CN106539825B - Kelp root product for reducing blood sugar and preparation method and application thereof - Google Patents
Kelp root product for reducing blood sugar and preparation method and application thereof Download PDFInfo
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- CN106539825B CN106539825B CN201510594980.7A CN201510594980A CN106539825B CN 106539825 B CN106539825 B CN 106539825B CN 201510594980 A CN201510594980 A CN 201510594980A CN 106539825 B CN106539825 B CN 106539825B
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Abstract
The invention relates to a kelp root product for reducing blood sugar, a preparation method and application thereof, comprising the following steps: extracting herba Zosterae Marinae with ethanol for several times, and adding antioxidant during the extraction process to obtain extract concentrate; and adding the extract liquor into the extract concentrate for extraction, adding an antioxidant during the extraction process, and drying to obtain the kelp root blood sugar reducing product. The kelp root used in the invention has sufficient raw materials, definite hypoglycemic effect and no toxic or side effect, and is a safe and effective hypoglycemic product; the hypoglycemic product obtained by the invention has obvious inhibition effect on alpha-glucosidase, IC50 reaches 140 mu g/ml, and the hypoglycemic product is a non-competitive inhibitor of alpha-glucosidase; compared with the prior art, the addition of the antioxidant vitamin C improves the blood sugar reducing effect IC50 by more than 1 time, and simultaneously improves the yield of the product by several times.
Description
Technical Field
The invention relates to the field of medicines, and in particular relates to a kelp root blood sugar reducing product as well as a preparation method and application thereof.
Background
Diabetes is a serious disease seriously harming human health, has many and serious complications and is difficult to cure radically. Diabetes patients in China reach tens of millions of people, and the diabetes mellitus is one of the most frequent countries in the world. At present, the clinical diabetes treatment medicines comprise sulfonylurea medicines, biguanide medicines and the like, but the medicines have serious side effects after long-term application, for example, the sulfonylurea medicines have hypoglycemia adverse reaction; biguanides have gastrointestinal side effects. The insulin substitute is a safe and effective medicament at the present stage, but is expensive and has limitation on storage conditions. Therefore, the important significance is to find the blood sugar reducing product which is safer and more effective, has less side effect and is small.
The invention discloses a hypoglycemic component in kelp Root and a preparation method thereof, wherein the preparation method comprises the steps of extracting kelp Root medicinal materials by using water and/or ethanol or ethyl acetate, concentrating to obtain a water extraction concentrate, an ethanol extraction concentrate, an ethyl acetate extraction concentrate or a corresponding concentrate thereof, extracting the corresponding concentrate by using ethyl acetate to obtain an ethyl acetate extract mainly containing polyphenol and terpenoid components, and carrying out chromatographic separation on the ethyl acetate extract to obtain a component which has an inhibitory effect on α -glucosidase, and has an IC50 of 380 mu g/ml.
Disclosure of Invention
In view of the above, the present invention aims to provide a kelp root hypoglycemic product, a preparation method and an application thereof, so as to solve the problem of low activity of the hypoglycemic component mentioned above.
The invention discloses a preparation method of a kelp root hypoglycemic product, which comprises the following steps: extracting herba Zosterae Marinae with ethanol for several times, and adding antioxidant during the extraction process to obtain extract concentrate; and adding the extract liquor into the extract concentrate for extraction, adding an antioxidant during the extraction process, and drying to obtain the kelp root blood sugar reducing product.
Further, the antioxidant is vitamin C.
Further, the amount of antioxidant added in the extraction process or the extraction process is 0-1%.
Further, the addition amount of the antioxidant added in the extraction process or the extraction process is 0.1%.
Further, the extraction process is carried out at least three times at a temperature of 60 ℃ or higher under vacuum reduced pressure.
Further, the ethanol concentration is at least 80% or more.
Further, the extraction process further comprises: the mixture was extracted at room temperature for 24 hours, and the extract was concentrated under reduced pressure.
Further, the amount of the extraction liquid is 5 times that of the extraction concentrate, and the extraction liquid is ethyl acetate.
The invention also discloses a kelp root blood sugar reducing product, which comprises the preparation method of the kelp root blood sugar reducing product.
The third purpose of the invention is to disclose the application of the kelp root hypoglycemic product in the preparation of hypoglycemic drugs.
The kelp root blood sugar reducing product, the preparation method and the application thereof provided by the invention have the following beneficial effects:
the kelp root used in the invention has sufficient raw materials, definite hypoglycemic effect and no toxic or side effect, and is a safe and effective hypoglycemic product; the hypoglycemic product obtained by the invention has obvious inhibition effect on alpha-glucosidase, IC50 reaches 140 mu g/ml, and the hypoglycemic product is a non-competitive inhibitor of alpha-glucosidase; compared with the prior art, the addition of the antioxidant vitamin C improves the blood sugar reducing effect IC50 by more than 1 time, and simultaneously improves the yield of the product by several times.
