CN110681183A - Study on purification process of total flavonoids of licorice with NKA-9 macroporous resin and its scavenging effect on nitrite - Google Patents
Study on purification process of total flavonoids of licorice with NKA-9 macroporous resin and its scavenging effect on nitrite Download PDFInfo
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- 229930003935 flavonoid Natural products 0.000 title claims abstract description 47
- 150000002215 flavonoids Chemical class 0.000 title claims abstract description 47
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 47
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 title claims abstract description 45
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 title claims abstract description 43
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 title claims abstract description 43
- 229940010454 licorice Drugs 0.000 title claims abstract description 43
- 239000011347 resin Substances 0.000 title claims abstract description 31
- 229920005989 resin Polymers 0.000 title claims abstract description 31
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 19
- 230000002000 scavenging effect Effects 0.000 title claims abstract description 18
- 238000000746 purification Methods 0.000 title abstract description 9
- 240000004670 Glycyrrhiza echinata Species 0.000 title 1
- 241000202807 Glycyrrhiza Species 0.000 claims abstract description 42
- 239000000287 crude extract Substances 0.000 claims abstract description 20
- 238000011068 loading method Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 239000002211 L-ascorbic acid Substances 0.000 claims description 3
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 2
- 244000303040 Glycyrrhiza glabra Species 0.000 claims 2
- 235000017443 Hedysarum boreale Nutrition 0.000 claims 2
- 235000007858 Hedysarum occidentale Nutrition 0.000 claims 2
- 239000001947 glycyrrhiza glabra rhizome/root Substances 0.000 claims 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims 1
- 229910017604 nitric acid Inorganic materials 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 241000411851 herbal medicine Species 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract 1
- 230000003000 nontoxic effect Effects 0.000 abstract 1
- 239000012264 purified product Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 22
- 238000002835 absorbance Methods 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000000523 sample Substances 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 5
- 238000012612 static experiment Methods 0.000 description 5
- 239000012085 test solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- LECSHJWIACEDPZ-UHFFFAOYSA-N ethane-1,2-diamine naphthalene hydrochloride Chemical compound C(CN)N.C1=CC=CC2=CC=CC=C12.Cl LECSHJWIACEDPZ-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 229950000244 sulfanilic acid Drugs 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003127 anti-melanomic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
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- 239000000284 extract Substances 0.000 description 1
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- 229940069445 licorice extract Drugs 0.000 description 1
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- 238000010992 reflux Methods 0.000 description 1
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Abstract
本发明公开了一种使用NKA‑9型大孔树脂对甘草总黄酮粗提物进行纯化的工艺，并研究了其对亚硝酸盐的清除作用，属于中草药中黄酮类化合物分离纯化技术领域。通过单因素实验得到如上样浓度、上样粗提液、上样体积、上样速度、洗脱体积、洗脱速度等工艺参数。经NKA‑9型大孔树脂纯化后的粗提物中甘草总黄酮含量由44.5%提高到57.8%，纯化后的甘草总黄酮对亚硝酸盐具有一定的清除作用。该方法易操作、工艺条件简单、使用的溶剂无毒无害，纯化后的产品对亚硝酸盐具有清除作用，具有较好的实际应用价值。The invention discloses a process for using NKA-9 macroporous resin to purify the crude extract of total flavonoids of licorice, and studies its scavenging effect on nitrite, belonging to the technical field of separation and purification of flavonoids in Chinese herbal medicines. Process parameters such as loading concentration, loading crude extract, loading volume, loading speed, elution volume and elution speed were obtained by single factor experiment. The content of total flavonoids of licorice in the crude extract purified by NKA-9 macroporous resin increased from 44.5% to 57.8%, and the purified total flavonoids of licorice had a certain scavenging effect on nitrite. The method is easy to operate, the process conditions are simple, the solvent used is non-toxic and harmless, the purified product has a scavenging effect on nitrite, and has good practical application value.
