Study method of drug antioxidant activity based on water lily rhizome extract

Abstract

The present invention discloses a method for studying the antioxidant activity of a drug based on a water lily rhizome extract, comprising the following steps: Step 1: Evaluation of DPPH free radical scavenging rate, specifically comprising the following steps: S1.1, preparation of the drug; S1.2, testing of the drug, testing the DPPH free radical scavenging ability; Step 2: Evaluation of total antioxidant capacity (T-AOC), specifically comprising the following steps: S2.1, preparation of the drug, including preparation of drugs from the water part of the ethanol extract, the ethyl acetate part of the ethanol extract, and the n-butanol part of the ethanol extract; S2.2, testing of the drug. The present invention adopts a stable detection method, has the advantages of reliable measurement results and high repeatability of detection data, and the measurement results can better reflect the antioxidant and free radical scavenging biological activities of the water lily rhizome extract, and has a wide range of uses in tracking the activity of biological products derived from the water lily rhizome extract, with broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical research, and in particular to a method for studying the antioxidant activity of pharmaceuticals based on water lily rhizome extracts. Background Art

[0002] Water lily (scientific name: Nymphaea tetragona The water lily was first mentioned in the "Compendium of Materia Medica" under the section on flowers. It states: "The water lily has several layers of leaves, which are large and resemble a shovel. Its flowers are five-colored. They bloom during the summer day and retract underwater at night, emerging again during the day, in contrast to the dream grass, which burrows into the water during the day and emerges at night. It is found in Guangzhou. A proverb says: 'Don't wear water lilies, as they make you treacherous.'" The "Illustrated Collection of Plant Names and Realities" lists it under the category of stone grasses, describing it as "a pi-bi flower, grown in marshes and ponds. Its leaves resemble a vase with forked branches, and a deep red back. In autumn, it blooms in green buds... with thousands of layers of white flowers... and long stems... When harvested and cooked, it is delicious in the garden." It is also known as "sleeping vegetable, water lily vegetable, water lily, and pi-bi flower." The water lily is known as the "Sleeping Beauty of Flowers" for its characteristic of curling up at night and unfurling during the day. It is a perennial aquatic herb with a short, thick rhizome. Its leaves grow in clusters and float on the water's surface. The auricles at the base are pointed or obtuse, with entire margins. The petioles are slender. The base of the calyx is square, with four sepals; the petals are 8-17, multi-layered; the stamens are numerous, 3-4 layers long, and the anthers are yellow; the berry is spherical, soft, and contains numerous small seeds. Embryonic acid is the most abundant substance in edible water lilies, earning them the nickname "the flower of purity and life." Water lily tea is specially made from whole edible water lilies. Analysis of the various nutritional components of water lilies revealed that water lilies contain over 17 amino acids, and water lily protein is a high-quality protein. The analysis also revealed that water lilies are rich in vitamin C, flavonoids, and the trace element zinc. These substances, when combined, exhibit a powerful lead-excretion function. Acute animal toxicity tests, micronucleus tests, and sperm aberration tests have demonstrated that water lilies are a reliable and safe food without any toxic side effects.

[0003] Oxidation is an important metabolic process for aerobic organisms, especially vertebrates and humans, but it leads to the formation of free radicals. Free radicals are intermediate products of normal metabolism (including but not limited to superoxide anions, hydrogen peroxide, hydroxyl radicals, etc.). Free radicals have strong oxidative reaction capabilities in the body, but excessive free radicals can easily react with various biological macromolecules in the body, causing oxidative damage to cells and tissues. Excessive free radicals in the body are an important cause of many diseases, so removing excessive free radicals has become an important way to prevent and treat many diseases. Under physiological conditions, free radicals in the human body are in a dynamic balance between production and removal, and this balance is mainly maintained by the body's antioxidant system. Excessive free radicals in the body are an important factor in the generation of oxidative stress and an important cause of disease, such as immune system-related diseases, neurodegenerative diseases, tumors, etc. Therefore, removing excessive free radicals in the body or preventing the generation of excessive free radicals has become an important way to prevent immune system and neurological diseases.

[0004] However, there is no in-depth study on the drug antioxidant capacity of water lily extracts in the prior art. Therefore, the present invention proposes a drug antioxidant research method based on water lily rhizome extracts. Summary of the Invention

[0005] 1. Technical problems to be solved

[0006] The purpose of the present invention is to solve the problem that the drug antioxidant capacity of water lily extract has not been deeply studied in the prior art, and to propose a drug antioxidant research method based on water lily rhizome extract.