The kelp root extract obtained by the invention is an alpha-glucosidase non-competitive inhibitor. The noncompetitive inhibitor is an inhibitor that the active substance contained in the extract does not bind to the active center of the enzyme, but binds to other sites of the enzyme, thereby inhibiting the binding of the enzyme to the substrate. The inhibition of enzyme activity by non-competitive inhibitors is not inhibited by substrate concentration compared to competitive inhibitors.
For the purposes of the foregoing and related ends, the one or more embodiments include the features hereinafter fully described and particularly pointed out in the claims. Other benefits and novel features will become apparent from consideration of the following detailed description, the disclosed embodiments are intended to include all such aspects and their equivalents.
Detailed Description
In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details.
The present invention will be described in detail below with reference to preferred embodiments.
Discloses a preparation method of a kelp root hypoglycemic product, which comprises the following steps: extracting herba Zosterae Marinae with ethanol for several times, and adding antioxidant during the extraction process to obtain extract concentrate; and adding the extract liquor into the extract concentrate for extraction, adding an antioxidant during the extraction process, and drying to obtain the kelp root blood sugar reducing product. Preferably, the antioxidant is vitamin C.
Wherein, the extracted active ingredients contained in the kelp root hypoglycemic substance are easy to oxidize, the antioxidant can be added in the extraction and separation process to obviously improve the content of the hypoglycemic substance, and the antioxidant can also be vitamin A, vitamin E and glutathione. The antioxidant vitamin C, IC50, was added during the extraction process.
In some illustrative embodiments, the extraction process or the extraction process adds an antioxidant in an amount of 0-1%. Preferably, the addition amount of the antioxidant added in the extraction process or the extraction process is 0.1%.
In some illustrative embodiments, the extraction process is performed at least three times at a temperature of 60 ℃ or higher under vacuum drying. The concentration of the ethanol is at least more than 80%.
In some illustrative embodiments, the extraction process further comprises: the mixture was extracted at room temperature for 24 hours, and the extract was concentrated under reduced pressure.
In some illustrative embodiments, the amount of the extraction liquid is 5 times the extraction concentrate and the extraction liquid is ethyl acetate.
Example 1
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving by a 60-mesh sieve, adding ethanol with the concentration of more than 80 percent and simultaneously adding vitamin C with the concentration of 0.1 percent according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering the extracting solution, extracting the filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the extract concentrate of the kelp roots, simultaneously adding 0.1% of vitamin C, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the kelp root blood sugar reducing product A.
Example 2
Taking 100kg of naturally air-dried kelp roots, drying at 60 ℃ for 24h, taking out, grinding into powder, sieving with a 60-mesh sieve, adding ethanol with the concentration of more than 80% according to the solid-liquid ratio of 1:5, carrying out reflux extraction at 60 ℃ for 2h, filtering the extracting solution, extracting filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the laminaria japonica thunb root, simultaneously adding 0.1% of vitamin C, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product B.
Example 3
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving by a 60-mesh sieve, adding ethanol with the concentration of more than 80 percent and simultaneously adding vitamin C with the concentration of 0.1 percent according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering the extracting solution, extracting the filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the kelp roots, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the kelp root blood sugar reducing product C.
Example 4
Taking 100kg of naturally air-dried kelp roots, drying at 60 ℃ for 24h, taking out, grinding into powder, sieving with a 60-mesh sieve, adding ethanol with the concentration of more than 80% according to the solid-liquid ratio of 1:5, carrying out reflux extraction at 60 ℃ for 2h, filtering the extracting solution, extracting filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the laminaria japonica thunb root, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product D.
Example 5
Measurement of alpha-glucosidase inhibitory Activity:
the samples of examples 1 to 4 were measured and prepared into test sample solutions with concentration gradients of 50, 100, 200, 400, 600, and 800. mu.g/ml, respectively. The inhibition of alpha-glucosidase was determined according to the following procedure.
1) Solution preparation: solution A (K3PO40.1mol/L, MgCL,3.2mmol/L, pH 6.8), solution B (K3PO40.5mol/L, MgCL,16mmol/L, pH 6.8); enzyme solution: 0.1unit/ml of alpha-glucosidase (product of Sigma Co., model I), dissolved in solution A, substrate solution: the substrate p-nitrophenyl-alpha-D-glucofuranose (Sigma) was dissolved in solution A.
