High-oxidation-resistance total flavonoids extract of p. sonchifolium and application thereof

Abstract

The application discloses high-antioxidant total flavonoids extract of Pediocactus silvestrii and application thereof. The extract is extracted from Pediocactus silvestrii by supercritical carbon dioxide extraction combined with ethanol entrainer, and the total flavonoids yield is 12.14±0.13 mg / g. The scavenging rate of the extract on DPPH free radicals is 39.38%±2.02% at a concentration of 1 mg / mL, the scavenging rate on ABTS+ free radicals is 52.53%±0.33%, and the scavenging rate on hydroxyl free radicals reaches 91.57%±1.32%, wherein the hydroxyl free radical scavenging capacity is close to that of vitamin C. The extract has the characteristics of green safety, high purity and significant antioxidant activity, and can be used for preparing food, health food, medicine, daily chemical product or animal functional feed additive with antioxidant function. The application clearly defines the core antioxidant index of the total flavonoids extract of Pediocactus silvestrii, and provides a basic raw material with excellent performance for the development of natural antioxidant products.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and bioactivity technology, specifically to a total flavonoid extract of *Sargassum fusiforme* with high antioxidant activity, and the application of the extract in food, health products, pharmaceuticals, daily chemical products and feed additives. Background Technology

[0002] Sand mustard ( Pugionium cornutum (L.) Gaertn *Gynostemma pentaphyllum*, a herbaceous plant belonging to the genus *Gynostemma pentaphyllum* in the Brassicaceae family, is endemic to the sandy areas of Inner Mongolia, Shaanxi, and Ningxia Hui Autonomous Regions, and is known as "desert ginseng." *Gynostemma pentaphyllum* not only possesses various pharmacological effects such as promoting gastrointestinal motility, lowering blood lipids, protecting the liver, and relieving cough and pain, but its rich flavonoids also exhibit excellent free radical scavenging capabilities. As a natural antioxidant, flavonoids have broad application prospects in functional foods, health products, and feed additives.

[0003] Traditional flavonoid extraction methods often employ hot reflux extraction with organic solvents, which suffers from problems such as cumbersome processes, long processing times, low extraction rates, organic solvent residues, and environmental pollution. Supercritical carbon dioxide extraction, as a green extraction technology, offers advantages such as low extraction temperatures, no solvent residue, and one-step extraction and separation, making it particularly suitable for the extraction of heat-sensitive natural products. However, due to the moderate polarity of flavonoids, the extraction efficiency of supercritical carbon dioxide alone is limited, necessitating the addition of polar entrainers (such as ethanol) to improve the extraction rate.

[0004] While existing research reports on supercritical carbon dioxide extraction of flavonoids from *Strombus haematomarginatus* exist, most are preliminary explorations of process conditions and lack systematic characterization of the extract's antioxidant activity and development of product applications. In particular, there is a lack of patents regarding a specific process-derived total flavonoid extract from *Strombus haematomarginatus* with clearly defined and excellent antioxidant activity indicators, as well as patents related to the product's uses. Summary of the Invention

[0005] The purpose of this invention is to provide a high antioxidant total flavonoid extract of *Sargassum fusiforme* and its applications, in order to fill the gaps in the prior art.

[0006] In a first aspect, the present invention provides a high antioxidant total flavonoid extract of *Strombax cuneata*.

[0007] The extract described is a total flavonoid extract obtained from *Strombus haematomarginatus* raw material through a combination of supercritical carbon dioxide extraction and ethanol entrainer. This extract possesses the following key antioxidant activity indicators: The scavenging rate of DPPH free radicals was 39.38% ± 2.02% at a concentration of 1 mg / mL; For ABTS +The free radical scavenging rate was 52.53% ± 0.33% at a concentration of 1 mg / mL; hydroxyl radical (OH) - The clearance rate of ) was 91.57% ± 1.32% at a concentration of 1 mg / mL.

[0008] In a preferred embodiment of the present invention, the total flavonoid yield of the extract is 12.14 ± 0.13 mg / g (based on the dry weight of the raw material), indicating that the extract maintains high extraction efficiency while achieving high activity and has good economic efficiency for mass production.

