Preparation method of total flavones of tribulus terrestris leaves and application thereof

Abstract

This invention discloses a method for preparing total flavonoids from Tribulus terrestris leaves and its applications. The method includes: extracting Tribulus terrestris leaves by reflux with 20% ethanol; adsorbing the extract onto AB-8 macroporous adsorption resin; eluting with 0.1% NaOH solution, neutralizing, undergoing secondary AB-8 resin adsorption, eluting with 90% ethanol, and drying to obtain total flavonoids. The total content of five flavonoid monomers (J1~J5) in the obtained total flavonoids reaches 34.94%, of which J2 content is 13.90%. This invention also establishes an HPLC-UV method for simultaneously determining the content of the five flavonoid monomers. Activity experiments show that the total flavonoids from Tribulus terrestris leaves and monomer J1 have significant scavenging effects on DPPH and ABTS free radicals, with an IC50 scavenging effect on DPPH free radicals. 50 The concentrations were 1.266 µg / mL and 1.128 µg / mL, respectively, which are superior to those of vitamin C. The method of this invention exhibits good selectivity and high purity, and the total flavonoids prepared can be applied in the fields of antioxidant functional foods, health foods, or cosmetics.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and food science and technology, specifically relating to a method for preparing total flavonoids from Tribulus terrestris leaves and their application in the preparation of antioxidant products. Background Technology

[0002] Tribulus terrestris ( Tribulus terrestris L. Tribulus terrestris (Zanthoxylum bungeanum) is an annual or perennial herb belonging to the genus Tribulus of the family Zygophyllaceae. Its roots, stems, leaves, flowers, and fruits are all used medicinally, making it a traditional food and medicine plant. Modern pharmacological studies have shown that Tribulus terrestris possesses various biological activities, including anti-inflammatory, antioxidant, cardiovascular protective, anti-tumor, and hypoglycemic effects. The main active components in Tribulus terrestris include flavonoids, steroidal saponins, and alkaloids, among which flavonoids have attracted attention due to their broad pharmacological effects, especially their significant antioxidant activity. Studies have shown that the flavonoid content in Tribulus terrestris leaves is significantly higher than that in the stems and fruits; therefore, Tribulus terrestris leaves are an important part for obtaining flavonoid active components.

[0003] In the field of natural products, the isolation of flavonoids with antioxidant activity from different plants has been reported. For example, Chinese patent CN113087752A discloses a flavonoid glycoside with antioxidant activity isolated from pomelo, namely apigenin-4′-O-β-D-glucosyl-7-O-α-L-rhamnosyl-(1→2)-[6″-O-(3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside, and verifies its oxygen free radical scavenging ability. Chinese patent CN102659876A discloses a flavonoid glycoside extracted from *Cephalotaxus fortunei*, namely kaempferol-3-O-[2″-O-β-D-glucopyranoside-6″-O-(2″-O-feruloyl-β-D-glucosyl)]-β-D-glucopyranoside, and finds that it has an immunomodulatory effect that promotes the proliferation of splenic lymphocytes and thymic lymphocytes. Chinese patent CN110251552A discloses a method for preparing total flavonoids from *Gnaphalium affine* leaves. The method involves extraction with 95% ethanol, petroleum ether extraction, and ODS reduced-pressure column chromatography to obtain total flavonoids. Flavonoid compounds such as kaempferol-3-O-β-D-gentiobiglycoside and quercetin-3-O-β-D-gentiobiglycoside are isolated from the flavonoids, and their α-glucosidase inhibitory activity and cell safety are verified.

[0004] However, the flavonoids in the aforementioned existing technologies originate from pomelo, *Cephalotaxus fortunei*, and *Tribulus terrestris* leaves, respectively. Their chemical compositions differ from those of *Tribulus terrestris* leaves, indicating variations in their chemical composition profiles. Existing literature reports on the extraction and purification of flavonoids from *Tribulus terrestris* leaves primarily employs conventional methods, such as alcohol extraction followed by single-stage purification with macroporous adsorption resins. For example, D101 macroporous resin is used, with elution using a gradient of different ethanol concentrations to obtain total flavonoids. These methods have the following drawbacks: firstly, they lack selectivity, making it difficult to effectively separate flavonoids from other compounds with similar polarity, such as saponins; secondly, the content of target flavonoid glycosides in the obtained total flavonoids is low, resulting in unsatisfactory enrichment; and thirdly, there is a lack of systematic optimization of the preparation process for specific flavonoid glycosides (such as kaempferol-3-O-β-D-gentiobiglycoside) from *Tribulus terrestris* leaves. Furthermore, there are few reports on methods for simultaneous determination of multiple flavonoid glycosides in total flavonoids from *Tribulus terrestris* leaves, or on systematic evaluation of the antioxidant activity of monomeric flavonoid compounds.

[0005] Therefore, developing a method for efficiently and selectively enriching flavonoids from Tribulus terrestris leaves, especially one that can significantly increase the content of target flavonoid glycosides, and establishing corresponding quality control methods and antioxidant activity evaluation systems, is of great practical significance for the in-depth development and utilization of Tribulus terrestris leaf resources. Summary of the Invention

[0006] To address the shortcomings of existing methods for preparing total flavonoids from Tribulus terrestris leaves, such as low selectivity, difficulty in effectively separating flavonoids from compounds with similar polarity (e.g., saponins), and low content of target flavonoid glycosides, this invention provides a method for preparing total flavonoids from Tribulus terrestris leaves with good selectivity, high enrichment efficiency, and simple operation, as well as its application in the field of antioxidants.

[0007] In a first aspect, the present invention provides a method for preparing total flavonoids from Tribulus terrestris leaves, comprising the following steps performed sequentially: (a) Crushing and extraction: Crushing the Tribulus terrestris leaves, adding an ethanol aqueous solution with a volume fraction of 10% to 30%, and extracting by heating and refluxing 2 to 4 times at a material-to-liquid ratio of 1:20 to 1:30 (g / mL), and combining the extracts; (b) Concentration and dilution: The extract obtained in step (a) is subjected to reduced pressure to recover ethanol until no alcohol odor is detected, and then diluted with water to obtain a diluted solution; (c) First macroporous adsorption resin adsorption: The diluted solution obtained in step (b) is passed through an AB-8 type macroporous adsorption resin column at a loading rate of 2 to 6 BV / h to adsorb flavonoid components onto the resin, and then eluted with distilled water until the effluent is colorless. (d) Alkaline elution: Elute the resin column obtained in step (c) with a 0.05% to 0.2% NaOH solution at a volume of 6 to 10 times the column volume, and collect the alkaline eluent; (e) Neutralization: Neutralize the alkaline eluent obtained in step (d) to a pH of 6.5–7.5 with a 5%–15% HCl solution; (f) Secondary macroporous adsorption resin adsorption: The neutralized solution obtained in step (e) is passed through the AB-8 macroporous adsorption resin column again at a loading rate of 2-6 BV / h, and eluted with distilled water until the effluent is neutral. (g) Alcohol elution: Elute the resin column obtained in step (f) with an ethanol solution of 70% to 95% by volume at a volume of 7 to 11 column volumes, and collect the alcohol eluent; (h) Drying: The solvent in the alcohol eluent obtained in step (g) is recovered under reduced pressure and dried to obtain total flavonoids from Tribulus terrestris leaves.