2) And (3) adding 2.82ml of distilled water into 1.02ml of the solution B, adding 2ml of the prepared substrate solution, and uniformly mixing to obtain a detection solution.
3) Add 58.4. mu.l of the above test solution to a 96-well plate, add the sample obtained in example 1, and set two controls (one is blank control, no enzyme and sample to be tested; the other group was negative control, enzyme was added but no test sample was added), and incubated in a 37 ℃ water bath for 5 min.
4) The 96-well plate of the water bath was removed, 20. mu.l of the enzyme solution prepared above was added to none of the wells, and 20. mu.l of solution A was added to the control group, and incubated at 37 ℃ for 30 minutes.
5) Mu.l of glycine solution (0.4mol/L) was added to each well to terminate the reaction.
6) The absorbance A was measured at 405nm using a microplate reader (Thermo, Varioskan flash).
7) And (3) calculating an inhibition rate: inhibition rate (absorbance of sample well-absorbance of blank control well)/absorbance of negative control well.
The results are shown in Table 1.
TABLE 1 examples 1-4 inhibition of alpha-glucosidase by a laminarin hypoglycemic product
The test result shows that the kelp root blood sugar reducing product A of example 1 inhibits the IC50 value of alpha-glucosidase to be 140 mu g/ml; the kelp root hypoglycemic product B of example 2 has an IC50 value of 395 mu g/ml for inhibiting alpha-glucosidase; the kelp root hypoglycemic product C of example 3 inhibits the IC50 value of alpha-glucosidase to be 382 mug/ml; the kelp root hypoglycemic product D of example 4 inhibits alpha-glucosidase with an IC50 value of 468. mu.g/ml; the inhibition type is non-competitive inhibition. The activity of the kelp root blood sugar reducing product A is improved by more than 2 times compared with the activity of the kelp root blood sugar reducing product B and the activity of the kelp root blood sugar reducing product C; the activity of the product D is improved by more than 3 times compared with that of the kelp root blood sugar reducing product D.
The noncompetitive inhibitor is an inhibitor that the active substance contained in the extract does not bind to the active center of the enzyme, but binds to other sites of the enzyme, thereby inhibiting the binding of the enzyme to the substrate. The inhibition of enzyme activity by non-competitive inhibitors is not inhibited by substrate concentration compared to competitive inhibitors.
Example 6
Effect of vitamin C addition on marine blood glucose lowering product yield:
taking 1kg of kelp root, preparing the hypoglycemic product according to the steps of the above examples 1, 2, 3 and 4, drying and weighing. The weight of the kelp root hypoglycemic products obtained in examples 1, 2, 3 and 4 are respectively as follows: 5.38, 3.85, 3.72 and 2.78 grams, with yields of 0.538%, 0.39%, 0.37% and 0.28%, respectively. The vitamin C is added in the extraction and extraction processes, so that the yield of the marine blood sugar reducing product can be obviously improved.
Example 7
60 Kunming mice are taken, streptozotocin is injected in the abdominal cavity according to 100mg/kg body weight, an equal volume of citric acid solution is given to a control group, 5 mice are extracted after 48 hours to measure blood sugar, and whether molding is successful is determined. And after the molding is successful, randomly grouping: negative control group (saline only injection); kelp root hypoglycemic products (taking kelp root hypoglycemic product A in example 1 as an example); positive control group (beiglipium), 10 mice per group, was gavaged once daily for 3 consecutive days, fasted 2h after the last administration (without water). After blood is taken in the frame, the fasting blood glucose is measured by a full-automatic biochemical analyzer. The blood glucose results for each group are shown in table 2.
TABLE 2 influence of Haematococcus japonicus root hypoglycemic products on blood glucose concentration in model mice
(**P＜0.01)
Table 2 shows that the kelp root blood sugar reducing product can obviously reduce the blood sugar of a model mouse by intragastric administration at 100mg/kg of body weight, and has extremely obvious difference with a negative control. The application of the kelp root hypoglycemic product in preparing the hypoglycemic drug is a safe and effective hypoglycemic product.
Example 8
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving by a 60-mesh sieve, adding ethanol with the concentration of more than 80 percent and simultaneously adding vitamin A with the concentration of 0.1 percent according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering the extracting solution, extracting the filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the laminaria japonica thunb root, simultaneously adding 0.1% of vitamin A, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product E.
Example 9
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving with a 60-mesh sieve, adding ethanol with the concentration of more than 80% and simultaneously adding 0.1% of glutathione according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering an extracting solution, extracting filter residues twice according to the method, combining filtrates, and drying under reduced pressure to obtain an extracted concentrate of the kelp roots.
