Anti-aging herba epimedii extract as well as preparation method and application thereof

Abstract

The invention discloses an anti-aging herba epimedii extract. The anti-aging herba epimedii extract is prepared by performing microbial fermentation on herba epimedii powder. The invention further discloses a preparation method of the anti-aging herba epimedii extract and application of the anti-aging herba epimedii extract in preparation of anti-aging drugs, food and health care products. According to the invention, through special plant lactobacillus and a fermentation process, efficient conversion of main glycoside components in herba epimedii is realized, the contents of icariin and epimedin C are reduced, the contents of secondary glycoside I and secondary glycoside II are remarkably increased, and new icaritin is generated. The herba epimedii extract disclosed by the invention has the effects of resisting aging and prolonging the service life.

Description

Technical Field

[0001] This invention belongs to the field of food and health products, specifically relating to an anti-aging epimedium extract, its preparation method, and its application. Background Technology

[0002] Aging is a complex physiological phenomenon. With increasing age, the morphology and function of the body's organs and tissues decline to varying degrees; it is an inevitable biological process. Aging contributes to a series of degenerative diseases, weakens tissues and impairs cellular function, and promotes proliferative disorders such as cancer. Although aging is unavoidable, research into its mechanisms is of great significance for preventing age-related diseases, improving the quality of life for the elderly, and saving on medical costs.

[0003] Epimedium includes the plant Epimedium (Berberis vulgaris) of the Berberidaceae family. Epimedium brevicornu Maxim., Epimedium sarcodactylis ( Epimedium sagittatum (Sieb. et Zucc.) Maxim.), Epimedium pubescens ( Epimedium pubescens Maxim.) or Korean Epimedium ( Epimedium koreanum Epimedium (such as Nakai) and its extracts primarily contain flavonoids, including icariin and icariin C. Modern pharmacological studies have shown that Epimedium and its extracts possess various biological activities, including antioxidant, anti-inflammatory, immunomodulatory, and bone metabolism-improving effects, demonstrating potential value in the anti-aging field. However, the main active flavonoids in Epimedium exist primarily in glycoside form (such as icariin and icariin C), which have large molecular weights and high polarity, resulting in generally low oral bioavailability and severely limiting their in vivo biological activity and practical application effects.

[0004] Currently, strategies to improve the bioavailability of glycoside-type flavonoids such as icariin mainly include physicochemical methods (such as nanotechnology and phospholipid complexes) and biotransformation methods (such as enzymatic hydrolysis and microbial fermentation). Among these, microbial fermentation has attracted attention due to its advantages such as mild conditions, high transformation specificity, and environmental friendliness. Existing studies have attempted to ferment Epimedium using specific microorganisms (such as certain fungi or bacteria), aiming to hydrolyze glycosides into aglycones or secondary glycosides through enzymes produced by the microorganisms (such as β-glucosidase). However, in existing technologies, the selection of fermentation strains and the optimization of fermentation process parameters (such as temperature, time, and culture medium composition) have significantly different effects on the yield, transformation efficiency, and bioavailability of the target transformation products (such as highly active secondary glycoside II and icariin), and systematic in vitro and in vivo efficacy verification is lacking. Furthermore, screening for efficient, safe, and specific probiotic strains suitable for Epimedium transformation and establishing a stable and controllable fermentation process remain key challenges in enhancing the anti-aging application value of Epimedium.

[0005] Therefore, developing a method for preparing Epimedium extract that can efficiently convert glycoside flavonoids in Epimedium, significantly improve its oral bioavailability, and has a clear anti-aging effect has important application prospects and market value. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a method for preparing an epimedium extract that can efficiently convert glycoside flavonoids in epimedium, significantly improve its oral bioavailability, and has a clear anti-aging effect.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an anti-aging epimedium extract, which is obtained by microbial fermentation of epimedium powder.

[0008] Secondly, the present invention provides a method for preparing an anti-aging epimedium extract, which includes the following steps: S1: Obtain fresh Epimedium, remove impurities and twigs, wash, air dry naturally, and grind or pulverize into Epimedium powder; S2: Add water to the Epimedium powder, stir well, and sterilize at high temperature to obtain the fermentation substrate; S3: Add fermentation broth to the fermentation substrate to carry out microbial fermentation; S4: High-temperature sterilization, filtration, collection of filtrate, freeze-drying or spray drying to obtain Epimedium extract.

