kit (Nanjing Jiancheng Bioengineering Institute); glacial acetic acid (Chengdu Cologne Chemical Co., Ltd.) was chromatographic grade; acetonitrile (Anhui Tiandi High Purity Solvent Co., Ltd.) and methanol (Sigma-Aldrich Trading Co., Ltd.) were chromatographic grade; water was ultrapure water, and other reagents were analytical grade. Four batches of Smilax glabra slices were purchased from Guangxi, Hunan, Guizhou, and Sichuan, and were identified as the dried rhizome of Smilax glabra Roxb., a plant of the Liliaceae family, by Associate Professor Xu Runchun of the School of Pharmacy of Chengdu University of Traditional Chinese Medicine.

[0033] Example 1

[0034] This embodiment provides a method for preparing a smilax glabra extract, comprising the following steps:

[0035] Step S1: Take 2 kg of Smilax glabra slices and soak them in 8 times the amount of water for 30 minutes;

[0036] Step S2: directly extract twice in a water bath at 70°C, each time for 2 hours, to obtain a drug solution;

[0037] Step S3: Pour out the liquid medicine, filter through a 120-mesh filter, add 6 times the amount of water and boil for 2 hours, pour out the liquid medicine, filter through a 120-mesh filter. Combine the filtrate, concentrate to 1.8L, and dry into powder.

[0038] Comparative Example 1

[0039] This comparative example provides a method for preparing a Smilax glabra extract, using Smilax glabra slices from the same batch as in Example 1. The only difference from Example 1 is that step S2 is replaced by reflux extraction twice, with each extraction time being 2 hours to obtain a medicinal solution. During the reflux extraction process, the medicinal solution is kept slightly boiling at a temperature of about 100°C.

[0040] Comparative Example 2

[0041] This comparative example provides a method for preparing a Smilax glabra extract, using Smilax glabra slices from the same batch as in Example 1, and the only difference from Example 1 is that step S2 is to boil and then extract in a water bath at 80° C. for 2 hours each time to obtain a medicinal solution.

[0042] Comparative Example 3

[0043] This comparative example provides a method for preparing a Smilax glabra extract, using Smilax glabra slices from the same batch as in Example 1, and the only difference from Example 1 is that step S2 is replaced by directly extracting in a water bath at 80° C. for 2 times, each time for 2 hours, to obtain a medicinal solution.

[0044] Comparative Example 4

[0045] This comparative example provides a method for preparing a Smilax glabra extract, using Smilax glabra slices from the same batch as in Example 1, and the only difference from Example 1 is that step S2 is replaced by boiling and then extracting in a water bath at 70°C for two times, each time for 2 hours, to obtain a medicinal solution.

[0046] Experimental Example 1: Solubility Investigation

[0047] Since precipitation still occurs after adding hot water and stirring for 5 minutes, it is difficult to quantitatively analyze the precipitation amount and solubility by naked eye observation. Although the method of calculating the proportion of insoluble matter after filtering and drying the precipitation can be used for quantitative analysis, it is time-consuming and cumbersome. The Turbiscan analyzer can quickly, accurately and quantitatively react to the solubility and precipitation of the sample by the change of light intensity during the dissolution of the Smilax glabra extract. Therefore, this study uses the Turbiscan analyzer to quantitatively analyze the solubility of the extract.