Adding ethyl acetate in an amount which is 5 times that of the extract concentrate of the laminaria japonica thunb root, adding 0.1% glutathione, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product F.
Example 10
Alpha-glucosidase inhibitory activity was measured for examples 1, 8 and 9, as detailed in example 5, and the results are shown in Table 3.
TABLE 3 inhibition of alpha-glucosidase by the laminarin hypoglycemic products of examples 1, 8, 9
The determination result shows that the activity of the kelp root blood sugar reducing product A is improved by more than 2 times compared with the kelp root blood sugar reducing product E and the kelp root blood sugar reducing product F.
Example 11
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving with a 60-mesh sieve, adding ethanol with the concentration of more than 80% and simultaneously adding vitamin C with the concentration of 1% according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering an extracting solution, extracting filter residues twice according to the method, combining filtrates, and drying under reduced pressure to obtain an extraction concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the laminaria japonica thunb root, simultaneously adding 0.1% of vitamin C, stirring and extracting at room temperature for 24h, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product G.
Example 12
Taking 100kg of naturally air-dried kelp roots, drying for 24h at 60 ℃, taking out, grinding into powder, sieving by a 60-mesh sieve, adding ethanol with the concentration of more than 80 percent and simultaneously adding vitamin C with the concentration of 0.1 percent according to the solid-liquid ratio of 1:5, carrying out reflux extraction for 2h at 60 ℃, filtering the extracting solution, extracting the filter residues twice according to the method, combining the filtrates, and drying under reduced pressure to obtain the extract concentrate of the kelp roots.
Adding 5 times of ethyl acetate into the obtained extract concentrate of the laminaria japonica thunb root, simultaneously adding 1% of vitamin C, stirring and extracting at room temperature for 24H, siphoning to collect the upper layer solution, extracting the lower layer precipitate for 2 times by the same method, combining the extract solutions, and drying under reduced pressure to obtain the laminaria japonica thunb root blood sugar reducing product H.
Example 13
Alpha-glucosidase inhibitory activity was measured for examples 1, 11, and 12, as detailed in example 5, and the results are shown in Table 4.
TABLE 4 examples 1, 11, 12 inhibition of alpha-glucosidase by a laminarin hypoglycemic product
The determination result shows that the activity of the kelp root blood sugar reducing product A is improved by more than 1 time than that of the kelp root blood sugar reducing product G and the kelp root blood sugar reducing product H. The vitamin C content of the kelp root blood sugar reducing product G in the extraction process is 1 percent, and the vitamin C content in the extraction process is 0.1 percent; the vitamin C content of the kelp root blood sugar reducing product H in the extraction process is 0.1 percent, the vitamin C content in the extraction process is 1 percent, and the activity is not greatly different.
In conclusion, the embodiment 1 is the best embodiment, the activity of the product is stronger than that of other kelp root hypoglycemic products, compared with the prior art, the hypoglycemic effect IC50 is improved by more than 1 time, and the yield of the product is improved by several times. The kelp root is sufficient in raw materials, has definite hypoglycemic effect and no toxic or side effect, is a safe and effective hypoglycemic product, and has obvious inhibition effect on alpha-glucosidase.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The preparation method of the kelp root hypoglycemic product is characterized by comprising the following steps: extracting radix laminariae with ethanol at a temperature of 60 deg.C or higher for at least three times, and adding antioxidant during the extraction process to obtain extract concentrate; adding the extract liquor into the extract concentrate for extraction, adding an antioxidant during the extraction process, drying to obtain the kelp root blood sugar reducing product,
wherein the antioxidant is vitamin C, and the addition amount of the antioxidant added in the extraction process or the extraction process is 0.1-1%;
the concentration of the ethanol is more than 80%;
the amount of the extraction liquid is 5 times of that of the extraction concentrate, and the extraction liquid is ethyl acetate;
the extraction process further comprises: the mixture was extracted at room temperature for 24 hours, and the extract was concentrated under reduced pressure.
2. The method according to claim 1, wherein the drying is vacuum drying under reduced pressure.
3. The kelp root hypoglycemic product prepared by the preparation method of claim 1 or 2.
4. The use of a kelp root hypoglycemic product as claimed in claim 3 in the preparation of hypoglycemic medicaments.
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| Title |
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| "海带根中alpha-葡萄糖苷酶抑制剂的分离纯化及其酶抑制作用动力学研究";卜同等;《中国海洋药物杂志》;20090630;第28卷(第3期);第13-16页 * |
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