[0007] 2. Technical solution

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The method for studying the antioxidant activity of drugs based on water lily rhizome extract includes the following steps:

[0010] Step 1: Evaluation of DPPH radical scavenging rate, specifically including the following steps:

[0011] S1.1, drug preparation, including preparation of the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract;

[0012] S1.2, drug testing, testing the DPPH free radical scavenging ability, specifically including the following steps:

[0013] 1) Preparation of test sample solutions: Prepare the drug sample to be tested into sample solutions at concentrations of 10, 25, 50, 75, 100, and 125 μg / mL. Dissolve DPPH in analytical grade methanol to a 0.1 mM DPPH free radical solution.

[0014] 2) Label six centrifuge tubes and add an equal volume of 470 μl of DPPH free radical solution. Then, add the above drug solutions of different concentrations in sequence. After reacting for 5 minutes at room temperature, measure the absorbance at 517 nm. The values ​​are blank control A0 and sample A1, respectively.

[0015] 3) Calculation of hydroxyl radical scavenging activity: Calculate the DPPH radical scavenging rate according to the following formula;

[0016] DPPH free radical scavenging rate (%) = (1-(A determination-A control) / A blank)*100%;

[0017] Step 2: Total antioxidant capacity (T-AOC) evaluation, including the following steps:

[0018] S2.1, drug preparation, including preparation of drugs from the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract;

[0019] S2.2, drug detection, using the ABTS method to test the drug sample, specifically including the following steps:

[0020] 1) Sample testing:

[0021] ① Dilute the 10mM Trolox standard solution to 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0mM; prepare the drug to be tested into a 1mg / mL solution with double-distilled water;

[0022] ② Take 200 μl of ABTS working solution (ABTS and oxidant K2S2O8 are prepared at a final concentration of 7mM:28mM). For the blank control, add 10 μl of double-distilled water or PBS. For the standard curve test, add 10 μl of Trolox standard solution of various concentrations. For the sample test, add 10 μl of drug solution. Mix gently. Incubate at room temperature for 2-6 minutes and then measure A735.

[0023] ③ Based on the measured A735 values ​​of the blank control and standard solution, draw a standard curve with the solution concentration on the horizontal axis and the A735 value on the vertical axis. Calculate the equimolar concentration of the sample and the Trolox standard solution based on the standard curve and the A735 value of the sample, and then calculate the total antioxidant activity of the sample using the following method;

[0024] 2) Calculation of total antioxidant activity of the sample: Expression of total antioxidant activity: When Trolox is used as a standard for antioxidant capacity detection, the antioxidant capacity of the sample is expressed as Trolox-Equivalent Antioxidant Capacity (TEAC). The calculation formula is TEAC = Trolox equivalent molar concentration / sample concentration (mol / g). The TEAC value is the total antioxidant activity.

[0025] Preferably, the preparation of the drug of the aqueous part of the ethanol extract in S1.1 includes the following steps: the drug is in the form of an extract, 17.4 mg of the extract is directly weighed, 2 ml of 80% methanol is added, ultrasonically dissolved at 70°C for 30 min, 11000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor for gradient dilution determination.

[0026] Preferably, the preparation of the drug from the ethyl acetate fraction of the ethanol extract in S1.1 comprises the following steps: grinding the drug, weighing 15.3 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolving at 70°C for 30 min, centrifuging at 11,000 r / min and 4°C for 10 min, taking the supernatant as the mother liquor, and performing gradient dilution determination.

[0027] Preferably, the preparation of the drug from the n-butanol part of the ethanol extract in S1.1 includes the following steps: grinding the drug, weighing 16.1 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolving at 70°C for 30 minutes, 11000 r / min, 4°C, centrifuging for 10 minutes, taking the supernatant as the mother liquor, and performing gradient dilution determination.

[0028] Preferably, the Nanjing Jiancheng test kit (Cat. No. A153-1-1) is used in S1.2, and the sample is added and tested according to the kit operating instructions.

[0029] Preferably, the preparation of the drug of the aqueous part of the ethanol extract in S2.1 includes the following steps: the drug is in the form of an extract, 32.7 mg of the extract is directly weighed, triple-distilled water is added for ultrasonic dissolution for 30 minutes, 11000 r / min, 4°C, centrifuged for 10 minutes, the supernatant is taken as the mother liquor, and gradient dilution is performed for determination.