[0008] Further, the volume fraction of the ethanol-water solution in step (a) is preferably 20%, the material-to-liquid ratio is preferably 1:25 (g / mL), the number of extractions is preferably 3, and the extraction time for each extraction is preferably 1.5 hours, 1 hour, and 0.5 hours, respectively.

[0009] Furthermore, the sample loading rate in steps (c) and (f) is preferably 4 BV / h, and the AB-8 macroporous adsorption resin is preferably pretreated by soaking in anhydrous ethanol for 24 hours before use.

[0010] Furthermore, the concentration of the NaOH solution in step (d) is preferably 0.1%, and the elution volume is preferably 8 times the column volume.

[0011] Furthermore, the concentration of the HCl solution in step (e) is preferably 10%, and the pH value after neutralization is preferably 7.0.

[0012] Furthermore, the volume fraction of the ethanol solution in step (g) is preferably 90%, and the elution volume is preferably 9 times the column volume.

[0013] Secondly, the present invention provides a method for determining the content of flavonoid components in the total flavonoids of Tribulus terrestris leaves prepared by the above method. The method employs high performance liquid chromatography-ultraviolet detection to simultaneously determine the content of quercetin-3-O-β-D-gentiobiglycoside (J1), kaempferol-3-O-β-D-gentiobiglycoside (J2), isorhamnetin-3-O-β-D-gentiobiglycoside (J3), kaempferol-3-O-β-D-(6″-p-coumaroyl)glucoside (J4) and kaempferol-3-O-β-D-gentiobiglycoside-7-O-β-D-glucopyranoside (J5) in the total flavonoids of Tribulus terrestris leaves.

[0014] Furthermore, the chromatographic conditions for the high performance liquid chromatography-ultraviolet detection method are as follows: the chromatographic column is a C-18 column (4.6×250 mm, 5 µm), the mobile phase is a methanol-water system with gradient elution, the detection wavelength is 330 nm, the flow rate is 1.0 mL / min, and the column temperature is 25℃.

[0015] Furthermore, the gradient elution program is as follows: methanol-water volume ratio 12:88 at 0 min, 30:70 at 45 min, 37:63 at 50 min, 43:57 at 57 min, 50:50 at 75 min, 50:50 at 78 min, and 100:0 at 100 min.

[0016] Thirdly, the present invention provides the application of the total flavonoids from Tribulus terrestris leaves prepared by the above method in the preparation of free radical scavengers or antioxidant products.

[0017] Furthermore, the free radical is a DPPH free radical or an ABTS cation free radical.

[0018] Fourthly, the present invention provides an antioxidant composition or functional food comprising total flavonoids from Tribulus terrestris leaves prepared by the above method and pharmaceutically or food-grade excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. High Selectivity in Preparation Method: This invention is the first to employ a combined process of "selective elution with low-concentration alkaline solution + neutralization + secondary macroporous resin adsorption," utilizing the difference in solubility between flavonoids and other similarly polar compounds such as saponins in alkaline solution to achieve highly selective enrichment of target flavonoid glycosides. Static adsorption experiments compared five macroporous adsorption resins (D-101, AB-8, HP-20, DM-301, and D4020). The results showed that AB-8 resin exhibited the highest static adsorption capacity (409.47 µg / g) for the target compound J2, along with a relatively high desorption rate (86.75%), demonstrating the best overall performance.

[0020] 2. High yield and purity of total flavonoids: Using optimized extraction conditions (20% ethanol, material-to-liquid ratio 1:25, extraction three times for 1.5h, 1h, and 0.5h respectively) and purification conditions (elution with 0.1% NaOH for 8 BV, secondary adsorption after neutralization, and elution with 90% ethanol for 9 BV), the total content of five representative flavonoid monomers (J1~J5) in the total flavonoids obtained from Tribulus terrestris leaves reached 34.94%, with J2 content as high as 13.90%, and the enrichment effect was significantly better than that of existing methods.

[0021] 3. Improved quality control methods: An HPLC-UV method for the simultaneous determination of five flavonoid monomers in total flavonoids from Tribulus terrestris leaves was established. Method validation results showed that each compound exhibited good linearity within a given concentration range (R0). 2 All values ​​were greater than 0.99. The RSDs of precision, repeatability, and stability were all less than 3%, and the recovery rates were between 94% and 101%. This method has high sensitivity and good reproducibility and can be used as a quality control standard for total flavonoids from Tribulus terrestris leaves.

[0022] 4. Significant antioxidant activity: DPPH free radical scavenging experiments showed that the IC50 values ​​of total flavonoids and monomeric compound J1 from Tribulus terrestris leaves were 1.266 µg / mL and 1.128 µg / mL, respectively, both superior to the positive control vitamin C (IC50). 50 =1.823 µg / mL); ABTS radical scavenging assay results show that J1's IC50 is 1.823 µg / mL. 50 The value was 0.028 mg / mL, and the IC50 of total flavonoids from Tribulus terrestris leaves was [value missing]. 50 The concentration was 0.111 mg / mL, and the scavenging effect was higher than that of vitamin C. The experimental data fully confirm that the total flavonoids in Tribulus terrestris leaves and the flavonoid monomers therein have good antioxidant activity and have the potential to be developed into natural antioxidants or functional foods. Attached Figure Description