[0009] Preferably, in step S2, the ratio of Epimedium powder to water is 1:2 to 2:1, more preferably 1:1.

[0010] Preferably, in steps S2 and S4, the high-temperature sterilization temperature is 121°C and the sterilization time is 15-20 minutes.

[0011] Preferably, in step S3, the mass ratio of fermentation substrate to fermentation broth is 1:1 to 2:1.

[0012] Preferably, in step S3, the fermentation temperature is 30-36℃ and the fermentation time is 24-36 hours.

[0013] Preferably, in step S3, the fermentation broth includes MRS modified culture medium and Lactobacillus plantarum GDMCC No. 64374.

[0014] Preferably, the inoculum size of *Lactobacillus plantarum* GDMCC No. 64374 in the fermentation broth is 1 × 10⁻⁶. 5 -2×10 5 CFU / mL.

[0015] Preferably, the composition of the MRS modified culture medium is as follows: 30 g / L peptone, 24 g / L yeast extract, 60 g / L glucose, 36 g / L calcium carbonate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, with water as the solvent.

[0016] Thirdly, the present invention provides the application of the aforementioned anti-aging Epimedium extract in the preparation of anti-aging drugs, foods, and health products.

[0017] This invention achieves highly efficient conversion of the main glycoside components in Epimedium through a special *Lactobacillus plantarum* and fermentation process. The contents of icariin and icariin C are significantly reduced, while highly active secondary metabolites are generated and enriched in large quantities. For example, secondary glycosides I and II are present from their absence, with a significant increase in the content of secondary glycoside II, and icariin is also newly generated. These secondary metabolites have smaller molecular weights and lower polarity. In vivo pharmacokinetic experiments confirm that the oral bioavailability of the core active component secondary glycoside II in the Epimedium extract prepared by this invention is significantly improved compared to the unfermented Epimedium extract, with a shorter time to peak concentration and faster absorption rate. The Epimedium extract of this invention can be used as a common excipient added to food and health products, and the formulation types can include, but are not limited to, solid beverages, compressed candies, baked goods, and beverages. Attached Figure Description

[0018] Figure 1 This is the result of SA-β-Gal staining of mouse epididymal fat. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0020] Unless otherwise specified, all instruments and reagents used in the examples are conventional instruments or reagents in the art and are commercially available products. Unless otherwise specified, all specific experimental operations involved in the text are understandable or known to those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0021] Lactobacillus plantarum in the embodiments of the present invention ( Lactiplantibacillus plantarum GDMCC No. 64374, selected independently, has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 64374, deposit date of February 5, 2024, and deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0022] The fermentation broth in this embodiment of the invention includes MRS modified medium and *Lactobacillus plantarum* GDMCC No. 64374. The inoculum size of *Lactobacillus plantarum* GDMCC No. 64374 in the fermentation broth is 1 × 10⁻⁶. 5 -2×10 5 CFU / mL.

[0023] The composition of the MRS modified culture medium is as follows: 30 g / L peptone, 24 g / L yeast extract, 60 g / L glucose, 36 g / L calcium carbonate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, with water as the solvent.

[0024] The following uses Korean Epimedium ( Epimedium koreanum The preparation process of Epimedium extract in this embodiment of the invention is illustrated using Nakai as an example. However, it should be noted that the preparation process of this invention is also applicable to other varieties of Epimedium, including but not limited to Epimedium sagittatum (Nakai). Epimedium sagittatum (Sieb. et Zucc.) Maxim.), Epimedium pubescens ( Epimedium pubescens Maxim.) or Epimedium ( Epimedium brevicornu Maximm. ).

[0025] Example 1 The steps for preparing Epimedium extract are as follows: (1) Obtain fresh Korean Epimedium, remove impurities and twigs, wash, air dry naturally, grind or crush and pass through a 60-mesh sieve to obtain Epimedium powder.

[0026] (2) Add water (mass ratio 1:1) to the Epimedium powder, stir evenly, sterilize at 121℃ for 20 minutes to obtain the fermentation substrate.

[0027] (3) Add fermentation broth (mass ratio 1:1) to the fermentation substrate for microbial fermentation at 36℃ for 36 hours. The inoculum size of *Lactobacillus plantarum* GDMCC No. 64374 in the fermentation broth is 1×10⁻⁶. 5 CFU / mL.

