CN117783405A - Screening and application of uric acid-lowering and hypoglycemic active ingredients in purified cactus fruit extracts - Google Patents

Abstract

The invention provides the screening and application of uric acid-lowering and hypoglycemic active ingredients in the purified extract of cactus fruit. The screening method includes: incubating the purified extract of cactus fruit with XOD/α-Glu, and bioaffinity ultrafiltration to remove the ligand-XOD The /α-Glu inhibitor complex is separated from the unbound complex and subsequently analyzed by UPLC-QTRAP-MS/MS method to identify and quantify the ligand compounds released from the ligand-XOD/α-Glu inhibitor complex. This invention uses bioaffinity ultrafiltration combined with UPLC-QTRAP-MS/MS technology to screen and identify XOD and α-Glu inhibitors from cactus fruit purified extracts for the first time, and elucidates the binding sites of inhibitors and enzymes based on molecular docking. The relevant mechanism of action provides theoretical and technical support for the preparation of natural drugs for lowering uric acid and blood sugar, and has great application prospects.

Description

Screening and application of uric acid-lowering and hypoglycemic active ingredients in purified cactus fruit extracts

Technical field

The invention belongs to the technical field of extracting plant active ingredients and relates to the screening and application of uric acid-lowering and hypoglycemic active ingredients in purified prickly pear fruit extracts.

Background technique

Hyperuricemia (HUA) is a metabolic syndrome caused by purine metabolism disorder. It is the main risk factor for gout. It is also responsible for obesity, hypertension, atherosclerosis, dyslipidemia, cardiovascular disease, chronic kidney disease and Major risk factors for diabetes. Xanthine oxidase (XOD) is a key molybdenum-containing enzyme that participates in the catabolism of purine nucleic acids, catalyzes the oxidation of xanthine to uric acid, and generates reactive oxygen species (ROS). XOD inhibitors inhibit the synthesis of uric acid. Lowering serum uric acid concentration can be used to prevent hyperuricemia. Allopurinol, an XOD inhibitor, is the most commonly prescribed drug for gout, but it has been associated with hypersensitivity reactions.

Diabetes mellitus (DM) is a chronic metabolic disease caused by abnormal carbohydrate metabolism. Postprandial hyperglycemia can cause severe damage, dysfunction and failure of multiple organs and tissues, accompanied by progressive metabolic complications. α-glucosidase (α-Glu) catalyzes the hydrolysis of carbohydrates in the diet and converts them into monosaccharides, which are then absorbed in the jejunum, which can easily cause diabetes and other related diseases. α-Glu inhibitors can delay the absorption of glucose and reduce postprandial blood glucose and insulin levels. Typical α-Glu inhibitors, such as acarbose and miglitol, can also cause gastrointestinal side effects.

Natural products with specific active skeletons, active molecules and good biological activities have played an important role in the long history of human resistance to metabolic diseases. Many enzyme inhibitors have been discovered and isolated from extracts of fruits, vegetables and herbs. Previous research reports have shown that polyphenols such as quercetin, kaempferol, isorhamnetin, rutin, hyperoside and quercetin were isolated and identified from Flos Sophorae Immaturus as potential inhibitors of XOD . In addition, rutin, isoquercetin, chlorogenic acid, quercetin and cinnamic acid were also screened and identified from Pyruspyrifolia Fruit as having certain inhibitory effects on α-Glu. Therefore, natural products extracted from plants can serve as effective inhibitors of XOD and α-Glu.

Opuntia ficus-indica fruit (OFI), also known as prickly pear, is a plant native to Mexico and belongs to the Cactaceae family. OFI not only has a large number of functional, nutritional and biological activities, but also has socio-economic, agricultural economic and ecological benefits. There are currently few published reports on the uric acid-lowering/hypoglycemic-related components of cactus fruit. Among them, the active ingredients and related action mechanisms of XOD and α-Glu inhibitors have not been reported, indicating that cactus fruit has uric acid-lowering/hypoglycemic activity. have not yet been fully exploited.