[0023] Figure 1 The graph shows the effect of ethanol concentration on the extraction rate of total flavonoids from Tribulus terrestris leaves. Figure 2 A comparative graph showing the effect of extraction methods on the extraction rate of total flavonoids from Tribulus terrestris leaves; Figure 3 The adsorption kinetics curve of AB-8 macroporous adsorption resin is shown. Figure 4 Desorption curve of 0.1% NaOH solution; Figure 5 The desorption curve of a 90% ethanol solution; Figure 6 The HPLC chromatogram of the mixed standard solution (J1-J5); Figure 7 HPLC chromatogram of the test solution of total flavonoids from Tribulus terrestris leaves; Figure 8 The standard curve of compound J1; Figure 9 This is the standard curve diagram for compound J2; Figure 10 The standard curve of compound J3; Figure 11 This is the standard curve diagram for compound J4; Figure 12The standard curve of compound J5; Figure 13 DPPH free radical scavenging rate curves of monomeric compounds J1, J2, J3, J4 and total flavonoids from Tribulus terrestris leaves at different concentrations; Figure 14 DPPH free radical scavenging rate curves of vitamin C at different concentrations as a positive control; Figure 15 ABTS free radical scavenging rate curves of monomeric compounds J1, J2, J3, J4 and total flavonoids from Tribulus terrestris leaves at different concentrations; Figure 16 ABTS free radical scavenging rate curves of vitamin C at different concentrations as a positive control; Figure 17 The chemical structural formulas of the monomeric compounds J1(Ⅰ), J2(Ⅱ), J3(Ⅲ), and J4(Ⅳ) are given. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All technical solutions implemented based on the above description of the present invention fall within the protection scope of the present invention.

[0025] Example 1 Preparation of total flavonoids from Tribulus terrestris leaves.

[0026] 1.1 Materials and Instruments Materials: Tribulus terrestris leaves were collected from the suburbs of Zhenlai County, Baicheng City, Jilin Province, and identified as dried leaves of Tribulus terrestris L. by Professor Zhang Minjing of the School of Pharmacy, Jilin University. Anhydrous ethanol (analytical grade) was purchased from Beijing Chemical Plant; methanol (chromatographic grade) was purchased from Shanghai Xingke High Purity Solvent Co., Ltd.; NaOH and HCl (analytical grade) were commercially available; purified water was a product of Wahaha Group. AB-8 macroporous adsorption resin was purchased from Beijing Future Technology Development Co., Ltd.; silica gel for column chromatography (200-300 mesh) was purchased from Qingdao Ocean Chemical Plant; ODS (70-100µm) was purchased from Beijing Future Technology Development Co., Ltd.

[0027] Instruments: Electronic balance (METTLER TOLEDO, Switzerland); Rotary evaporator (RE-201C, Gongyi Yingyu Yuhua Instrument Factory); Circulating water multi-purpose vacuum pump (SHZ-CB, Gongyi Yingyu Yuhua Instrument Factory); Dark box UV analyzer (ZF-20D, Gongyi Yuhua Instrument Co., Ltd.); High performance liquid chromatograph (ACCHROM S6000, Huapu Scientific Instruments Technology Co., Ltd.); Analytical column (Agilent SB C-18, 4.6×250 mm, 5 µm); Preparative high performance liquid chromatograph (LC3000, Beijing Innovation Tongheng Technology Co., Ltd.); Preparative column (Agilent SB C-18, 10×250 mm, 10 µm); Nuclear magnetic resonance spectrometer (BRUEKR AV500 / 600).

[0028] 1.2 Optimization of Extraction Process Conditions (1) Selection of extraction solvent Six portions of Tribulus terrestris leaf powder, each 1 g, were extracted with pure water, 20%, 40%, 60%, 80%, and pure ethanol, respectively, at a liquid-to-solid ratio of 20:1 (mL / g), under reflux for 2 h. After filtration, the solvent was recovered under reduced pressure. The residue was dissolved in 12% methanol aqueous solution (V / V) and brought to a final volume of 25 mL. The solution was filtered through a 0.22 µm filter, and 20 µL was analyzed by HPLC. The extraction efficiency was evaluated by the peak area of ​​compound J2 (kaempferol-3-O-β-D-gentiopicroside).

[0029] The results are as follows Figure 1 As shown, when the extraction solvent is a 20% ethanol aqueous solution, the peak area of ​​J2 is the largest, indicating that 20% ethanol has the highest extraction efficiency for flavonoid glycosides. The peak area of ​​pure water extraction is only about 20% of that of 20% ethanol, and the peak area also decreases significantly when extracted with high-concentration ethanol (80% and above). Therefore, a 20% ethanol aqueous solution is selected as the optimal extraction solvent.

[0030] (2) Selection of extraction method Four portions of Tribulus terrestris leaf powder, 1 g each, were added to a 20% ethanol aqueous solution at a liquid-to-solid ratio of 20:1. Four extraction methods were employed: shaking extraction, ultrasonic extraction, reflux extraction, and cold soaking extraction. The reflux, shaking, and ultrasonic extraction times were all 1.5 h, while the cold soaking extraction time was 24 h. The J2 peak area was then measured using the methods described above.

[0031] The results are as follows Figure 2 As shown, the J2 peak area of ​​the heating reflux extraction method is the highest, significantly higher than other methods. Cold maceration extraction for 24 hours is still less effective than reflux extraction for 1.5 hours. Therefore, the heating reflux extraction method was chosen.

[0032] (3) Selection of material-liquid ratio Take three portions of Tribulus terrestris leaf powder, each 1 g, and add 20% ethanol solution at material-to-liquid ratios of 1:20, 1:25, and 1:30 (g / mL), respectively. Reflux extraction is performed three times (90 min for the first extraction, 60 min for the second, and 30 min for the third). Combine the extracts and measure the J2 peak area after processing.

[0033] The results showed that the J2 peak area was the largest when the solid-liquid ratio was 1:25, and it decreased slightly when the ratio was 1:30. Considering the amount of solvent used and the cost of subsequent concentration, the optimal solid-liquid ratio was determined to be 1:25 (g / mL).

[0034] (4) Selection of extraction times Take 1g of Tribulus terrestris leaf powder, add 20% ethanol at a material-to-liquid ratio of 1:25, and reflux extract four times for extraction times of 1.5h, 1h, 0.5h, and 0.5h, respectively. Measure the J2 peak area after each extraction and calculate the cumulative extraction rate.

[0035] The results showed that the cumulative extraction rate was 80.1% after the first extraction (1.5 h), 96.4% after the second extraction (1 h), and 99.8% after the third extraction (0.5 h). The J2 peak area in the fourth extract was close to 0. Therefore, the number of extractions was determined to be 3, with extraction times of 1.5 h, 1 h, and 0.5 h, respectively.