[0028] (4) Sterilize at 121°C for 20 minutes, filter, take the filtrate, spray dry to obtain Epimedium extract.

[0029] Example 2 The steps for preparing Epimedium extract are as follows: (1) Obtain fresh Korean Epimedium, remove impurities and twigs, wash, air dry naturally, grind or crush and pass through a 60-mesh sieve to obtain Epimedium powder.

[0030] (2) Add water (mass ratio 1:1) to the Epimedium powder, stir evenly, sterilize at 121℃ for 15 minutes to obtain the fermentation substrate.

[0031] (3) Add fermentation broth (mass ratio 2:1) to the fermentation substrate for microbial fermentation at 30℃ for 24 hours. The inoculum size of *Lactobacillus plantarum* GDMCC No. 64374 in the fermentation broth is 2 × 10⁻⁶. 5 CFU / mL.

[0032] (4) Sterilize at 121°C for 15 minutes, filter, take the filtrate, spray dry to obtain Epimedium extract.

[0033] Example 3 Application of Epimedium Extract in Anti-aging Foods and Health Products (1) Solid beverage (by weight): 30 parts Epimedium extract, 10 parts Polygonatum powder, 10 parts Lycium barbarum powder, 10 parts resistant dextrin, 10 parts fructooligosaccharide, 5 parts inulin, 3 parts arginine, and 2 parts vitamin C.

[0034] Weigh each component, mix them evenly, and obtain an anti-aging Epimedium extract solid beverage.

[0035] (2) Compressed candy (by weight): 30 parts Epimedium extract, 10 parts Polygonatum powder, 10 parts Lycium barbarum powder, 10 parts resistant dextrin, 10 parts fructooligosaccharides, 10 parts microcrystalline cellulose, 5 parts sodium carboxymethyl starch, 5 parts inulin, 3 parts arginine, 2 parts vitamin C, 2 parts magnesium stearate, and 1 part silicon dioxide.

[0036] The above materials are mixed, wet granulated, boiled dry, and compressed into tablets to obtain anti-aging Epimedium extract compressed candies.

[0037] (3) Capsules (by weight): 30 parts Epimedium extract, 10 parts Polygonatum powder, 10 parts Lycium barbarum powder, 10 parts microcrystalline cellulose, 10 parts maltodextrin, 5 parts sodium carboxymethyl starch, 3 parts arginine, 2 parts vitamin C, 2 parts hydroxypropyl methylcellulose, 1 part magnesium stearate, 1 part silicon dioxide.

[0038] Epimedium extract, Polygonatum sibiricum powder, Lycium barbarum powder, microcrystalline cellulose, and maltodextrin are premixed, and 2% hydroxypropyl methylcellulose aqueous solution is added to make a soft mass. The mass is granulated by passing it through a 20-mesh sieve and dried by boiling at 50-60℃. Then, the remaining excipients are mixed and encapsulated to obtain anti-aging Epimedium extract capsules.

[0039] (4) Oral liquid (by weight): 30 parts Epimedium extract, 10 parts Polygonatum powder, 10 parts Lycium barbarum powder, 10 parts microcrystalline cellulose, 10 parts maltodextrin, 5 parts sodium carboxymethyl starch, 3 parts arginine, 2 parts vitamin C, 1 part Tween 80, 1 part steviol glycoside, 0.2 parts potassium sorbate, 0.1 parts glycerin, 0.1 parts citric acid, and water.

[0040] The components are mixed evenly to obtain an oral liquid containing epimedium extract, which has anti-aging properties.

[0041] Component analysis of Epimedium extract Detection method: HPLC-MS / MS Chromatographic column: C18 reversed-phase column (2.1 × 50 mm, 1.7 μm) Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B), gradient elution (0–5 min: 25% B, 5→15 min: 25%→60% B) Detection of ion pairs (ESI+): Glycoside I: m / z 529.2→369.1 M+H + Glycoside II: m / z 515.2→369.1 M+H + The results of component analysis are shown in Table 1 below.

[0042] Comparative sample: Fresh Korean Epimedium was obtained, impurities and residues were removed, it was washed, air-dried naturally, and ground or pulverized through a 60-mesh sieve to obtain Epimedium powder.