[0030] Preferably, the preparation of the drug of the ethyl acetate fraction of the ethanol extract in S2.1 is as follows: the drug is ground into powder, 19.2 mg of powder is weighed, 2 ml of triple-distilled water is added, ultrasonically dissolved for 30 min, 11000 r / min, 4°C, centrifuged for 10 min, the supernatant is taken as the mother liquor, and gradient dilution is performed for determination.

[0031] Preferably, the preparation of the drug of the n-butanol part of the ethanol extract in S2.1 is as follows: the drug is ground into powder, 17.5 mg of powder is weighed, 2 ml of triple-distilled water is added, ultrasonically dissolved for 30 min, 11000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor for gradient dilution determination.

[0032] Preferably, the S2.2 is performed using the Nanjing Jiancheng test kit (Cat. No. A05-2-1), and the test is performed according to the kit's operating instructions.

[0033] 3. Beneficial effects

[0034] Compared with the prior art, the advantages of the present invention are:

[0035] In the present invention, the detection method is stable and has the advantages of reliable measurement results and high repeatability of detection data. The measurement results can better reflect the antioxidant and free radical scavenging biological activities of the water lily rhizome extract, and has a wide range of uses in tracking the activity of biological products of the water lily rhizome extract, and has broad application prospects. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0037] Example 1:

[0038] The method for studying the antioxidant activity of drugs based on water lily rhizome extract includes the following steps:

[0039] Step 1: Evaluation of DPPH radical scavenging rate, specifically including the following steps:

[0040] S1.1, drug preparation, including preparation of the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract;

[0041] S1.2, drug testing, testing the DPPH free radical scavenging ability, S1.2 uses the Nanjing Jiancheng test kit (Cat. No.: A153-1-1), and the test is performed according to the kit's operating instructions. The specific steps include:

[0042] 1) Preparation of test sample solutions: Prepare the drug sample to be tested into sample solutions at concentrations of 10, 25, 50, 75, 100, and 125 μg / mL. Dissolve DPPH in analytical grade methanol to a 0.1 mM DPPH free radical solution.

[0043] 2) Label six centrifuge tubes and add an equal volume of 470 μl of DPPH free radical solution. Then, add the above drug solutions of different concentrations in sequence. After reacting for 5 minutes at room temperature, measure the absorbance at 517 nm. The values ​​are blank control A0 and sample A1, respectively.

[0044] 3) Calculation of hydroxyl radical scavenging activity: Calculate the DPPH radical scavenging rate according to the following formula;

[0045] DPPH free radical scavenging rate (%) = (1-(A determination-A control) / A blank)*100%;

[0046] Step 2: Total antioxidant capacity (T-AOC) evaluation, including the following steps:

[0047] S2.1, drug preparation, including preparation of drugs from the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract;

[0048] S2.2. Drug testing: The drug was tested using the ABTS method, using the Nanjing Jiancheng test kit (Cat. No. A05-2-1). The test was performed according to the kit's instructions, specifically including the following steps:

[0049] 1) Sample testing:

[0050] ① Dilute the 10mM Trolox standard solution to 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0mM; prepare the drug to be tested into a 1mg / mL solution with double-distilled water;

[0051] ② Take 200 μl of ABTS working solution (ABTS and oxidant K2S2O8 are prepared at a final concentration of 7mM:28mM). For the blank control, add 10 μl of double-distilled water or PBS. For the standard curve test, add 10 μl of Trolox standard solution of various concentrations. For the sample test, add 10 μl of drug solution. Mix gently. Incubate at room temperature for 2-6 minutes and then measure A735.

[0052] ③ Based on the measured A735 values ​​of the blank control and standard solution, draw a standard curve with the solution concentration on the horizontal axis and the A735 value on the vertical axis. Calculate the equimolar concentration of the sample and the Trolox standard solution based on the standard curve and the A735 value of the sample, and then calculate the total antioxidant activity of the sample using the following method;

[0053] 2) Calculation of total antioxidant activity of the sample: Expression of total antioxidant activity: When Trolox is used as a standard for antioxidant capacity detection, the antioxidant capacity of the sample is expressed as Trolox-Equivalent Antioxidant Capacity (TEAC). The calculation formula is TEAC = Trolox equivalent molar concentration / sample concentration (mol / g). The TEAC value is the total antioxidant activity.

[0054] In the present invention, the preparation of the drug of the aqueous part of the ethanol extract in S1.1 includes the following steps: the drug is in the form of an extract, 17.4 mg of the extract is directly weighed, 2 ml of 80% methanol is added, and ultrasonic dissolution is carried out at 70°C for 30 minutes, and then centrifuged at 11000 r / min and 4°C for 10 minutes. The supernatant is taken as the mother liquor and subjected to gradient dilution determination.