[0009] In another preferred embodiment of the present invention, the extract is obtained by a preparation method comprising the following steps: washing, air-drying, pulverizing, and passing the aerial parts of *Gnaphalium affine* through a 60-mesh sieve to obtain *Gnaphalium affine* powder; weighing the *Gnaphalium affine* powder and placing it into the extraction vessel of a supercritical carbon dioxide extraction device; setting the extraction temperature to 45.1°C, and introducing carbon dioxide after the temperature stabilizes; pressurizing to 24.8 MPa; using 90% (v / v) ethanol as an entrainer, and a flow rate of 1-2 mL / min, circulating and extracting for 2 hours; collecting the extract to obtain the final product.

[0010] In a second aspect, the present invention provides a composition comprising the above-mentioned high antioxidant total flavonoid extract of *Sargassum fusiforme*.

[0011] The composition may contain excipients or carriers that are acceptable in food science, health food science, pharmaceutical science or feed science, and may be prepared into various dosage forms such as tablets, capsules, granules, oral liquids, powders, solutions or emulsions.

[0012] Thirdly, the present invention provides a series of applications of the above-mentioned high antioxidant total flavonoid extract of *Sargassum fusiforme*.

[0013] Specifically, it includes: Applications in the preparation of foods, health foods, pharmaceuticals, or daily chemical products with antioxidant functions.

[0014] Applications in the preparation of products that scavenge DPPH free radicals, ABTS⁺ free radicals, or hydroxyl free radicals.

[0015] Application in the preparation of functional animal feed additives.

[0016] In the above applications, the extract exerts its free radical scavenging effect through its flavonoid active components. It is particularly noteworthy that its scavenging capacity against hydroxyl radicals (91.57% ± 1.32% at 1 mg / mL) is very close to that of the traditional strong antioxidant vitamin C (94.69% ± 1.17% at 1 mg / mL), demonstrating significant application potential.

[0017] Compared with the prior art, the present invention has the following outstanding advantages: 1. This invention is the first to define the total flavonoid extract of *Strombax ceiba* in three key antioxidant indicators (DPPH, ABTS). + The specific scavenging rate data on hydroxyl radicals, especially in the scavenging of hydroxyl radicals, showed activity close to that of vitamin C, providing clear quality standards and technical support for the functional application of the product.

[0018] 2. The extract of the present invention not only has high antioxidant activity, but also maintains a good yield of 12.14±0.13 mg / g, solving the common problems of "high activity but low yield" or "high yield but low activity", and achieving a balance between activity and yield.

[0019] 3. The extraction process uses supercritical carbon dioxide and food-grade ethanol, leaving no harmful organic solvent residues. The product conforms to the development trend of green, safe and environmentally friendly products, and can be directly applied to food, health food and feed and other fields.

[0020] 4. This invention builds an application system from functional raw materials to end products around the extract product itself, covering multiple industry directions such as food, health food, pharmaceuticals, daily chemicals and feed additives, and has extremely high commercial transformation value and market potential. Attached Figure Description

[0021] Figure 1 Example 1 of this invention: Scavenging ability curve of total flavonoid extract of *Solanum tuberosum* against DPPH free radicals (concentration 0.2-1.0 mg / mL, compared with vitamin C).

[0022] Figure 2 The total flavonoid extract of *Strombax ceiba* prepared in Example 1 of this invention is effective against ABTS. + Free radical scavenging capacity curves (concentrations 0.2-1.0 mg / mL, compared with Trolox).

[0023] Figure 3 The total flavonoid extract of *Strombax ceiba* prepared in Example 1 of this invention is effective against hydroxyl radicals (OH). - The scavenging capacity curve of ) (concentration 0.2-1.0 mg / mL, compared with vitamin C). Detailed Implementation

[0024] The present invention will be further illustrated below through specific embodiments and comparative data. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0025] Materials and Instruments: The *Sargassum fusiforme* was collected from Ordos City, Inner Mongolia Autonomous Region, and identified by the Inner Mongolia Grassland Research Institute as stem and leaf parts of *Sargassum fusiforme*.

[0026] Rutin (purity ≥98%, catalog number: 25090190, TMStandard); DPPH (purity ≥97%, catalog number: D9370, Solarbio); ABTS (purity ≥98%, catalog number: A9590, Solarbio); Salicylic acid, ferrous sulfate heptahydrate, potassium persulfate (purity ≥98%, catalog numbers: S817529, I8560, P816371, MACKLIN); Ascorbic acid (purity ≥99%, catalog number: A8101, Solarbio); Trolox (purity ≥98%, catalog number: 53188-07-1, Jiangxi Baicaoyuan Biotechnology Co., Ltd.); 30% hydrogen peroxide (catalog number: 20261008, Shanghai Lingfeng Chemical Reagent Co., Ltd.); The test water was ultrapure water.