[0043] Table 1. Detection results of components in Epimedium extract

[0044] As shown in Table 1, during the fermentation process, icariin was preferentially hydrolyzed into secondary glycoside I (retaining one glucose unit), and its content reached a peak (>1000 μg / g) in the middle of fermentation (12-18 hours), and was partially converted into secondary glycoside II in the later stage.

[0045] Efficacy Evaluation 1. Rat experiment: Bioavailability test (1) Laboratory animals Twelve healthy SD rats were acclimatized for one week at a room temperature of 23-26℃ and a relative humidity of 50-70%, with a 12-hour light / dark cycle and free access to food and water. They were randomly divided into two groups of six rats each: a control group and an experimental group. After a 12-hour fast, the rats were administered the drugs. The control group received 1 g / kg of the comparative example Epimedium powder via gavage, while the experimental group received 1 g / kg of Epimedium extract from Examples 1-2 via gavage. Plasma samples were collected before administration (0 h) and at 0.5, 1, 2, 4, 6, 7, 12, and 24 h after administration. Proteins were precipitated with acetonitrile, and the concentrations of hypoglycine II / I were determined by HPLC-MS / MS.

[0046] (2) Detection method: Chromatographic column: C18 reversed-phase column (2.1 × 50 mm, 1.7 μm) Mobile phase: A 0.1 formic acid water : B acetonitrile (0-5min 25% B, 5-8min 35%-60% B, 8-10min 60%-90% B, 10-12min 90% B, 12-15min 90%-25% B) Mass spectrometry: ESI positive ion mode, detection ion pair m / z 513→369 (glucosinolate II), detection ion pair m / z 529→369 (glucosinolate I) (3) Data analysis: Pharmacokinetic parameter calculation (DAS 3.0 software): AUC 0-24 (Area under the plasma concentration-time curve), Cmax (peak concentration), Tmax (time to peak), t 1 / 2 (half life) The formula for calculating relative bioavailability (Fr) is as follows:

[0047] The pharmacokinetic parameters of glucosinolate II in rats after oral administration of equal amounts of the extract are shown in Table 2.

[0048] Table 2. Pharmacokinetic parameters of glycoside II in rats after oral administration of equal amounts of the extract.

[0049] As shown in Table 2, when the same dose (1 g / kg) of extract was administered orally, the absolute exposure (AUC) of glucosinolate II in the experimental group was... 0-24 Compared with the control group, the peak concentration (Cmax) was significantly increased, and the time to peak concentration (Tmax) was significantly shortened. This fully demonstrates that the fermentation process greatly improves the bioavailability of glucosinolate II per unit weight of extract.

[0050] The pharmacokinetic parameters of glucosinolate I in rats after oral administration of equal amounts of the extract are shown in Table 3.

[0051] Table 3. Pharmacokinetic parameters of glycoside I in rats after oral administration of equal amounts of the extract.

[0052] As shown in Table 3, when the same dose (1 g / kg) of the extract was orally ingested, glucosinolate I was almost undetectable in the control group, while glucosinolate I showed good oral absorption characteristics in the experimental group, and its absolute exposure (AUC) was significantly higher. 0-24 The concentration reached 950 ng·h / mL, the peak concentration (Cmax) reached 175 ng / mL, and the time to peak (Tmax) was 1.5 hours. This directly proves that the fermentation process successfully converted the precursor into glucosinolate I, making it an active ingredient that can be effectively absorbed and utilized after oral administration.

[0053] 2. Fruit fly experiment: lifespan experiment Three-day-old female adult fruit flies were divided into eight groups of 50 each and housed in culture media containing different concentrations (0, 1, 10, and 100 μM) of Epimedium extract from Examples 1-2. They were maintained under standard temperature and humidity conditions. Every two days, the flies were transferred to bottles containing fresh culture media, and any deaths were recorded.

[0054] The experimental results are shown in Table 4.

[0055] Table 4. Results of the fruit fly lifespan experiment

[0056] Table 4 shows that different concentrations of Epimedium extract had varying effects on the lifespan of female fruit flies. However, compared to the control group, all groups supplemented with Epimedium extract significantly prolonged the lifespan of the fruit flies, with the 10 μM group showing the most significant effect. No negative effects were observed in any of the different concentration groups, indicating that Epimedium extract has no toxic effect on fruit flies.