[0036] 1.3 Optimization of purification process conditions (1) Screening of macroporous adsorption resins Five macroporous adsorption resins (D-101, AB-8, HP-20, DM-301, and D4020) were pretreated by soaking in anhydrous ethanol for 24 hours. Then, 25.0 mL of Tribulus terrestris leaf extract (containing 41.63 µg / mL J2 as determined by HPLC) was added, and the mixture was shaken at room temperature for 24 hours. The J2 content in the supernatant was measured, and the static adsorption capacity and adsorption rate were calculated. The supernatant was then discarded, and 25.0 mL of anhydrous ethanol was added. The mixture was shaken for 24 hours, and the J2 content in the desorption solution was measured. The static desorption capacity and desorption rate were calculated. The results are shown in Table 1.

[0037] Table 1: Static adsorption and desorption performance of J2 by ​​different macroporous adsorption resins.

[0038]

[0039] As shown in Table 1, AB-8 resin exhibits the highest static adsorption capacity for J2 (409.47 µg / g) and an adsorption rate of 98.36%, as well as the highest desorption rate (86.75%), demonstrating the best overall performance. Therefore, AB-8 macroporous adsorption resin was selected as the purification medium for this invention.

[0040] (2) Adsorption kinetic curves Take 2.5 g of AB-8 resin, add 25.0 mL of extraction solution, shake at room temperature, and collect the supernatant at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h to determine the J2 content. The results are as follows: Figure 3 As shown, the J2 concentration no longer decreased after 6 hours of adsorption, indicating that the resin reached adsorption saturation.

[0041] (3) Selection of alkaline elution concentration Five 2.5 mL saturated AB-8 resin columns were eluted with three column volumes (BV) of 0.05%, 0.1%, 0.15%, 0.20%, and 0.25% NaOH solutions, respectively, at a flow rate of 4 BV / h. The concentration of J2 in the eluent was measured. The results showed that the highest J2 concentration (19.86 µg / mL) was observed in the 0.1% NaOH eluent, while the concentration was only 12.35 µg / mL in the 0.05% NaOH eluent, and decreased to 14.27 µg / mL in the 0.25% NaOH eluent. Therefore, 0.1% NaOH was determined to be the optimal alkaline elution concentration.

[0042] (4) Desorption curve of NaOH solution Take the saturated AB-8 resin column, elute with 0.1% NaOH solution, collect the fraction per 1 BV, and determine the J2 content. Results are as follows: Figure 4 As shown, 8 BV of 0.1% NaOH solution can completely elute J2, and further increasing the elution volume yields no additional benefit. Therefore, the optimal elution volume is determined to be 8 BV.

[0043] (5) Selection of ethanol elution concentration A saturated AB-8 resin column was eluted with 3 BV of each of 15%, 30%, 45%, 60%, 75%, and 90% ethanol solutions at a flow rate of 4 BV / h. The concentration of J2 in the eluent was measured. The results showed that the highest J2 concentration (19.85 µg / mL) was found in the 90% ethanol eluent, while the concentration in the 15% ethanol eluent was only 2.35 µg / mL. Therefore, 90% ethanol was determined to be the optimal desorption solvent.

[0044] (6) Desorption curve of ethanol solution Take a saturated AB-8 resin column, elute with 90% ethanol solution, collect the fraction per 1 BV, and determine the J2 content. Results are as follows: Figure 5 As shown, 90% ethanol at 9 BV can completely elute J2. Therefore, the ethanol elution volume is determined to be 9 BV.

[0045] 1.4 Optimal Preparation Method Take 25 kg of Tribulus terrestris leaves, crush them, add 20% (v / v) ethanol aqueous solution (solid-liquid ratio 1:25, g / mL), and extract three times by heating and reflux for 1.5 h, 1 h, and 0.5 h respectively. Combine the extracts. Remove ethanol from the extract under reduced pressure until no alcohol odor remains, and dilute with 20 L of water. Pass the diluted solution through an AB-8 macroporous adsorption resin column (pre-treated with anhydrous ethanol for 24 h) at a loading rate of 4 BV / h, and elute with distilled water until the eluent is colorless. Elute with 0.1% NaOH solution at 8 BV, and collect the alkaline eluent. Neutralize with 10% HCl solution to pH 7.0. Pass the neutralized solution through an AB-8 macroporous adsorption resin column again at a loading rate of 4 BV / h, and elute with distilled water until the eluent is neutral. The solution was eluted with 90% ethanol solution at 9 BV, the ethanol eluent was collected, the solvent was recovered under reduced pressure, and the solution was dried to obtain 120 g of total flavonoids from Tribulus terrestris leaves, with a yield of approximately 0.48% (based on the dry weight of the raw material).

[0046] Example 2 Determination of the content of five flavonoid monomers in total flavonoids from Tribulus terrestris leaves.

[0047] 2.1 Chromatographic conditions Chromatographic column: Agilent SB C-18 (4.6 × 250 mm, 5 µm); mobile phase: methanol-water system, gradient elution program as shown in Table 2; detection wavelength: 330 nm; flow rate: 1.0 mL / min; column temperature: 25℃; injection volume: 20 µL (for determination of J1, J3, J4, and J5) or 10 µL (for determination of J2, due to its higher content). Typical HPLC chromatograms of the mixed standard and the total flavonoids from Tribulus terrestris leaf test solution are shown below. Figure 6 and Figure 7 .

[0048] Table 2: Gradient elution procedure.

[0049]

[0050] 2.2 Preparation of reference solution Accurately weigh 10.00 mg each of reference standards J1, J3, J4, and J5, and 20.00 mg of reference standard J2. Dissolve each in methanol and dilute to 10 mL volumetric flasks to prepare single reference standard stock solutions. Accurately measure appropriate amounts of each stock solution, mix them, and dilute with methanol-water (70:30, V / V) to prepare mixed reference standard solutions of various concentrations for use in standard curve plotting. The concentrations of J1, J3, J4, and J5 in the mixed reference standard solutions are all 1.00 mg / mL, and the concentration of J2 is 2.00 mg / mL.

[0051] 2.3 Preparation of the test solution Accurately weigh 40 mg of total flavonoids from Tribulus terrestris leaves prepared in Example 1, place them in a 5 mL volumetric flask, add methanol-water (70:30, V / V) to dissolve and dilute to the mark, shake well, filter through a 0.22 µm microporous membrane, and take the filtrate as the test solution.

[0052] 2.4 Methodological Examination 2.4.1 Linear Relationship Inject 20 µL of each of the series of mixed reference solutions at various concentrations and plot the standard curve with the injection volume (µg) on ​​the x-axis and the peak area (mAU) on the y-axis. The regression equations, linear ranges, and correlation coefficients for J1–J5 are shown in Table 3, and the standard curve plots are shown below. Figures 8-12 The results showed that the compounds exhibited good linearity within a given range (R0). 2 All are greater than 0.999.