[0048] The Turbiscan analyzer is based on the principle of multiple light scattering. It uses a pulsed near-infrared light source (λ=880nm) and two synchronous optical detectors for transmitted light and backscattered light to scan the sample to be tested vertically from bottom to top in a cylindrical glass test bottle. The transmitted light detector receives the transmitted light (T, 0° with the incident angle) that passes through the sample, and is used to study clear and transparent samples; the backscattered light detector receives the reflected light (BS, 135° with the incident angle) reflected by the sample, and is used to study opaque samples with a concentration of up to 95%. The detector scans along the sample height (up to 55mm), and collects transmitted light and backscattered light data every 40μm height. The data collection time can be set according to the change rate of the sample, and can be collected at least once every 0.1 second. The curve obtained by scanning gives the relationship between the transmitted light and backscattered light at different time points and the sample height, which can intuitively, quickly and accurately reflect the changes in particle size in the sample and the dynamic changes of particle floating, precipitation, aggregation / flocculation, etc. In addition, Turbiscan can also process data through Turbiscan Easysoft software to obtain parameters such as stability index (TSI) and peak thickness. When there are particle migration and precipitation phenomena in the solution, the transmitted light and backscattered light change with the particle migration speed, and the TSI value will also increase accordingly. The higher the TSI, the more unstable the solution; the peak thickness also increases with the increase of the precipitation layer thickness (see the literature Huang Haozhou, Lin Junzhi, Wei Xichuan, Ma Hongyan, Liu Haiyan, Fan Sanhu, Zou Wenquan, Han Li, Zhang Dingkun. Pharmaceutical mechanism and regulation strategy for stable storage of ellagic acid molecules in traditional Chinese medicine oral solution [J]. Acta Pharmaceutics, 2019, 54(04): 737-745. and the literature Hao-zhou Huang, Sheng-yu Zhao, Xiu-mei Ke, Jun-zhi Lin, Shu-sen Huang, Run-chun Xu, Hong-yan Ma, Yi Zhang, Li Han, Ding-kun Zhang. Study on the stability control strategy of Triphala solution based on the balance of physical stability and chemical stabilities [J]. Journal of Pharmaceutical and Biomedical Analysis, 2018, 158.).

[0049] The extracts of Smilax glabra obtained in the examples and comparative examples were used as test samples. 1 g of each test sample was added to 200 mL of hot water, stirred for 5 min, and immediately poured into a glass test bottle, which was then placed in the scanning pool of the Turbiscan analyzer. The data were scanned every 30 s in the vertical direction for 10 min to obtain the transmission light curve data. Figure 2 Afterwards, the scanned sample was filtered with quantitative filter paper, the solid was dried, and the insoluble matter ratio (%) was calculated. The insoluble matter ratio is the percentage of the mass of the solid after drying to the mass of the test sample. The results are shown in Figure 3 As shown, Example 1 is 4.03%, while Comparative Examples 1-4 are 34.93%, 26.67%, 24.48% and 29.65% respectively.

[0050] Results Figure 2-6 As shown in FIG. 1 , the Smilax glabra extract solution is a transparent liquid, so the transmitted light intensity (T) is selected for analysis. Figure 4 The time-dependent changes in the intensity of transmitted light of each Smilax glabra extract show that the intensity of transmitted light of sample solutions A to D gradually increases with time. The intensity of transmitted light at the bottom of the sample bottle (0-10 mm) is relatively low, while the intensity of transmitted light at the middle (10-30 mm) and top (30-40 mm) is higher than that at the bottom, and increases in a gradient, indicating that the samples prepared in Comparative Examples 1-4 precipitate at the bottom of the bottle, and the particles in the solution continue to settle within 10 minutes of standing, so that the solution from the middle to the top gradually clarifies. Figure 4 -E's overall transmitted light intensity changes slightly, indicating that the sample prepared in Example 1 is relatively stable and no obvious precipitation occurs. Figure 5 The figure is a trend chart of TSI values ​​of five samples changing with time. It can be seen that the TSI values ​​of the five samples gradually increase with time, but the TSI value of the sample prepared by Example 1 changes the least, indicating that it has the best stability.