Contents of the invention

The purpose of the present invention is to provide screening and application of uric acid-lowering and hypoglycemic active ingredients in a purified cactus fruit extract. For the first time, potential inhibitors for lowering uric acid and hypoglycemic substances are screened out from a purified cactus fruit extract, thereby preparing a method for lowering uric acid and hypoglycemic agents. Natural medicine provides theoretical and technical support and has great application prospects.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

The invention provides a method for screening uric acid-lowering and hypoglycemic active ingredients in a purified prickly pear fruit extract, which includes: incubating the purified prickly pear fruit extract with XOD/α-Glu, and bioaffinity ultrafiltration to remove the ligand -XOD/α- The Glu inhibitor complex is separated from the unbound complex and subsequently analyzed by UPLC-QTRAP-MS/MS method to identify and quantify the ligand compound released from the ligand-XOD/α-Glu inhibitor complex.

Preferably, 10 mg/mL cactus fruit purified extract is mixed with 5 U/mL, pH 7.4 XOD solution and 10 U/mL, pH 6.8 α-Glu solution respectively according to the volume ratio of 1:2 in a 30kDa ultrafiltration tube at a constant temperature of 37°C. Incubate for 30 minutes.

Preferably, ultrafiltration centrifugation is performed at 12000×g for 10 minutes using an ultrafiltration tube, and the compounds not specifically bound to XOD/α-Glu are eluted by washing three times with 0.2M/0.1M phosphate buffer, and then dissociated with 90% methanol to elute the substances bound to the enzyme in the ultrafiltration tube to obtain an eluate.

Preferably, the biological affinity of the inhibitor and XOD/α-Glu is calculated according to the following formula:

Among them, C1, C2 and C0 represent the concentrations of the screened compounds in the purified extract of cactus fruit with active XOD/α-Glu, inactive XOD/α-Glu and no XOD/α-Glu, respectively.

Preferably, the target compound is chromatographed by a Waters UPLC liquid chromatography column, the liquid chromatography phase A is an aqueous solution containing 0.1% formic acid, the phase B is acetonitrile with a flow rate of 0.8 mL/min, and the gradient elution program is: 0-5 min, 0-15% A; 5-20 min, 15-25% A; 20-40 min, 25-50% A; 40-55 min, 50-80% A; 55-60 min, 15% A, the column temperature is set to 40°C, the automatic sampler temperature is set to 4°C, and the injection volume is 2 μL.

Preferably, mass spectrometry analysis is performed in multiple reaction monitoring mode, and the ion source parameters are as follows: IonSpray Voltage: +5500/-4500V, Curtain Gas: 35psi, Temperature: 400°C, Ion Source Gas 1:60psi, IonSource Gas 2:60psi, DP :+100V.

Preferably, seven XOD inhibitor components are screened out from the purified cactus fruit extract: dihydromyricetin, kaempferol, methyl gallate, isorhamnetin, chlorogenic acid, naringenin, and dihydroquercetin. and eight α-Glu inhibitor ingredients: kaempferol, isorhamnetin, vetch glycoside, rutin, isoquercitrin, naringenin, chlorogenic acid, and dihydroquercetin.

Preferably, the purified cactus fruit extract is prepared by the following method: freshly picked cactus fruits are seeded, vacuum freeze-dried, ground into powder, passed through a 60-mesh sieve, the fruit powder is fully mixed with 60% ethanol, and ultrasonic-assisted extraction is performed. Then centrifuge to collect the supernatant, and the residue is extracted once again according to the same method as mentioned above, and the supernatants extracted twice are combined. After the supernatant is concentrated with a rotary evaporator, it is purified by AB-8 macroporous adsorption resin filled chromatography column. The effluent was washed with distilled water until colorless, and then eluted with 95% ethanol. The ethanol-eluted eluate was concentrated and freeze-dried to obtain a purified polyphenol extract.

The present invention also provides the use of the uric acid and blood sugar lowering active ingredients obtained by the above screening method in the preparation of uric acid and blood sugar lowering products.

Preferably, the product includes a drug.