[0053] Table 3: Linear Relationship between J1 and J5

[0054] 2.4.2 Limit of Detection and Limit of Quantification A low-concentration reference solution was taken and serially diluted with methanol-water (70:30, V / V), and then injected for determination. The injection volume with a signal-to-noise ratio (S / N) ≥ 3 was used as the limit of detection (LOD), and the injection volume with an S / N ≥ 10 was used as the limit of quantitation (LOQ). The results are shown in Table 4.

[0055] Table 4: Limits of Detection and Limits of Quantification for J1~J5

[0056] 2.4.3 Precision Test A mixed reference solution (J1 0.0978 mg / mL, J2 0.0985 mg / mL, J3 0.0982 mg / mL, J4 0.0972 mg / mL, J5 0.0979 mg / mL) was injected six times consecutively, and the intra-day precision was calculated. Inter-day precision was calculated after three consecutive days of measurements. The results showed that the intra-day RSDs of the peak areas of J1–J5 were 1.06%, 1.31%, 1.16%, 1.04%, and 1.67%, respectively, and the inter-day RSDs were 0.43%, 0.81%, 0.14%, 1.14%, and 1.38%, respectively, all less than 2%, indicating good instrument precision.

[0057] 2.4.4 Repeatability Test Six samples of total flavonoids from the same batch of Tribulus terrestris leaves, each 40 mg, were taken. Test solutions were prepared according to the method described in section "2.3", and the contents of J1 to J5 were calculated. The average contents of J1 to J5 were 4.80%, 14.86%, 6.05%, 7.01%, and 2.75%, respectively, with RSDs of 2.18%, 0.88%, 2.71%, 1.75%, and 1.78%, respectively, all less than 3%, indicating good repeatability of the method.

[0058] 2.4.5 Stability Test The same test solution was taken and placed at room temperature for 0, 2, 4, 6, 8, and 10 h before being injected and measured. The results showed that the RSD of the retention time of J1 to J5 was less than 0.7%, and the RSD of the peak area was less than 2.5%, indicating that the test solution was stable within 10 h.

[0059] 2.4.6 Recovery Test Six portions of total flavonoids from Tribulus terrestris leaves with known content, each containing 40 mg, were divided into three groups of two portions each. Reference solutions equivalent to 0.8, 1.0, and 1.2 times the content of J1–J5 in the sample were added to each group, respectively. Test solutions were prepared according to the method described in section “2.3”, injected, and the recoveries were calculated. The average recoveries of J1–J5 were 98.5%, 97.8%, 97.6%, 99.1%, and 97.3%, respectively, with RSDs of 1.56%, 1.64%, 2.17%, 1.14%, and 2.52%, indicating good method accuracy.

[0060] 2.5 Content determination results Three batches of total flavonoids from Tribulus terrestris leaves prepared in Example 1 were taken, and the contents of J1 to J5 were determined according to the above method. The average value of the results is shown in Table 5.

[0061] Table 5: Content of J1~J5 in total flavonoids of Tribulus terrestris leaves (%, n=3)

[0062] Example 3 DPPH free radical scavenging activity of total flavonoids and flavonoid monomers from Tribulus terrestris leaves.

[0063] 3.1 Experimental Materials DPPH (2,2-diphenyl-1-picrylhydrazyl, purity ≥97%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (batch number S30629-1g); Vitamin C (VC, purity ≥99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (batch number B21293-100mg); Anhydrous ethanol (analytical grade) was purchased from Beijing Chemical Plant. Total flavonoids from Tribulus terrestris leaves were prepared according to Example 1; monomeric compounds J1, J2, J3, and J4 were isolated and purified from total flavonoids from Tribulus terrestris leaves according to the method in Example 5, and their purities, determined by HPLC area normalization, were 97.8%, 98.5%, 98.2%, and 97.2%, respectively.

[0064] 3.2 Experimental Methods Solution preparation: DPPH ethanol solution: Accurately weigh 2.0 mg of DPPH powder, dissolve it in anhydrous ethanol, and dilute to a final volume of 50 mL in a brown volumetric flask to prepare 0.10 mmol·L⁻¹ DPPH solution. -1 The DPPH ethanol solution should be stored away from light.

[0065] Sample solutions: Accurately weigh 2.0 mg each of total flavonoids from Tribulus terrestris leaves and J1, J2, J3, and J4, dissolve them in 50% ethanol aqueous solution and bring the volume to 100 mL to obtain a stock solution of 20 µg / mL. Then, serially dilute the stock solution to obtain a series of concentration solutions of 18, 14, 10, 5, 3, 2, 0.7, 0.4, 0.21, and 0.07 µg / mL.

[0066] VC standard solution: Accurately weigh 2.0 mg of VC powder, dissolve it in anhydrous ethanol and dilute to 2 mL in a brown volumetric flask to prepare a 1 mg / mL stock solution, and then dilute it to prepare a series of concentration solutions of 50, 25, 15, 10, 7, 4 and 2 µg / mL.

[0067] Scavenging rate determination: Take 1.5 mL of sample solutions of different concentrations into test tubes, add 0.5 mL of DPPH ethanol solution (0.10 mmol·L⁻¹). -1 Mix well and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Each concentration was measured in triplicate, and the average value was taken. Simultaneously, 1.5 mL of 50% ethanol + 0.5 mL of DPPH solution was used as a blank control (A0), and 1.5 mL of sample solution + 0.5 mL of anhydrous ethanol was used as the sample background (A2). Calculate the DPPH free radical scavenging rate using the following formula: Clearance rate (%) = [1 - (A1– A2) / A0] × 100% Where A1 is the absorbance of the sample + DPPH solution, A2 is the absorbance of the sample + anhydrous ethanol, and A0 is the absorbance of the blank control.

[0068] 3.3 Experimental Results The scavenging rate of DPPH radicals by each sample increased with increasing concentration, showing a clear dose-dependent relationship. The scavenging rate curves are shown below. Figure 13 (Sample) and Figure 14 (VC). The half-maximal inhibitory concentration (IC50) was calculated using software. 50 The results are shown in Table 6.

[0069] Table 6: IC50 of each sample against DPPH radicals 50 value

[0070] The results showed that the DPPH free radical scavenging activities of J1 and total flavonoids from Tribulus terrestris leaves were superior to those of the positive control vitamin C, with J1 exhibiting the strongest activity and IC50. 50 The value was only 1.128 µg / mL. The order of activity strength was: J1 > total flavonoids from Tribulus terrestris leaves > VC > J3 > J2 > J4.