[0051] Figure 6 The overall TSI values, average light intensity at the top and bottom of the bottle, and peak thickness of the five samples at the 10th minute calculated by Turbiscan Easysoft software can accurately and quantitatively reflect the changes in each solution. In the figure, the average top light intensity of the samples prepared in Comparative Examples 1-4 is greater than 15%, and the average bottom light intensity is less than 0, and the average top light intensity is significantly higher than that at the bottom; while the average top and bottom light intensity of the samples prepared in Example 1 are both about 2%, with a small difference, further indicating that the samples prepared in Comparative Examples 1-4 have precipitates and poor solubility. The peak thickness reflects the height of the aqueous phase of the solution during the stratification process. The thicker the peak thickness, the thicker the precipitate. Figure 6It can be seen from the average value of the bottom peak thickness of the solution that the average peak thickness of the sample solutions prepared by extraction comparison examples 1, 2, 3, and 4 is greater than 2 mm, and comparison example 1> comparison example 4> comparison example 2> comparison example 3, while the average peak thickness of the sample solution prepared by example 1 is almost 0, indicating that the sample prepared by example 1 has very little or almost no precipitation, and the samples prepared by comparison examples 1-4 have more precipitation, and the sample prepared by comparison example 1 has the most precipitation, followed by comparison examples 4, comparison examples 2 and comparison examples 3.

[0052] Based on the above indicators, it can be seen that lowering the extraction temperature can improve the solubility of the Smilax glabra extract, among which Extraction Example 1 has the most obvious improvement on the solubility of the Smilax glabra extract.

[0053] Experimental Example 2 Characteristic Spectrum

[0054] Preparation of test solution: Take the Smilax glabra extract prepared in the example and the comparative example as the test sample, grind it into powder, take about 0.1g of each test sample, put it in a stoppered conical flask, add 100mL of 70% ethanol, ultrasonically treat (power 300W, frequency 45kHz) for 40min, take out and cool, shake well, filter, and take the filtrate to obtain.

[0055] Preparation of reference solution: Take 0.8010g of Smilax glabra reference medicinal material, place it in a stoppered conical flask, add 100mL of 60% methanol, heat and reflux for 60min, take it out and let it cool, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Take appropriate amount of astilbin reference substance and cyperus flask reference substance, accurately weigh them, add 60% methanol to prepare a mixed solution containing 50.00μg / mL of astilbin and 51.25μg / mL of cyperus flask, as the reference substance solution.

[0056] 1 μL of the reference solution and the test solution were taken respectively, and the characteristic spectrum was determined under the following chromatographic conditions: ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: acetonitrile as mobile phase A, 0.2% glacial acetic acid solution as mobile phase B, gradient elution; gradient elution conditions: 0-2 min, 13% A; 2-5 min, 13%-17% A; 5-14 min, 17%-23% A; flow rate: 0.4 mL min -1 ; Column temperature: 30℃; Detection wavelength: 291nm; Injection volume: 1μL. Record the chromatogram and retention time. Use the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition" to match the chromatogram, and use the reference herbal material chromatogram of Smilax glabra (reference) as a reference to calculate the similarity between the UPLC chromatogram of each extract and the reference chromatogram. The results are as follows Figure 7 shown.

[0057] The experimental results showed that the UPLC characteristic spectra of the Smilax glabra extracts prepared by the five methods and the chromatograms of the reference medicinal materials all detected 5 characteristic common peaks, and the chromatogram similarities were all greater than 0.96. In addition, the retention times of the astilbin peaks and astragaloside peaks in the characteristic spectra of each Smilax glabra extract corresponded to the retention times of the reference peaks of the corresponding reference substances, indicating that the components of each Smilax glabra extract did not change significantly, and lowering the extraction temperature had no effect on the components.

[0058] Experimental Example 3 Determination of Astilbin Content

[0059] Preparation of reference solution: Take an appropriate amount of astilbin reference substance, weigh accurately, and add 60% methanol to prepare a reference solution containing 53.00 μg / mL of astilbin.

[0060] Preparation of test solution: Take the Smilax glabra extract prepared in the examples and comparative examples as the test sample, grind it into powder, take 0.1 g of the test sample respectively, accurately weigh it, put it in a stoppered conical flask, accurately add 100 mL of 60% methanol, weigh it, ultrasonically treat it (power 300 W, frequency 45 kHz) for 40 min, cool it, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it through a 0.22 μm filter membrane, and get the test solution.