This invention uses bioaffinity ultrafiltration combined with UPLC-QTRAP-MS/MS technology to screen and identify XOD and α-Glu inhibitors from cactus fruit purified extracts for the first time. In the ultrafiltration system, the mixture of target substances and extracts passes through Ultrafiltration membranes, natural product components that specifically bind to high molecular weight target molecules are trapped on the membrane, while unbound mixture components are removed. The method is simple, reliable and fast. The present invention further elaborates on inhibitors and enzymes based on molecular docking. The binding sites and related mechanisms of action provide theoretical and technical support for the preparation of natural drugs or health products for lowering uric acid and blood sugar, which is conducive to the further development and utilization of cactus fruit and has great application prospects.

Description of drawings

Figure 1 shows the antioxidant activity and enzyme inhibition IC ₅₀ value of the purified cactus fruit extract of the present invention.

Figure 2 is a chromatogram of enzyme inhibitors screened by bioaffinity ultrafiltration of the present invention.

Figure 3 shows the biological binding degree and the enzyme inhibition IC ₅₀ value of the standard product for ultrafiltration screening of enzyme inhibitors of the present invention.

Figures 4 and 5 are surface diagrams and 2D diagrams of the molecular docking between the inhibitor of the present invention and xanthine oxidase.

Figures 6 and 7 are surface diagrams and 2D diagrams of docking molecules of the inhibitor of the present invention and α-glucosidase.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. Those skilled in the art should understand that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of the present invention.

Example

1. Preparation of purified extract of cactus fruit

Freshly picked cactus fruits were seeded, vacuum freeze-dried, and grinded into powder. Passed through a 60-mesh sieve, the fruit powder was thoroughly mixed with 60% ethanol (w/v=1:30), and ultrasonic-assisted extraction was carried out at 40°C and 320W. 20 min, then centrifuge at 10000g for 10 min at 4°C and collect the supernatant. The residue is extracted once again in the same way as above, and the supernatants extracted twice are combined. After the supernatant was concentrated with a rotary evaporator, it was purified by filling a chromatography column with AB-8 macroporous adsorption resin, and the effluent was washed with distilled water until colorless, and then eluted with about 2BV 95% ethanol. Finally, the ethanol-eluted eluate was concentrated using a rotary evaporator and freeze-dried to obtain the purified polyphenol extract.

2. Determination of total phenolic content

The total polyphenol content was determined using the Folin-Ciocalteu colorimetric method, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample in dry mass (DM), with the unit being mg GAE/g DW.

The total polyphenol content of the extract purified by macroporous resin was 355.03 mg GAE/g DW, which was increased by approximately 86 times compared with the crude extraction, indicating that prickly pear fruit extract is rich in bioactive substances.

3. Determination of antioxidant activity

The antioxidant activity of purified cactus fruit extracts was evaluated according to four different antioxidant methods: DPPH, ABTS ⁺ , CUPRAC, and FRAR. Trolox was used as a positive control and a standard curve was drawn. The DPPH/ABTS ⁺ scavenging activity value was expressed in μmol Trolox (TE)/g DM sample.

Among them: Ac is the absorbance of the DPPH/ABTS ⁺ solution and the sample solvent mixture, Ai is the absorbance of the DPPH/ABTS solution and the sample solution mixture, and Aj is the absorbance of the solvent and sample solution mixture.

Iron ion reducing ability (FRAP) and copper ion reducing ability (CUPRAC) used Trolox as a positive control and drew a standard curve. FRAP and CUPRAC were expressed in μmol Trolox (TE)/g DW.

The health beneficial effects of prickly pear fruit polyphenols may depend on their antioxidant and free radical scavenging activities. The results of measuring the antioxidant activities of ABTS+, FRAP, DPPH and CUPRAC are shown in Figure 1 (left picture), which shows that the extract has certain antioxidant activity, among which FRAP (1091.40mmol TE/100g DW) has the most significant effect.