[0071] Example 4 ABTS free radical scavenging activity of total flavonoids and flavonoid monomers from Tribulus terrestris leaves.

[0072] 4.1 Experimental Materials The ABTS free radical scavenging ability assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Vitamin C (VC, purity ≥99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and anhydrous methanol (analytical grade) was purchased from Beijing Chemical Plant. The total flavonoids and monomers J1~J4 from Tribulus terrestris leaves were obtained from the same sources as in Example 3.

[0073] 4.2 Experimental Methods Solution preparation: ABTS + Working solution: Dissolve 4.10 mg of ABTS solid powder in 1 mL of distilled water to obtain ABTS stock solution; dissolve 0.70 mg of potassium persulfate in 1 mL of distilled water to obtain potassium persulfate stock solution. Mix equal volumes of the two stock solutions and react at room temperature in the dark for 12 h. Then add anhydrous methanol (stock solution: anhydrous methanol = 1:55, V / V), mix well, and obtain ABTS. + The working solution requires an absorbance of 0.70 ± 0.02 at 734 nm.

[0074] Sample solutions: Accurately weigh 2.0 mg each of total flavonoids from Tribulus terrestris leaves and J1, J2, J3, and J4, dissolve them in 50% ethanol aqueous solution and dilute to 1 mL to obtain a stock solution of 2.0 mg / mL, and then dilute to a series of concentration solutions of 1.8, 1.4, 1.0, 0.8, 0.6, 0.4, 0.1, 0.05, and 0.02 mg / mL.

[0075] VC standard solution: Accurately weigh 3.52 mg of VC, dissolve it in anhydrous ethanol and dilute to 2 mL in a brown volumetric flask to prepare a 1.76 mg / mL stock solution, and then dilute it to prepare a series of concentration solutions of 0.176, 0.070, 0.035, 0.018, 0.009, 0.004 and 0.002 mg / mL.

[0076] Scavenging rate determination: Add the reagents according to Table 7 to a 96-well plate, mix well, react at room temperature in the dark for 6 min, and measure the absorbance at 405 nm. Each concentration was measured in triplicate, and the average value was taken.

[0077] Table 7: Sample loading table for ABTS free radical scavenging experiment.

[0078]

[0079] Calculate the clearance rate using the following formula: Clearance rate (%) = [A blank - (A assay - A control)] / A blank × 100% 4.3 Experimental Results The scavenging rate of ABTS radicals for each sample increased with increasing concentration, as shown in the scavenging rate curves. Figure 15 (Sample) and Figure 16 (VC). IC 50 The values ​​are shown in Table 8.

[0080] Table 8: IC50 of each sample against ABTS radicals 50 value

[0081] The results showed that J1 and total flavonoids from Tribulus terrestris leaves had significant scavenging activity against ABTS free radicals, with J1 having the highest IC50 value. 50 The value was 0.028 mg / mL, and the IC50 of total flavonoids from Tribulus terrestris leaves was [value missing]. 50 The value was 0.111 mg / mL. J2, J3, and J4 showed weak activity in this experimental system, with clearance rates still below 50% at the highest tested concentration of 2.0 mg / mL.

[0082] 4.4 Structure-Property Relationship Analysis The chemical structures of compounds J1, J2, J3, and J4 are as follows: Figure 17As shown. J1 is quercetin-3-O-β-D-gentiobiglycoside, with a 3′,4′-dihydroxy structure on ring B; J2 is kaempferol-3-O-β-D-gentiobiglycoside, with only one 4′-hydroxy group on ring B; J3 is isorhamnetin-3-O-β-D-gentiobiglycoside, with a 3′-methoxy-4′-hydroxy group on ring B; J4 is kaempferol-3-O-β-D-(6″-O-p-coumaryl)glucoside, with a p-coumaryl group attached to the sugar chain.

[0083] IC50 from DPPH and ABTS free radical scavenging experiments 50 The values ​​show that the number of hydroxyl groups on ring B of the compound has a decisive influence on its antioxidant activity: J1 (three hydroxyl groups on ring B) has the strongest activity, J2 and J3 (one hydroxyl group on ring B or hydroxyl groups replaced by methoxy groups) have significantly reduced activity, and J4 (coumaroyl group replaced on the sugar chain) has the weakest activity. This indicates that the ortho-dihydroxyl structure on ring B is the key pharmacophore for enhancing free radical scavenging activity, while acylation modification on the sugar chain weakens the activity. The total flavonoids from Tribulus terrestris leaves prepared in this invention have excellent overall antioxidant activity due to their rich content of highly active flavonoid glycosides such as J1 and J2.

[0084] Example 5: Isolation and structural identification of monomeric compounds from total flavonoids in Tribulus terrestris leaves.

[0085] This embodiment illustrates the method for isolating and purifying the main monomeric compounds in total flavonoids from Tribulus terrestris leaves, as well as the results of structural identification.

[0086] Separation process: 120 g of total flavonoids from Tribulus terrestris leaves prepared in Example 1 were subjected to silica gel column chromatography (eluting agent I: ethyl acetate-ethanol-water = 18:2:1, gradient elution), and the fractions were combined by TLC to obtain 15 fractions A~O.

[0087] Fragments E and F were combined and subjected to ODS column chromatography (eluting solvent III: methanol-water = 1:1, gradient elution) to obtain E1 and E2. E1 was subjected to silica gel column chromatography (eluting solvent IV: ethyl acetate-ethanol-water = 13:1:1, gradient elution) to obtain E1-1 to E1-9, among which E1-7 was identified as compound J2 (kaempferol-3-O-β-D-gentiopicroside), with a mass of 343.2 mg.

[0088] Fraction I was subjected to ODS column chromatography (eluent III: methanol-water = 1:1, gradient elution) to obtain I1~I5. I3 and I4 were combined and subjected to silica gel column chromatography (eluent IV: ethyl acetate-ethanol-water = 13:1:1, gradient elution) to obtain I3-1~I3-6. I3-1 was purified by preparative high performance liquid chromatography (mobile phase V: methanol-water = 37:63, isocratic elution) to obtain compound J1 (quercetin-3-O-β-D-gentiobiglycoside), with a mass of 80.3 mg. I3-5 was purified by preparative high performance liquid chromatography (mobile phase XI: methanol-water = 69:31, isocratic elution) to obtain compound J5 (kaempferol-3-O-β-D-gentiobiglycoside-7-O-β-D-glucopyranoside), with a mass of 12.3 mg.