Description
技术领域technical field
本发明属于中草药中黄酮类化合物分离纯化技术领域，具体涉及一种使用NKA-9型大孔树脂对甘草黄酮粗提物进行分离纯化的工艺，并对亚硝酸盐进行清除的研究。The invention belongs to the technical field of separation and purification of flavonoids in Chinese herbal medicines, and in particular relates to a process for separating and purifying crude extracts of licorice flavonoids by using NKA-9 macroporous resin, and research on removing nitrite.
背景技术Background technique
近些年来大量研究显示甘草中的黄酮类化合物有着重要的生理活性，在抗HIV、抗肿瘤、抗炎、抗菌、镇静、镇痛、抗黑色素瘤等方面均具有重要的药理作用。由于甘草所含的天然成分种类复杂，因此在提取过程中不可避免的引入一系列杂质，导致甘草黄酮粗提物中总黄酮的含量较低，无法直接应用于实际生产之中，大大限制其发展。近些年大孔树脂纯化技术发展迅速，其原理是利用化合物与大孔树脂之间吸附力的不同及不同物质分子量大小的差异来进行分离，因此被广泛应用于各类天然产物的分离纯化。利用大孔树脂纯化甘草总黄酮的报道很多，相关专利有：（1）专利号：CN 103623041 B，名称：大孔树脂对甘草提取液中黄酮分离纯化方法，该专利正处于有效期；（2）专利号：CN 108261438 A，名称：一种甘草黄酮组分的提取及纯化方法，该专利正处于有效期。与以上相关专利不同，本专利首次选用NKA-9型大孔树脂对甘草总黄酮进行纯化，成功将甘草总黄酮纯度提高到57.8%。并研究了其对亚硝酸盐的清除作用，结果表明甘草总黄酮对亚硝酸盐有很好的清除作用，甚至在低浓度时清除效果要优于对照的L-抗坏血酸。In recent years, a large number of studies have shown that flavonoids in licorice have important physiological activities, and have important pharmacological effects in anti-HIV, anti-tumor, anti-inflammatory, antibacterial, sedative, analgesic, and anti-melanoma. Due to the complex types of natural components contained in licorice, a series of impurities are inevitably introduced in the extraction process, resulting in a low content of total flavonoids in the crude extract of licorice flavonoids, which cannot be directly used in actual production, which greatly limits its development. . In recent years, the purification technology of macroporous resin has developed rapidly. There are many reports on the purification of total flavonoids of licorice by macroporous resin, and the related patents are: (1) Patent No.: CN 103623041 B, name: separation and purification method of flavonoids in licorice extract by macroporous resin, the patent is in the validity period; (2) Patent number: CN 108261438 A, name: a method for extracting and purifying flavonoids of licorice, the patent is in the validity period. Different from the above related patents, this patent selects NKA-9 macroporous resin for the first time to purify the total flavonoids of licorice, and successfully increases the purity of total flavonoids of licorice to 57.8%. And studied its scavenging effect on nitrite, the results show that the total flavonoids of licorice have a good scavenging effect on nitrite, even at low concentrations, the scavenging effect is better than the control L-ascorbic acid.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种利用NKA-9型大孔树脂对甘草总黄酮粗提物进行纯化的方法，通过优化工艺参数，得到纯化后的甘草总黄酮产品，并对其清除亚硝酸盐能力进行了研究。其步骤为：The invention provides a method for purifying the crude extract of total flavonoids of licorice by using NKA-9 macroporous resin. By optimizing the process parameters, the purified total flavonoids product of licorice is obtained, and its ability to remove nitrite is tested. Research. The steps are:
一、甘草总黄酮粗提物的制备1. Preparation of crude extract of total flavonoids of licorice
精确称取甘草适量，用15～25倍量的60%～80%乙醇，回流提取2～3 h，得到甘草总黄酮粗提液，加入一定体积乙酸乙酯进行萃取，收集上层油相，减压浓缩，所得粗提物干燥收集。Accurately weigh an appropriate amount of licorice, use 15 to 25 times the amount of 60% to 80% ethanol, and reflux for extraction for 2 to 3 hours to obtain a crude extract of total flavonoids of licorice, add a certain volume of ethyl acetate for extraction, collect the upper oil phase, reduce Concentrated under pressure, and the obtained crude extract was collected by drying.