4. Determination of XOD and α-Glu inhibitory activity

Take 50 μL of sample in a 96-well plate, add 50 μL of XOD solution (0.1U), shake for 10 seconds and incubate at 37°C for 5 minutes. Then add 150 μL of xanthine solution (0.2 mM), measure the absorbance at 290 nm every 20 seconds, record the change of absorbance within 10 minutes, and replace the sample solution with 50 μL of phosphate buffer (0.2 M, pH 7.5) as blank. Take allopurinol as the positive control and calculate the XOD inhibitory activity according to the following formula:

Where, (dA/dt)blank: blank reaction rate; (dA/dt)sample: sample reaction rate.

100 μL 1U/ml α-Glu was dissolved in 0.1M phosphate buffer (PBS, pH 6.8), incubated with 100 μL polyphenol extract at 37°C for 10 min, then 100 μL 5mM PNPG solution was added to react at 37°C for 20 min, and 500 μL 1M Na ₂ CO ₃ solution was added to terminate the reaction. The absorbance was measured at 405 nm. Acarbose and PBS were used as positive and blank controls, and the α-Glu inhibitory activity was calculated according to the following formula:

Where A1, A0, B1 and B0 represent the absorbance of the blank test group (containing PBS buffer and enzyme), blank control group (containing only PBS buffer), sample test group (containing sample extract, PBS buffer solution and enzyme) and sample control group (containing sample extract and PBS buffer), respectively.

The inhibitory activity of the extract against XOD and α-Glu was evaluated by in vitro enzyme inhibition experiments. As shown in Figure 1 (right), the purified extract had a certain inhibitory effect on XOD (IC ₅₀ =199.00 μg/mL) and α-Glu (IC ₅₀ =159.67 μg/mL), among which allopurinol and acarbose were positive controls for XOD and α-Glu, respectively. The inhibitory activity of the extract against α-Glu was better than that against acarbose (IC ₅₀ =345.67 μg/mL).

5. Screening and identification of potential inhibitors

Screen the XOD and α-Glu inhibitors in the extract. The specific steps are as follows: add 100 μL of 10 mg/mL extract solution and 200 μL of XOD (5 U/mL, pH 7.4)/α-Glu (10 U/mL, pH 6.8) in Incubate in a 30kDa ultrafiltration tube at 37°C for 30 minutes, then centrifuge and filter the reaction solution, finally capture the ligand-XOD/α-Glu inhibitor complex at 37°C, and centrifuge at 12000×g for 10 minutes. Then wash three times with 200 μL 0.2M/0.1M phosphate buffer, and finally add 200 μL 90% methanol for dissociation. Repeat twice to release the ligand from the inhibitor complex. The dissociation solution was quantified by UPLC-QTRAP-MS/MS to quantify the released ligand. The same ultrafiltration step was performed using inactivated XOD/α-Glu as a negative control. Calculate the biological affinity of the inhibitor and XOD/α-Glu according to the formula.

Among them, C1, C2 and C0 represent the concentrations of screened compounds in active XOD/α-Glu, inactive XOD/α-Glu and XOD/α-Glu-free OFI extracts, respectively.

6. UPLC-QTRAP-MS/MS determination

This application uses EXION LC System (SCIEX) ultra-high performance liquid chromatography and a Waters UPLC liquid chromatography column (Waters Acquity UPLC HSS T3, 1.8 μm, 2.1 × 100 mm) to perform chromatographic separation of the target compound. Phase A of the liquid chromatography is an aqueous solution containing 0.1% formic acid, phase B is acetonitrile, the flow rate is 0.8mL/min, and the gradient elution program is: 0 to 5 min, 0 to 15% A; 5 to 20 min, 15 to 25% A; 20 ~40min, 25~50%A; 40~55min, 50~80%A; 55~60min, 15%A. The column temperature was set to 40°C, the autosampler temperature was set to 4°C, and the injection volume was 2 μL.

Sciex QTrap 6500 mass spectrometer instrument parameter settings:

This application uses a SCIEX 6500QTRAP+ triple quadrupole mass spectrometer equipped with an IonDrive Turbo V ESI ion source to perform mass spectrometry analysis in multiple reaction monitoring (MRM) mode. The ion source parameters are as follows: IonSpray Voltage: +5500/-4500V, Curtain Gas: 35psi, Temperature: 400℃, Ion Source Gas 1:60psi, IonSource Gas 2:60psi, DP: +100V.