[0089] Fragment D was subjected to ODS column chromatography (eluent VI: methanol-water = 2:1, gradient elution) to obtain D1~D8. D5 was subjected to silica gel column chromatography (eluent VII: ethyl acetate-ethanol-water = 26:2:3, gradient elution) to obtain D5-1 and D5-2. D5-1 was purified by preparative high performance liquid chromatography (mobile phase VIII: methanol-water = 48:52, isocratic elution) to obtain compounds J4 (kaempferol-3-O-β-D-(6″-p-coumaroyl)glucoside, 51.9 mg) and J6 (kaempferol-3-O-β-D-(6″-O-feruloyl)glucopyranoside, 12.3 mg).

[0090] Structural assessment: Compound J1: Yellow powder, ESI-MS m / z 649.13685 [M+Na] + Molecular formula C 27 H 30 O 17 . 1 H NMR and 13 The C10 NMR data were consistent with the literature reports, and the product was identified as quercetin-3-O-β-D-gentiopicoside.

[0091] Compound J2: Yellow powder, ESI-MS m / z 633.14208 [M+Na] + Molecular formula C 27 H 30 O 16 . 1 H NMR and 13 The C NMR data were consistent with the literature reports, and it was identified as kaempferol-3-O-β-D-gentiopicoside.

[0092] Compound J3: Yellow powder, ESI-MS m / z 663.15269 [M+Na] + Molecular formula C 28 H32 O 17 . 1 H NMR and 13 The C10 NMR data were consistent with the literature reports, and the product was identified as isorhamnetin-3-O-β-D-gentiopicoside.

[0093] Compound J4: Yellow powder, ESI-MS m / z 633.09989 [M+K] + Molecular formula C 30 H 26 O 13 . 1 H NMR and 13 The C NMR data were consistent with the literature reports, and it was identified as kaempferol-3-O-β-D-(6″-p-coumaroyl)glucoside.

[0094] Compound J5: Yellow powder, ESI-MS m / z 795.19475 [M+Na] + Molecular formula C 33 H 40 O 21 . 1 H NMR and 13 The C18 NMR data were consistent with the literature reports, and it was identified as kaempferol-3-O-β-D-gentiobiglycoside-7-O-β-D-glucopyranoside.

[0095] Compound J6: Yellow powder, ESI-MS m / z 623.14260 [M+H] + Molecular formula C 31 H 26 O 14 . 1 H NMR and 13 The C NMR data were consistent with the literature reports, and it was identified as kaempferol-3-O-β-D-(6″-O-feruloyl)glucopyranoside.

[0096] Comparative Example 1 Comparison of adsorption and desorption properties of different macroporous adsorption resins.

[0097] This comparative example was used to verify the superiority of the AB-8 type resin. The experimental method was the same as in Example 1, “(1) Screening of macroporous adsorption resins”, and the results are shown in Table 1. The AB-8 type resin had the highest static adsorption capacity (409.47 µg / g) and desorption rate (86.75%), while DM301 had a relatively high adsorption rate but a significantly lower desorption rate (65.27%), and D4020 had the lowest adsorption capacity. Therefore, the AB-8 type resin is the best choice for this invention.

[0098] Comparative Example 2 The effect of alkaline elution step on the purity of total flavonoids.

[0099] This comparative example demonstrates the necessity of the "alkali elution-neutralization-secondary adsorption" process. A control group was set up: the saturated AB-8 resin column was eluted directly with 90% ethanol without alkali elution (9 BV), the eluent was collected, dried, and the total flavonoid sample A was obtained. The experimental group was prepared according to the method in Example 1 (alkali elution → neutralization → secondary adsorption → 90% ethanol elution) to obtain the total flavonoid sample B. The J2 content in both groups was determined (according to the method in Example 2). The results showed that the J2 content in the control group was 5.28%, while the J2 content in the experimental group was 13.90%, indicating that the alkali elution process increased the purity of the target flavonoid glycosides by approximately 2.6 times. Simultaneously, the product color of the experimental group was light yellow, while that of the control group was dark yellowish-brown, indicating that alkali elution effectively removed impurities such as pigments.

[0100] Comparative Example 3 Comparison of extraction solvents.

[0101] This comparative example was used to verify that 20% ethanol is the optimal extraction solvent. The experimental method was the same as in Example 1, "(1) Selection of extraction solvent", and the results are as follows: Figure 1 As shown, the J2 peak area was the highest (607.8 mAU) when extracted with 20% ethanol, compared to only 125.3 mAU with pure water and only 76.5 mAU with anhydrous ethanol. This indicates that an appropriate concentration of ethanol-water mixed solvent provides the highest extraction efficiency for flavonoid glycosides, while excessively high or low ethanol concentrations are detrimental to extraction.

[0102] Comparative Example 4 Comparison of extraction methods.

[0103] This comparative example is used to verify that the heating reflux method is the optimal extraction method. The experimental method is the same as in Example 1, "(2) Selection of Extraction Method", and the results are as follows: Figure 2 As shown, the J2 peak area was the highest (607.8 mAU) with heating reflux extraction, while it was only 415.8 and 380.2 mAU with ultrasonic and oscillation extraction, respectively, and only 320.5 mAU with cold soaking extraction for 24 h. Therefore, heating reflux extraction is the most efficient and time-saving method.

[0104] Based on the verification results of the above embodiments and comparative examples, the present invention has the following advantages: 1. The validation results of the extraction and purification process are significant. Through system optimization and comparison in Example 1 and Comparative Examples 1-4, the optimal process parameters were determined as follows: 20% ethanol, material-to-liquid ratio 1:25, and reflux extraction three times. Figure 1 , Figure 2AB-8 macroporous adsorption resin was the optimal purification medium (adsorption capacity 409.47 µg / g, desorption rate 86.75%). Comparative Example 2 demonstrated that after using the process of "0.1% NaOH alkaline elution-neutralization-secondary adsorption-90% ethanol elution", the content of the target flavonoid glycoside J2 in the total flavonoids was significantly increased from 5.28% in the conventional method to 13.90%, the purity increased by about 2.6 times, and the product appearance changed from dark yellowish-brown to light yellow, confirming the superiority of this combined process in removing impurities such as saponins and pigments.