二、甘草总黄酮粗提物的纯化2. Purification of crude extract of total flavonoids of licorice
NKA-9型大孔吸附树脂的静态实验：Static experiment of NKA-9 macroporous adsorption resin:
（1）取甘草总黄酮粗品溶解后定容，取溶液5份，调节至不同pH，再分别加入一定量大孔树脂，置于恒温摇床反应，测定溶液吸光度（总黄酮含量采用NaNO2-Al(NO3)3-NaOH显色法测定，下同）。(1) Dissolve the crude licorice total flavonoids and dilute to volume, take 5 parts of the solution, adjust to different pH values, then add a certain amount of macroporous resin respectively, place it on a constant temperature shaker for reaction, and measure the absorbance of the solution (the total flavonoid content adopts NaNO 2 - Al(NO 3 ) 3 -NaOH colorimetric method, the same below).
（2）取一定体积已知浓度甘草黄酮粗提物溶液，加入一定量大孔树脂，置于恒温摇床反应，每隔一定时间取1 mL溶液测定吸光度。(2) Take a certain volume of licorice flavonoid crude extract solution of known concentration, add a certain amount of macroporous resin, place it on a constant temperature shaker for reaction, and take 1 mL of the solution at regular intervals to measure the absorbance.
（3）取（2）中吸附饱和的树脂若干，均分为五份，分别加入相同体积的不同浓度的乙醇进行解吸，测定解吸溶液吸光度。(3) Take some resins saturated with adsorption in (2), divide them into five parts, add the same volume of ethanol with different concentrations respectively for desorption, and measure the absorbance of the desorption solution.
（4）配制一定浓度的甘草总黄酮粗提物溶液，再稀释为一系列浓度梯度的待测液，各加入一定量大孔树脂，置于恒温摇床反应一定时间，测定溶液吸光度。(4) Prepare a solution of licorice total flavonoids crude extract with a certain concentration, and then dilute it into a series of concentration gradients to be tested, add a certain amount of macroporous resin to each, and place it on a constant temperature shaker to react for a certain period of time, and measure the absorbance of the solution.
NKA-9型大孔吸附树脂的动态实验：Dynamic experiment of NKA-9 macroporous adsorption resin:
（1）取一定量大孔树脂处理后湿法装柱（层析柱30 mm×16 mm)，树脂床柱体积为1 BV。使用SK-500Ⅲ注射泵控制上样速度，每0.5 BV收集一个样品，测定吸光度（以上样溶液浓度的1/10为泄露标准），绘制不同上样速度下的大孔树脂泄露曲线。(1) Take a certain amount of macroporous resin and pack it into a wet column (
（2）取一定量大孔树脂按上述动态实验（1）中得到的最佳工艺条件下装柱、上样，使用SK-500Ⅲ注射泵控制洗脱速度，每0.5 BV收集一个样品，测定吸光度。(2) Take a certain amount of macroporous resin and load the column and sample under the optimal process conditions obtained in the above dynamic experiment (1), use the SK-500Ⅲ syringe pump to control the elution speed, collect a sample every 0.5 BV, and measure the absorbance .