[0055] In the present invention, the preparation of the drug of the ethyl acetate part of the ethanol extract in S1.1 includes the following steps: grinding the drug, weighing 15.3 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolving at 70°C for 30 minutes, 11000 r / min, 4°C, centrifuging for 10 minutes, taking the supernatant as the mother liquor, and performing gradient dilution determination.

[0056] In the present invention, the preparation of the drug of the n-butanol part of the ethanol extract in S1.1 includes the following steps: grinding the drug, weighing 16.1 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolving at 70°C for 30 minutes, 11000 r / min, 4°C, centrifuging for 10 minutes, taking the supernatant as the mother liquor, and performing gradient dilution determination.

[0057] In the present invention, the preparation of the drug of the aqueous part of the ethanol extract in S2.1 includes the following steps: the drug is in the form of an extract, 32.7 mg of the extract is directly weighed, triple-distilled water is added for ultrasonic dissolution for 30 minutes, 11000 r / min, 4°C, centrifuged for 10 minutes, and the supernatant is taken as the mother liquor for gradient dilution determination.

[0058] In the present invention, the preparation of the drug of the ethyl acetate part of the ethanol extract in S2.1 is as follows: the drug is ground and crushed, 19.2 mg of powder is weighed, 2 ml of triple-distilled water is added, ultrasonically dissolved for 30 min, 11000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor for gradient dilution determination.

[0059] In the present invention, the preparation of the drug of the n-butanol part of the ethanol extract in S2.1 is as follows: the drug is ground and crushed, 17.5 mg of powder is weighed, 2 ml of triple-distilled water is added, ultrasonically dissolved for 30 min, 11000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor for gradient dilution determination.

[0060] Example 2:

[0061] It has the implementation content of the above embodiment, wherein, for the specific implementation of the above embodiment, reference can be made to the above description, and the embodiment here will not be repeated in detail; and in the embodiment of the present application, it is different from the above embodiment in that:

[0062] Analysis of DPPH free radical scavenging rate experimental results:

[0063] Table 1: DPPH radical scavenging rate of drugs in water part

[0064]

[0065] Table 2: DPPH radical scavenging rate of drugs in ethyl acetate site

[0066]

[0067] Table 3: DPPH radical scavenging rate of drugs in n-butanol site

[0068]

[0069] Results: Tables 1-3 show that all three drugs showed significant DPPH free radical scavenging activity. However, at a concentration of 0.034 mg / mL, the water fraction had a scavenging rate of only 20.44%. At a concentration of 0.0299 mg / mL, the ethyl acetate fraction still had a scavenging rate of 90.44%. At a concentration of 0.03145 mg / mL, the n-butanol fraction had a scavenging rate of only 21.29%. Overall, the ethyl acetate fraction showed the greatest DPPH free radical scavenging activity.

[0070] Example 3:

[0071] It has the implementation content of the above embodiment, wherein, for the specific implementation of the above embodiment, reference can be made to the above description, and the embodiment here will not be repeated in detail; and in the embodiment of the present application, it is different from the above embodiment in that:

[0072] Analysis of drug total antioxidant capacity results:

[0073] The line equation is: Y = -1.49 * X + 1.475, R² = 0.9912, where X is the molar concentration of Trolox and Y is the absorbance. The total antioxidant capacity of each polar fraction is expressed as a multiple of its antioxidant capacity compared to Trolox.

[0074] Table 4: Total antioxidant capacity of drugs in water fraction

[0075]

[0076] Table 5: Total antioxidant capacity of drugs in ethyl acetate fraction

[0077]

[0078] Table 6: Total antioxidant capacity of drugs in n-butanol fraction

[0079]

[0080] Results: Tables 4-6 show that all three drugs have significant total antioxidant capacity. However, at a concentration of 0.255 mg / mL, the water fraction had a total antioxidant capacity equivalent to 0.041 mM Trolox; at a concentration of 0.15 mg / mL, the ethyl acetate fraction had a total antioxidant capacity equivalent to 0.394 mM Trolox; and at a concentration of 0.137 mg / mL, the n-butanol fraction had a total antioxidant capacity equivalent to 0.011 mM Trolox. Overall, the ethyl acetate fraction exhibited the greatest total antioxidant activity.

[0081] Conclusion: Both DPPH free radical scavenging ability and ABTS method showed that the ethyl acetate fraction of ethanol extract had the best antioxidant effect.