[0061] The reference solution and the test solution were taken separately for content determination according to the following chromatographic conditions: ACQUITYUPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm); mobile phase: methanol (A)-0.1% glacial acetic acid solution (B); elution conditions: 0-10 min: 30% A; flow rate: 0.4 mL·mL -1 ; Column temperature: 30℃; Detection wavelength: 291nm; Injection volume: 2μL.

[0062] The content of astilbin in each group of Smilax glabra extract and its transfer rate were calculated (the transfer rate of astilbin in Smilax glabra extract = the content of astilbin in Smilax glabra extract * the amount of Smilax glabra decoction slice extract / the content of astilbin in Smilax glabra decoction slices * the amount of medicinal materials). According to the experimental results and referring to the National Drug Standard YBZ-PFKL-2021123 of the State Food and Drug Administration, the peak positions of neoastilbin, astilbin, neoisoastilbin and isoastilbin were identified, and the total flavonoid area was calculated. The results are shown in the following table.

[0063] Table 1 Astilbin content and transfer rate of Smilax glabra extract ( n=3)

[0064]

[0065] Note: Total flavonoids peak area = new astilbin peak area + astilbin peak area + new isoastilbin peak area + isoastilbin peak area

[0066] The experimental results show that compared with comparative examples 1-4, the astilbin content and its transfer rate, as well as the total flavonoids content of the Smilax glabra extract obtained in Example 1 of the present invention are significantly improved.

[0067] Experimental Example 4: Multi-batch verification experiment

[0068] Three more batches of Smilax glabra slices were used to prepare extracts using the method of Example 1. The solubility (ratio of insoluble matter) was measured according to the above experiment, and the paste rate was calculated.

[0069] Table 2 The yield of extracts from different batches of Smilax glabra extract and the proportion of insoluble matter ( n=3)

[0070] serial number Cream yield (%) Insoluble matter ratio S2 17.09±0.28 6.9％ S3 14.01±0.56 6.9％ S4 14.70±0.60 6.4％

[0071] The results showed that different batches of Smilax glabra extracts prepared in Example 1 had good solubility, indicating that the extraction method can effectively improve the solubility of Smilax glabra extracts from different origins and batches. In addition, the paste yield met the requirements of the National Drug Standard YBZ-PFKL-2021123 of the State Food and Drug Administration (13%-24%).

[0072] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a smilax glabra extract, characterized in that: The method comprises taking a piece of Smilax glabra decoction and soaking it in water for 20-40 minutes, then directly performing water bath extraction at a temperature of 65-75°C to obtain a medicinal solution, the number of water bath extractions is 2-3 times, each water bath extraction time is 2 hours, after the medicinal solution is filtered, boiling it in water for 2-4 hours, filtering, combining the filtrate, concentrating the filtrate, and drying to obtain the medicinal solution.

2. The method for preparing a Smilax glabra extract according to claim 1, characterized in that: The water bath extraction was performed twice.

3. The method for preparing the Smilax glabra extract according to claim 1, characterized in that: The water bath extraction temperature was 70°C.

4. The method for preparing the Smilax glabra extract according to claim 1, characterized in that: Before water bath extraction, add 6-10 times the amount of water as the Smilax glabra slices for soaking.

5. The method for preparing a Smilax glabra extract according to any one of claims 1 to 4, characterized in that: The mass of water added during the decoction process is 5-8 times the mass of the Smilax glabra slices.

6. The method for preparing a Smilax glabra extract according to any one of claims 1 to 4, characterized in that: The ratio of the volume of the concentrated medicinal solution to the mass of the Smilax glabra slices is 1.5-2L:2kg.

7. A Smilax glabra extract obtained by the method for preparing the Smilax glabra extract according to any one of claims 1 to 6.

8. A smilax glabra formula granule, characterized in that: The invention comprises a Smilax glabra extract prepared by the method for preparing the Smilax glabra extract according to any one of claims 1 to 6.

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