三、甘草总黄酮对亚硝酸盐的清除能力研究3. Study on the scavenging ability of total flavonoids of licorice to nitrite
准确称量一定质量纯化后的甘草总黄酮配制已知浓度的待测液，然后稀释为一定浓度梯度的待测液。取上述1 mL不同浓度的待测液于试管中，分别加入5μg/mL的NaNO2溶液1mL，随后将其置于37℃水浴30 min后取出，加入0.4%对氨基苯磺酸1 mL，放置5 min后加入0.2%盐酸萘乙二胺0.5 mL，静置15 min后，以蒸馏水为参比，在波长λ=545 nm下测定溶液吸光度A1。对照组用蒸馏水代替NaNO2溶液测定溶液吸光度A2，用蒸馏水代替甘草总黄酮待测液测定溶液吸光度A0，计算不同浓度下待测液对亚硝酸盐的清除率。Accurately weigh a certain mass of purified licorice total flavonoids to prepare a test solution of known concentration, and then dilute to a test solution with a certain concentration gradient. Take the above-mentioned 1 mL of different concentrations of the solution to be tested in a test tube, add 1 mL of 5 μg/mL NaNO 2 solution respectively, then place it in a 37 °C water bath for 30 min, take it out, add 1 mL of 0.4% p-aminobenzenesulfonic acid, and leave it for After 5 minutes, 0.5 mL of 0.2% naphthalene ethylenediamine hydrochloride was added, and after standing for 15 minutes, the absorbance A 1 of the solution was measured at a wavelength of λ=545 nm using distilled water as a reference. In the control group, distilled water was used instead of NaNO 2 solution to measure the absorbance A 2 of the solution, and distilled water was used to replace the total flavonoids of licorice to measure the absorbance A 0 of the solution.
亚硝酸盐清除率计算：Nitrite removal rate calculation:
亚硝酸盐清除率/%=(A0+A2-A1)/A0×100%Nitrite removal rate/%=(A 0 +A 2 -A 1 )/A 0 ×100%
式中A0代表用蒸馏水代替甘草总黄酮待测液时溶液的吸光度；A1代表加入不同浓度甘草总黄酮待测液时溶液的吸光度；A2代表蒸馏水代替NaNO2溶液时溶液的吸光度。In the formula, A 0 represents the absorbance of the solution when distilled water is used to replace the test solution of total flavonoids of licorice; A 1 represents the absorbance of the solution when adding different concentrations of total flavonoids of licorice to the test solution; A 2 represents the absorbance of the solution when distilled water replaces the NaNO solution.
附图说明：Description of drawings:
图1为甘草总黄酮含量测定标准曲线；Fig. 1 is a standard curve for the determination of total flavonoids in licorice;
图2为静态实验（1）中溶液不同pH对树脂吸附量的影响；Figure 2 shows the effect of different pH of the solution on the adsorption capacity of the resin in the static experiment (1);
图3为静态实验（2）中NKA-9大孔树脂的吸附饱和时间测定；Figure 3 shows the adsorption saturation time measurement of NKA-9 macroporous resin in static experiment (2);
图4为静态实验（3）中不同浓度乙醇对吸附饱和大孔树脂洗脱效果的影响；Figure 4 shows the effect of different concentrations of ethanol on the elution effect of the adsorption-saturated macroporous resin in the static experiment (3);
图5为静态实验（4）中NKA-9大孔树脂在室温下的吸附等温曲线测定；Figure 5 shows the adsorption isotherm curve measurement of NKA-9 macroporous resin at room temperature in static experiment (4);
图6为动态试验（1）不同上样速度和上样体积对大孔树脂装柱的影响；Figure 6 shows the dynamic test (1) the effect of different loading speeds and loading volumes on the packing of macroporous resin;
图7为动态试验（2）不同洗脱速度和洗脱体积对大孔树脂纯化甘草总黄酮粗提物的影响；Figure 7 is the dynamic test (2) the effect of different elution speeds and elution volumes on the crude extract of total flavonoids of licorice purified by macroporous resin;
图8为发明内容三中不同浓度甘草总黄酮对亚硝酸盐的清除能力。Figure 8 shows the scavenging ability of total flavonoids of licorice with different concentrations to nitrite in the third content of the invention.