[0027] DFE-650M Supercritical Carbon Dioxide Extraction System (Tianjin Shianjia Biotechnology Co., Ltd.); InfiniteE Plex Full-Function Microplate Reader (Shanghai Diken Experimental Equipment Co., Ltd.); N-1300V-W Rotary Evaporator (Shanghai Ailang Instrument Co., Ltd.); Milli-Q EQ 7008 Ultrapure Water System (Shanghai Merck Chemical Technology Co., Ltd.); ME4002 Precision Electronic Analytical Balance (Shanghai Mettler Toledo Instrument Co., Ltd.).

[0028] Example 1: Preparation of total flavonoid extract of highly antioxidant sand mustard greens 1. Raw material pretreatment: Harvest the above-ground parts of sand mustard, wash and drain them, air dry them in a ventilated place, crush them and pass them through a 60-mesh sieve to obtain sand mustard powder, seal and refrigerate for later use.

[0029] 2. Supercritical CO2 Extraction: A supercritical carbon dioxide extraction device (such as the DFE-650M model) is used. Accurately weigh 50 g of sand mustard powder and place it into the extraction vessel. Start the cold circulation, setting the extraction vessel temperature to 45.1℃ and the separation vessel temperature to 45℃. After the temperature stabilizes, introduce CO2, simultaneously adjusting the back pressure valve to gradually increase the extraction pressure to 24.8 MPa and maintain it stable. Turn on the entrainer pump, using 90% (v / v) ethanol as the entrainer, injecting 100 mL of ethanol at a flow rate of 1-2 mL / min for circulation extraction. The extraction time is 2 hours.

[0030] 3. Product collection: After extraction, turn off the pump and air inlet valve, open the discharge valve at the bottom of the separator, collect the extract, filter it, and obtain the total flavonoid extract of sand mustard.

[0031] Example 2: Determination of total flavonoid yield and antioxidant activity of extract 1. Determination of total flavonoid yield: The sodium nitrite-aluminum nitrate colorimetric method was used, with rutin as the standard (standard curve: y=5.8636x+0.0909, R²=0.9993). The absorbance of the extract obtained in Example 1 was measured at a wavelength of 510 nm. The yield was calculated according to the formula. The results of three parallel experiments were: 12.163 mg / g, 12.050 mg / g, and 12.186 mg / g, with an average value of 12.14±0.13 mg / g.

[0032] 2. DPPH free radical scavenging rate determination: Following the method of Chen et al., extract solutions of Example 1 at different concentration gradients (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) were prepared and reacted with DPPH solution. The absorbance at 517 nm was measured. Vitamin C was used as a positive control. The results showed that at a concentration of 1.0 mg / mL, the scavenging rate reached 39.38% ± 2.02%, and exhibited a clear concentration dependence (see [link to relevant documentation]). Figure 1 ).

[0033] 3. ABTS + Free radical scavenging rate determination: Following the method of Chen et al., ABTS working solution was reacted with the above-mentioned gradient concentration extract solutions, and the absorbance at 734 nm was measured. Trolox was used as a positive control. The results showed that at a concentration of 1.0 mg / mL, the scavenging rate reached 52.53% ± 0.33% (see...). Figure 2 ).

[0034] 4. Hydroxyl radical (OH) - Scavenging rate determination: Following the method of Chen et al., the Fenton reaction system was used, and the absorbance at 510 nm was measured. Vitamin C was used as a positive control. The results showed that at a concentration of 1.0 mg / mL, the scavenging rate reached 91.57% ± 1.32%, which was very close to the 94.69% ± 1.17% of vitamin C (see...). Figure 3 ).

[0035] Comparative Example: Comparison of extract yield and activity under different extraction conditions Total flavonoid extracts of *Strombax ceiba* were prepared in parallel according to the three sets of extraction conditions shown in Table 1, and the total flavonoid yield was determined.