[0082] In the present invention, the detection method is stable and has the advantages of reliable measurement results and high repeatability of detection data. The measurement results can better reflect the antioxidant and free radical scavenging biological activities of the water lily rhizome extract, and has a wide range of uses in tracking the activity of biological products of the water lily rhizome extract, and has broad application prospects.

[0083] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for studying the antioxidant activity of drugs based on water lily rhizome extract, characterized in that: The following steps are involved: Step 1: Evaluation of DPPH radical scavenging rate, specifically including the following steps: S1.1, drug preparation, including preparation of the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract; S1.2, drug testing, testing the DPPH free radical scavenging ability, specifically including the following steps: 1) Preparation of test sample solutions: Prepare the drug sample to be tested into sample solutions at concentrations of 10, 25, 50, 75, 100, and 125 μg / mL. Dissolve DPPH in analytical grade methanol to a 0.1 mM DPPH free radical solution. 2) Label six centrifuge tubes and add an equal volume of 470 μl of DPPH free radical solution. Then, add the sample solutions of different concentrations described above in sequence. After reacting for 5 minutes at room temperature, measure the absorbance at 517 nm. The values ​​are blank control A0 and sample A1, respectively. 3) Calculation of hydroxyl radical scavenging activity: Calculate the DPPH radical scavenging rate according to the following formula; DPPH free radical scavenging rate (%) = (1-(A determination-A control) / A blank)*100%; Step 2: Total antioxidant capacity (T-AOC) evaluation, including the following steps: S2.1, drug preparation, including preparation of drugs from the aqueous portion of the ethanol extract, the ethyl acetate portion of the ethanol extract, and the n-butanol portion of the ethanol extract; S2.2, drug detection, using the ABTS method to test the drug sample, specifically including the following steps: 1) Sample testing: ① Dilute the 10mM Trolox standard solution to 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0mM; prepare the drug to be tested into a 1mg / mL solution with double-distilled water; ② Take 200 μl of ABTS working solution and 10 μl of double-distilled water or PBS as blank control. For standard curve detection, add 10 μl of Trolox standard solution of various concentrations. For sample detection, add 10 μl of drug solution. Mix gently and incubate at room temperature for 2-6 minutes before measuring A735. The ABTS and oxidant K2S2O8 are prepared at a final concentration of 7mM:28mM. ③ Based on the measured A735 values ​​of the blank control and standard solution, draw a standard curve with the solution concentration on the horizontal axis and the A735 value on the vertical axis. Calculate the equimolar concentration of the sample and the Trolox standard solution based on the standard curve and the A735 value of the sample, and then calculate the total antioxidant activity of the sample using the following method; 2) Calculation of total antioxidant activity of the sample: When Trolox is used as a standard for antioxidant capacity testing, the antioxidant capacity of the sample is expressed as TEAC. The calculation formula is TEAC = Trolox equivalent molar concentration / sample concentration, in mol / g. The TEAC value is the total antioxidant activity. The preparation of the drug from the water part of the ethanol extract in S1.1 comprises the following steps: the drug is in the form of an extract, 17.4 mg of the extract is directly weighed, 2 ml of 80% methanol is added, ultrasonically dissolved at 70°C for 30 min, 11,000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed. The preparation of the drug from the ethyl acetate part of the ethanol extract comprises the following steps: grinding the drug, weighing 15.3 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolved at 70°C for 30 min, 11,000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed. The preparation of the drug from the n-butanol part of the ethanol extract comprises the following steps: grinding the drug, weighing 16.1 mg of powder, adding 2 ml of 80% methanol, ultrasonically dissolved at 70°C for 30 min, 11,000 r / min, 4°C, centrifuged for 10 min, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed. The preparation of the drug of the water part of the ethanol extract in S2.1 includes the following steps: the drug is in the form of an extract, 32.7 mg of the extract is directly weighed, and triple-distilled water is added for ultrasonic dissolution for 30 minutes, 11000 r / min, 4°C, and centrifuged for 10 minutes, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed; the preparation of the drug of the ethyl acetate part of the ethanol extract in S2.1 includes grinding the drug, weighing 19.2 mg of the powder, adding 2 ml of triple-distilled water for ultrasonic dissolution for 30 minutes, 11000 r / min, 4°C, and centrifuged for 10 minutes, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed; the preparation of the drug of the n-butanol part of the ethanol extract in S2.1 includes grinding the drug, weighing 17.5 mg of the powder, adding 2 ml of triple-distilled water for ultrasonic dissolution for 30 minutes, 11000 r / min, 4°C, and centrifuged for 10 minutes, and the supernatant is taken as the mother liquor, and gradient dilution determination is performed.