具体实施方式：Detailed ways:
以下结合实施例对本发明做具体的说明：Below in conjunction with embodiment, the present invention is described in detail:
实施例1：NKA-9型大孔树脂纯化前后粗提物中甘草总黄酮含量的变化Example 1: Changes in the content of total flavonoids of licorice in crude extracts before and after NKA-9 macroporous resin purification
（1）精密称取甘草适量，再以75%乙醇为提取溶剂，按料液比1：20，提取时间3 h进行提取得到甘草黄酮粗提液，加入适量体积乙酸乙酯进行萃取，收集上层油相，减压浓缩，所得粗提物在80℃下干燥，收集。(1) Precisely weigh an appropriate amount of licorice, and then use 75% ethanol as the extraction solvent, extract the crude licorice flavonoids at a ratio of 1:20 and extraction time of 3 h to obtain a crude extract of licorice flavonoids, add an appropriate volume of ethyl acetate for extraction, and collect the upper layer. The oil phase was concentrated under reduced pressure, and the resulting crude extract was dried at 80°C and collected.
（2）取一定量上述甘草黄酮粗提物溶解于15%乙醇溶液，超声溶解过滤后，调节pH至5.0，得到浓度为2.8 mg/mL的上样溶液待用；(2) Dissolve a certain amount of the above-mentioned licorice flavonoid crude extract in 15% ethanol solution, after ultrasonic dissolution and filtration, adjust the pH to 5.0 to obtain a sample loading solution with a concentration of 2.8 mg/mL for use;
（3）精确称量5.0 g经过预处理的NKA-9大孔树脂，湿法装柱（层析柱规格300mm×16mm），柱高8.5 cm；(3) Accurately weigh 5.0 g of pretreated NKA-9 macroporous resin, and pack it into a wet column (chromatographic column size 300mm×16mm), with a column height of 8.5 cm;
（4）使用SK-500Ⅲ注射泵控制上样速度2.0 BV/h，上样体积为3.5 BV。再使用80%乙醇对其进行洗脱，洗脱速度为2.5 BV/h，洗脱体积为8.0 BV。经NKA-9大孔树脂纯化后粗提物中甘草总黄酮含量由44.5%变化为57.8%。(4) The SK-500Ⅲ syringe pump was used to control the sample loading speed of 2.0 BV/h and the sample loading volume to be 3.5 BV. It was then eluted with 80% ethanol at an elution rate of 2.5 BV/h and an elution volume of 8.0 BV. After purification by NKA-9 macroporous resin, the total flavonoid content of licorice in the crude extract changed from 44.5% to 57.8%.
实施例2：纯化后的甘草总黄酮对亚硝酸盐的清除作用研究Example 2: Study on the scavenging effect of purified licorice total flavonoids on nitrite
NaNO2在弱酸性条件下，与对氨基苯磺酸重氮化后，再与盐酸萘乙二胺偶合生成红色化合物，用紫外分光光度计测出该化合物的吸光度，就可知道反应液中NaNO2 含量多少。因此可通过测定相同条件下NaNO2含量的变化来反映甘草总黄酮溶液清除能力亚硝酸盐能力的强弱，按实验内容三中的步骤进行甘草总黄酮溶液对亚硝酸盐消除能力的测定。从图8可以看出，甘草总黄酮对亚硝酸盐有一定的清除能力，且随着浓度的提高其清除作用得到增强，在低浓度时甘草总黄酮的清除效果要优于其对照L-抗坏血酸。NaNO 2 is diazotized with p-aminobenzenesulfonic acid under weakly acidic conditions, and then coupled with naphthalene ethylenediamine hydrochloride to form a red compound. The absorbance of this compound is measured with an ultraviolet spectrophotometer, and the NaNO in the reaction solution can be known. 2 how much content. Therefore, the change of NaNO 2 content under the same conditions can be measured to reflect the nitrite scavenging ability of licorice total flavonoids solution. It can be seen from Figure 8 that the total flavonoids of licorice have a certain scavenging ability to nitrite, and the scavenging effect is enhanced with the increase of the concentration. At low concentrations, the scavenging effect of total flavonoids of licorice is better than its control L-ascorbic acid .
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