In order to better determine the uric acid-lowering and hypoglycemic components of the purified OFI extract, a non-targeted metabolomic approach based on UPLC-MS was adopted in this application. By comparing the substance database with standards such as retention time and ion mass-to-charge ratio, substances can be qualitatively identified. As shown in Figure 2, a total of 16 chemical components were identified in the purified OFI extract. It includes a total of 5 phenolic acids and their derivatives (peaks 1, 2, 3, 8, 12) and 11 flavonoids (peaks 4 to 7, 9 to 11, 13 to 16). Among them, five phenolic acids and their derivatives were identified as chlorogenic acid, methyl gallate, gentisic acid, ferulic acid and methyl caffeate, and peaks 4 to 7, 9 to 11, and 13 to 16 were identified respectively. It is dihydromyricetin, isoquercetin, rutin, kaempferol, kaempferol-3-O-rutinoside, dihydroquercetin, vetch glycoside, quercetin, naringenin, kaempferol, Isorhamnetin.

Through bioaffinity ultrafiltration screening, seven XOD inhibitor components and eight α-Glu inhibitor components were screened out from the purified extract (Figure 2). Analysis of the biological affinities of the identified and isolated inhibitors with the enzyme showed (Figure 3) that the affinities with XOD were dihydromyricetin (53.13%), kaempferol (49.21%), and methyl gallate (28.19 %), isorhamnetin (13.30%), chlorogenic acid (7.56%), naringenin (6.10%), dihydroquercetin (3.25%). The affinity with α-Glu is kaempferol (54.75%), isorhamnetin (24.07%), vetch glycoside (19.00%), rutin (17.31%), isoquercitrin (16.06%), naringin Cortin (9.55%), chlorogenic acid (7.85%), dihydroquercetin (3.23%). The results show that dihydromyricetin and kaempferin have stronger affinity for XOD, while kaempferol and isorhamnetin have greater affinity for α-Glu than other inhibitors. And there are significant differences in the affinity between different compounds and enzymes. These differences may be due to different competitive binding relationships between biologically active ingredients and different enzymes.

To further analyze the correlation between affinity and inhibitors of the two enzymes, the inhibitory activity of a single inhibitor standard against the two enzymes was measured separately (Figure 3). The results also show that the screened inhibitors of the two enzymes have a certain inhibitory effect on them, indicating that ultrafiltration screening technology can be used to quickly screen natural inhibitors.

7. Molecular docking

Use AutoDock Vina software (http://vina.scripps.edu/) to prepare the ligands and proteins required for molecular docking. For the target protein, its crystal structure needs to be preprocessed, including removing hydrogenation, modifying amino acids, optimizing energy and Adjust the force field parameters so that the low-energy conformation of the ligand structure is satisfied. Finally, the target structure was molecularly docked with the active ingredient structure, and the vina inside the pyrx software was used for docking. Pymol was used for visual analysis, and the 2D diagram was visualized using Discovery Studio 2020 Client.

In order to elucidate the inhibition mechanism of potential inhibitors and enzymes, molecular docking technology was used for verification. It is generally believed that when the molecular docking binding energy is <0, it indicates that the two molecules have spontaneous binding ability, and when the molecular docking binding energy is <-5.0kcal/mol, it indicates that the two molecules have good binding activity. The docking binding energies of the seven inhibitors and XOD ranged from -10.6 to -7.4kcal/mol, all less than -5.0kcal/mol, indicating that these inhibitors all showed good binding ability. In the docking of eight inhibitors with α-Glu, the docking binding energies ranged from -8.4 to -6.9 kcal/mol, which also showed that the selected inhibitors all had inhibitory effects on α-Glu. Further analysis found that the screened inhibitors mainly inhibit the activity of the enzyme by docking with the active center of the enzyme through hydrogen bonds and hydrophobic interactions (Figure 4-Figure 7). In summary, it can be seen from the above analysis that the active compounds screened by ultrafiltration can be well embedded in the active pocket of the key enzyme and show significant interactions with the key amino acid residues of the enzyme.