[0036] Table 1 Different comparative extraction conditions

[0037] As can be seen from the comparison, the extract prepared under the optimal conditions of this invention (24.8 MPa, 45.1℃, 2 h, 90% ethanol) has a significantly higher total flavonoid yield than other combinations of conditions, being 1.5 to 3.4 times higher than that of Comparative Examples 1-3. Furthermore, combined with antioxidant activity data (Example 2), this extract, while maintaining the highest yield, also exhibits excellent free radical scavenging ability, demonstrating the superior overall quality of the extract product of this invention.

[0038] Example 3: Composition and Application Examples Health food tablets: Take 100 g of the total flavonoid extract of *Sargassum fusiforme* prepared in Example 1, add 150 g of microcrystalline cellulose, 50 g of starch and 5 g of magnesium stearate, mix evenly, and granulate and compress using conventional wet granulation to make tablets containing 50 mg of total flavonoid extract of *Sargassum fusiforme* per tablet, which can be used for daily antioxidant health care.

[0039] Functional feed additive: Take 100 g of the total flavonoid extract of *Gnaphalium affine* prepared in Example 1, mix it evenly with 900 g of carrier (such as corn cob powder or wheat bran) to make 1 kg of premix. Add it to livestock and poultry diets at a ratio of 0.1%-0.5% to improve the antioxidant capacity of animals and improve meat quality.

[0040] Antioxidant skin care lotion: Take 1 g of the total flavonoid extract of sand mustard prepared in Example 1, add 2 g of cetearyl alcohol, 5 g of glycerin, 0.1 g of hyaluronic acid and deionized water to 100 g, emulsify and homogenize to make a skin care lotion with antioxidant and anti-aging effects.

[0041] In summary, this invention provides a total flavonoid extract of *Strombax ceiba* with clearly defined high antioxidant activity. Its preparation process is green and efficient, the product has excellent performance, a wide range of applications, and good industrial promotion value and commercialization prospects.

Claims

1. A high-antioxidant total flavonoid extract of *Strombax cuneata*, characterized in that, The extract is a total flavonoid extract obtained by supercritical carbon dioxide extraction combined with ethanol entrainer extraction of sand mustard raw material, wherein: (a) The extract showed a DPPH radical scavenging rate of 39.38% ± 2.02% at a concentration of 1 mg / mL; (b) The extract for ABTS + The free radical scavenging rate was 52.53% ± 0.33% at a concentration of 1 mg / mL; (c) The extract has an effect on hydroxyl radicals (OH-). - The clearance rate of ) was 91.57% ± 1.32% at a concentration of 1 mg / mL.

2. The extract according to claim 1, characterized in that, The total flavonoid yield of the extract was 12.14 ± 0.13 mg / g, calculated based on the dry weight of the raw materials.

3. The extract according to claim 1 or 2, characterized in that, The extract is obtained by a preparation method including the following steps: (a) Dry, pulverize, and sieve the raw material of sand mustard to obtain sand mustard powder; (b) Place the sand mustard powder in a supercritical carbon dioxide extraction device and extract it with ethanol as an entrainer; (c) Collect the extract to obtain the extract; The extraction pressure was 24.8 MPa, the extraction temperature was 45.1℃, the extraction time was 2 hours, and the concentration of the ethanol entrainer was 90% (v / v).

4. A composition, characterized in that, It comprises the high antioxidant total flavonoid extract of *Sargassum fusiforme* as described in any one of claims 1-3, and excipients or carriers acceptable in food science, health food science, pharmaceutical science, or feed science.

5. The composition according to claim 4, characterized in that, The dosage form of the composition is selected from one or more of tablets, capsules, granules, oral liquids, powders, solutions, or emulsions.

6. The use of the high antioxidant total flavonoid extract of *Solanum lyratum* according to any one of claims 1-3 in the preparation of food, health food, pharmaceutical or daily chemical products with antioxidant function.

7. The use of the high antioxidant total flavonoid extract of *Solanum lyratum* according to any one of claims 1-3 in the preparation of products that scavenge DPPH free radicals, ABTS⁺ free radicals or hydroxyl free radicals.

8. The use of the high antioxidant total flavonoid extract of *Sinomenium acutum* according to any one of claims 1-3 in the preparation of functional animal feed additives.

9. The application according to any one of claims 6-8, characterized in that, The extract exerts its free radical scavenging effect through its flavonoid active components, and its hydroxyl radical scavenging ability is close to that of vitamin C.

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