[0057] 3. Mouse experiments: Anti-aging experiments Forty C57BL / 6 mice, aged 16 weeks, were acclimatized for one week at a room temperature of 23-26℃ and a relative humidity of 50-70%, with a 12-hour light / dark cycle and free access to food and water. They were randomly divided into four groups of ten mice each: a control group, an doxorubicin-induced aging model group (DOXO), a low-dose intervention group (dxorubicin-induced aging model + low-dose Epimedium extract from Example 1 (DOXO + Example 1 (L)), doxorubicin-induced aging model + low-dose Epimedium extract from Example 2 (DOXO + Example 2 (L))), and a high-dose intervention group (dxorubicin-induced aging model + high-dose Epimedium extract from Example 1 (DOXO + Example 1 (H)), doxorubicin-induced aging model + high-dose Epimedium extract from Example 2 (DOXO + Example 2 (H))).

[0058] Animal model establishment: Mice were given a first intraperitoneal injection of doxorubicin (10 mg / kg) at week 16, followed by a second intraperitoneal injection of doxorubicin (10 mg / kg) 10 days later. Subsequently, the experimental group was administered Epimedium extract (low dose 20 mg / kg, high dose 80 mg / kg) by gavage every 2 days for 30 consecutive days.

[0059] After the intervention period, the patient was fasted and deprived of water for 12 hours. The cervical dislocation was treated, and epididymal fat was harvested via dissection. SA-β-Gal staining was then performed according to the kit instructions. The epididymal fat was fixed by immersion in galactosidase fixative and incubated at 37°C in the dark for 12 hours. The tissue block was rinsed three times with PBS for 10 minutes each time. The staining results were observed.

[0060] Senescence-associated β-galactosidase (SA-β-Gal) staining uses X-Gal as a substrate and produces a deep blue product under the catalysis of senescence-specific β-galactosidase. It is a commonly used method for detecting cellular senescence, especially in adipose tissue studies to identify senescent cells.

[0061] Observation under an optical microscope revealed that, for example Figure 1 As shown, compared with the blank group, the epididymal adipose tissue of mice in the aging model group DOXO group showed obvious blue staining deposition. After intervention with low-dose and high-dose Epimedium extract, the SA-β-Gal staining intensity of the epididymal adipose tissue of aging mice was significantly reduced, indicating that the Epimedium extracts in Examples 1-2 of this invention all have the effect of regulating cell senescence.

[0062] The results of the anti-aging experiment on mice with Epimedium extract in Example 2 were similar to those of Epimedium extract in Example 1.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An anti-aging epimedium extract, characterized in that, The anti-aging epimedium extract is obtained by microbial fermentation of epimedium powder.

2. A method for preparing the anti-aging Epimedium extract as described in claim 1, characterized in that, Includes the following steps: S1: Obtain fresh Epimedium, remove impurities and twigs, wash, air dry naturally, and grind or pulverize into Epimedium powder; S2: Add water to the Epimedium powder, stir well, and sterilize at high temperature to obtain the fermentation substrate; S3: Add fermentation broth to the fermentation substrate to carry out microbial fermentation; S4: High-temperature sterilization, filtration, collection of filtrate, freeze-drying or spray drying to obtain Epimedium extract.

3. The method according to claim 2, characterized in that, In step S2, the ratio of Epimedium powder to water is 1:

1.

4. The method according to claim 2, characterized in that, In steps S2 and S4, the high-temperature sterilization temperature is 121°C, and the sterilization time is 15-20 minutes.

5. The method according to claim 2, characterized in that, In step S3, the mass ratio of fermentation substrate to fermentation broth is 1:1 to 2:

1.

6. The method according to claim 2, characterized in that, In step S3, the fermentation temperature is 30-36℃ and the fermentation time is 24-36 hours.

7. The method according to claim 2, characterized in that, In step S3, the fermentation broth includes MRS modified medium and Lactobacillus plantarum GDMCC No. 64374.

8. The method according to claim 2, characterized in that, The inoculum size of *Lactobacillus plantarum* GDMCC No. 64374 in the fermentation broth was 1 × 10⁻⁶. 5 -2×10 5 CFU / mL.

9. The method according to claim 2, characterized in that, The composition of the MRS modified culture medium is as follows: 30 g / L peptone, 24 g / L yeast extract, 60 g / L glucose, 36 g / L calcium carbonate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, with water as the solvent.

10. The application of the anti-aging Epimedium extract as described in claim 1 in the preparation of anti-aging drugs, foods, and health products.

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