The screening results showed that the main types of the two enzyme inhibitors were phenolic and flavonoid compounds. These compounds, as a natural active ingredient, have good biological activity and antioxidant activity and can be used to prepare anti-hyperuricemia or anti-diabetic drugs and health products.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Screening method for uric acid-lowering and hypoglycemic active ingredients in the purified extract of cactus fruit, including: incubating the purified extract of cactus fruit with XOD/α-Glu, and inhibiting the ligand-XOD/α-Glu by bioaffinity ultrafiltration. The agent complex is separated from the unbound complex and subsequently analyzed by UPLC-QTRAP-MS/MS method to identify and quantify the ligand compound released from the ligand-XOD/α-Glu inhibitor complex. 2. The screening method according to claim 1 is characterized in that 10 mg/mL of the purified cactus fruit extract is incubated with 5 U/mL, pH 7.4 XOD solution and 10 U/mL, pH 6.8 α-Glu solution in a volume ratio of 1:2 in a 30 kDa ultrafiltration tube at 37°C for 30 minutes. 3. The screening method according to claim 2, characterized in that, using an ultrafiltration tube to centrifuge at 12000×g for 10 minutes to perform ultrafiltration centrifugation, and washing with 0.2M/0.1M phosphate buffer three times without XOD during elution. /α-Glu specifically binds compounds, and then uses 90% methanol to dissociate, and the substances bound to the enzyme in the ultrafiltration tube are eluted to obtain the eluent. 4. The screening method according to claim 1, characterized in that the biological affinity of the inhibitor and XOD/α-Glu is calculated according to the following formula: Wherein C1, C2 and C0 represent the concentrations of the screened compounds in the purified extracts of cactus fruit with active XOD/α-Glu, inactive XOD/α-Glu and no XOD/α-Glu, respectively. 5. The screening method according to claim 1, characterized in that the target compound is chromatographically separated by a Waters UPLC liquid chromatography column, the liquid chromatography phase A is an aqueous solution containing 0.1% formic acid, and the phase B is acetonitrile with a flow rate of 0.8mL. /min, the gradient elution program is: 0~5min, 0~15%A; 5~20min, 15~25%A; 20~40min, 25~50%A; 40~55min, 50~80%A; 55 ~60 min, 15% A, the column temperature is set to 40°C, the autosampler temperature is set to 4°C, and the injection volume is 2 μL. 6. The screening method according to claim 5, characterized in that mass spectrometry analysis is performed in multiple reaction monitoring mode, and the ion source parameters are as follows: IonSpray Voltage: +5500/-4500V, Curtain Gas: 35psi, Temperature: 400°C, Ion Source Gas 1:60psi, Ion Source Gas 2:60psi, DP:+100V. 7. The screening method according to claim 1, characterized in that seven XOD inhibitor components are screened out from the purified cactus fruit extract: dihydromyricetin, kaempferol, methyl gallate, and isorhamnetin. , chlorogenic acid, naringenin, dihydroquercetin and eight α-Glu inhibitor ingredients: kaempferol, isorhamnetin, vetch glycoside, rutin, isoquercitrin, naringenin, green Orthoacid, dihydroquercetin. 8. The screening method according to claim 1 is characterized in that the purified cactus fruit extract is prepared by the following method: freshly picked cactus fruit is deseeded, vacuum freeze-dried, ground into powder, passed through a 60-mesh sieve, the fruit powder is fully mixed with 60% ethanol, ultrasonic-assisted extraction is performed, and then the supernatant is collected by centrifugation, the residue is extracted once again according to the same method as above, and the supernatants of the two extractions are combined, the supernatant is concentrated by a rotary evaporator, purified by a chromatography column filled with AB-8 macroporous adsorption resin, and the effluent is washed with distilled water until it is colorless, and then eluted with 95% ethanol, the eluate eluted with ethanol is concentrated, and freeze-dried to obtain a purified polyphenol extract. 9. Application of the urate-lowering and hypoglycemic active ingredients obtained by the screening method according to any one of claims 1 to 8 in the preparation of urate-lowering and hypoglycemic products. 10. Use according to claim 9, characterized in that the product includes a drug.