[0105] 2. Sufficient validation data for the content determination method. Example 2 established an HPLC-UV method for the simultaneous determination of five flavonoid monomers. Figure 6 , Figure 7 Methodological validation showed that all compounds exhibited good linearity in the range of 0.196–19.700 µg (R0). 2 >0.999, Figures 8-12 The detection limit is as low as 0.0011~0.0037 µg, and the precision, repeatability, and stability RSD are all less than 3%, with a recovery rate of 94%~101%, fully meeting the requirements for quantitative analysis. The sensitivity, accuracy, and repeatability of this method are superior to conventional colorimetric methods or single-component determination methods reported in the literature.

[0106] 3. The experimental data on antioxidant activity are outstanding. Example 3 DPPH free radical scavenging experiment ( Figure 13 , Figure 14 This indicates that the IC50 of monomer J1 and total flavonoids... 50 The concentrations were 1.128 µg / mL and 1.266 µg / mL, respectively, both superior to the positive control vitamin C (1.823 µg / mL). Example 4: ABTS free radical scavenging experiment ( Figure 15 , Figure 16 This indicates that J1's IC 50 The concentration of total flavonoids was 0.028 mg / mL, and the concentration of total flavonoids was 0.111 mg / mL. Structure-activity relationship analysis ( Figure 17 This study confirmed that the number of hydroxyl groups on the B ring is a key factor determining antioxidant activity. J1 (three hydroxyl groups on the B ring) showed the strongest activity, while J2 and J3 (one hydroxyl group or methoxy group on the B ring) showed significantly reduced activity, and J4 (glycosylated) showed the weakest activity. This finding provides a theoretical basis for the structural optimization of natural antioxidants.

[0107] 4. Comprehensive advantages of industrial applicability Tribulus terrestris leaves are widely distributed and inexpensive in my country. The method of this invention is simple to operate and uses conventional equipment (extraction tank, resin column, rotary evaporator). The reagents used are all common industrial solvents (ethanol, alkali, acid), and the ethanol can be recycled. In Example 1, 25 kg of raw material can produce 120 g of total flavonoids, with a yield of about 0.48%, which is suitable for large-scale production.

[0108] In summary, through systematic verification by examples and comparative examples, this invention successfully solves the problems of poor selectivity, low purity, imperfect quality control methods, and unsystematic evaluation of antioxidant activity in the extraction and purification of flavonoids from Tribulus terrestris leaves in the prior art, providing reliable technical support and experimental basis for the in-depth development and utilization of Tribulus terrestris leaf resources.

Claims

1. A method for preparing total flavonoids from Tribulus terrestris leaves, characterized in that, The following steps are performed sequentially: (a) Crushing and extraction: Crushing the Tribulus terrestris leaves, adding an ethanol aqueous solution with a volume fraction of 10% to 30%, and extracting by heating and refluxing 2 to 4 times at a material-to-liquid ratio of 1:20 to 1:30 g / mL, and combining the extracts; (b) Concentration and dilution: The extract obtained in step (a) is subjected to reduced pressure to recover ethanol until no alcohol odor is detected, and then diluted with water to obtain a diluted solution; (c) First macroporous adsorption resin adsorption: The diluted solution obtained in step (b) is passed through an AB-8 type macroporous adsorption resin column at a loading rate of 2 to 6 BV / h to adsorb flavonoid components onto the resin, and then eluted with distilled water until the effluent is colorless. (d) Alkaline elution: Elute the resin column obtained in step (c) with a 0.05% to 0.2% NaOH solution at a volume of 6 to 10 times the column volume, and collect the alkaline eluent; (e) Neutralization: Neutralize the alkaline eluent obtained in step (d) to a pH of 6.5–7.5 with a 5%–15% HCl solution; (f) Secondary macroporous adsorption resin adsorption: The neutralized solution obtained in step (e) is passed through the AB-8 macroporous adsorption resin column again at a loading rate of 2-6 BV / h, and eluted with distilled water until the effluent is neutral. (g) Alcohol elution: Elute the resin column obtained in step (f) with an ethanol solution of 70% to 95% by volume at a volume of 7 to 11 column volumes, and collect the alcohol eluent; (h) Drying: The solvent in the alcohol eluent obtained in step (g) is recovered under reduced pressure and dried to obtain total flavonoids from Tribulus terrestris leaves.

2. The preparation method according to claim 1, characterized in that, The volume fraction of the ethanol-water solution in step (a) is 20%, the material-to-liquid ratio is 1:25 g / mL, the extraction is performed 3 times, and the extraction time for each extraction is 1.5 hours, 1 hour and 0.5 hours respectively.

3. The preparation method according to claim 1, characterized in that, The loading rate in steps (c) and (f) is 4 BV / h, and the AB-8 macroporous adsorption resin is pretreated by soaking in anhydrous ethanol for 24 hours before use.

4. The preparation method according to claim 1, characterized in that, The concentration of the NaOH solution in step (d) is 0.1%, and the elution volume is 8 times the column volume.

5. The preparation method according to claim 1, characterized in that, The concentration of the HCl solution in step (e) is 10%, and the pH value after neutralization is 7.

0.

6. The preparation method according to claim 1, characterized in that, The volume fraction of the ethanol solution in step (g) is 90%, and the elution volume is 9 times the column volume.

7. A method for determining the flavonoid content in total flavonoids from Tribulus terrestris leaves prepared by the method according to any one of claims 1 to 6, characterized in that, The contents of quercetin-3-O-β-D-gentiopicroside, kaempferol-3-O-β-D-gentiopicroside, isorhamnetin-3-O-β-D-gentiopicroside, kaempferol-3-O-β-D-(6″-p-coumaroyl)glucoside and kaempferol-3-O-β-D-gentiopicroside-7-O-β-D-glucopyranoside in total flavonoids from Tribulus terrestris leaves were determined simultaneously using high performance liquid chromatography-ultraviolet detection.

8. The method according to claim 7, characterized in that, The chromatographic conditions for the high-performance liquid chromatography-ultraviolet detection method are as follows: C-18 column, methanol-water mobile phase, gradient elution, detection wavelength of 330 nm, flow rate of 1.0 mL / min, and column temperature of 25 °C. The gradient elution program is as follows: methanol-water volume ratio 12:88 at 0 min, 30:70 at 45 min, 37:63 at 50 min, 43:57 at 57 min, 50:50 at 75 min, 50:50 at 78 min, and 100:0 at 100 min.

9. The use of total flavonoids from Tribulus terrestris leaves prepared by the method according to any one of claims 1 to 6 in the preparation of DPPH free radical scavengers or ABTS cationic free radical scavengers.

10. An antioxidant composition or functional food, characterized in that, It comprises total flavonoids from Tribulus terrestris leaves prepared by the method according to any one of claims 1 to 6, and pharmaceutically or food-grade excipients.

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