Cistanche tubulosa fermentation liquor for improving energy and cell aging and preparation method thereof

Abstract

The application provides a cistanche fermention liquid for improving energy and cell aging and a preparation method, and belongs to the technical field of medical preparations. The method comprises the following steps: (1) mixing cistanche with deionized water, performing hot water extraction, and obtaining cistanche water extract; (2) performing homogenization treatment on the cistanche water extract, and obtaining a cistanche mixture; (3) adding cellulase and pectinase to the cistanche mixture for enzymolysis, and obtaining a cistanche enzymolysis liquid; (4) adding a carbon source to the cistanche enzymolysis liquid; (5) sterilizing the cistanche enzymolysis liquid after the carbon source is added, and then cooling to a fermentation temperature; (6) inoculating lactic acid bacteria strains in the cooled cistanche enzymolysis liquid for fermentation, and obtaining a cistanche fermentation liquid; (7) performing solid-liquid separation on the cistanche fermentation liquid, and obtaining a cistanche fermentation supernatant; and (8) sterilizing the cistanche fermentation supernatant, and obtaining the cistanche fermentation liquid.

Description

Technical Field

[0001] This invention relates to a Cistanche deserticola fermentation broth that enhances energy and improves cell aging, and its preparation method, belonging to the field of medical preparation technology. Background Technology

[0002] With the fast pace of modern life and increasing environmental pressure, male reproductive health issues are becoming increasingly prominent. Decreased testosterone levels have become a significant factor affecting male fertility, sexual function, and overall health. Cistanche deserticola, a traditional Chinese medicine for tonifying the kidneys and strengthening yang, has been proven to improve reproductive function, provide antioxidant effects, and combat fatigue. Its main active ingredients include phenylethyl glycosides (such as echinacoside and verbascoside), polysaccharides, and iridoid compounds.

[0003] However, traditional water or alcohol extraction processes for Cistanche deserticola extract suffer from low component dissolution rates and insufficient bioavailability, limiting their practical application. In recent years, microbial fermentation technology has been widely used in the extraction of traditional Chinese medicine. Enzymatic transformation can significantly increase the release of active ingredients and generate new metabolites, thereby enhancing pharmacological activity. Therefore, utilizing microbial fermentation technology to improve the extraction rate and bioactivity of echinacoside and verbascoside from Cistanche deserticola has become a research hotspot in the field of deep processing of Cistanche deserticola.

[0004] However, existing Cistanche fermentation technology still has some problems, such as the selection of fermentation strains not being optimized enough and the fermentation conditions not being simple enough. These will affect the quality and biological activity of Cistanche fermentation liquid. Summary of the Invention

[0005] This invention provides a Cistanche deserticola fermentation broth that enhances energy and improves cell aging, as well as its preparation method, which can effectively solve the above-mentioned problems.

[0006] This invention provides a method for preparing Cistanche deserticola fermentation broth that enhances energy and improves cell aging, comprising the following steps:

[0007] (1) Extraction: Mix Cistanche deserticola with deionized water and extract with hot water to obtain Cistanche deserticola aqueous extract.

[0008] (2) Homogenization: The water extract of Cistanche deserticola is homogenized to obtain a mixture of Cistanche deserticola and water extract.

[0009] (3) Enzymatic hydrolysis: Cellulase and pectinase are added to the Cistanche deserticola mixture for enzymatic hydrolysis to obtain Cistanche deserticola hydrolysate.

[0010] (4) Add carbon source: Add carbon source to the Cistanche deserticola enzymatic hydrolysate.

[0011] (5) Sterilization before fermentation: Sterilize the Cistanche hydrolysate after adding carbon source, and then cool it to the fermentation temperature.

[0012] (6) Fermentation: Lactic acid bacteria are inoculated into the cooled Cistanche hydrolysate to ferment and obtain Cistanche fermentation broth; the lactic acid bacteria are Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68.

[0013] The *Lactobacillus paracasei* B20, Latin scientific name: Lacticaseibacillus paracasei The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 32434. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 31, 2024.

[0014] Lactobacillus plantarum MPs-68, Latin scientific name: Lactiplantibacillus plantarum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 31976. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: September 14, 2024.

[0015] (7) Solid-liquid separation: The fermentation liquid of Cistanche deserticola is subjected to solid-liquid separation to obtain the fermentation supernatant of Cistanche deserticola.

[0016] (8) Sterilization after fermentation: Sterilize the supernatant of the fermentation of Cistanche deserticola to obtain the fermentation liquid of Cistanche deserticola.

[0017] In some embodiments, the lactic acid bacteria strain is obtained by mixing Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 0.8 to 1.1:1 to 1.2.

[0018] In some embodiments, the lactic acid bacteria strain is obtained by mixing Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 at a mass ratio of 0.9:1.2.

[0019] In some embodiments, in step (6), the amount of lactic acid bacteria added is 0.005–0.015 wt% of the Cistanche deserticola enzymatic hydrolysate system; the viable count of the Lactobacillus paracasei B20 bacterial powder is 1–5 × 10⁻⁶. 12 CFU / g; the viable count of the *Lactobacillus plantarum* MPs-68 powder is 1–5 × 10⁻⁶. 11 CFU / g; the fermentation conditions are 36-38℃ for 15-20 h.

[0020] In some embodiments, in step (1), the mass ratio of Cistanche deserticola to deionized water is 1:15-25; the hot water extraction conditions are water extraction at 90-100℃ for 60-80 min.

[0021] In some embodiments, in step (3), 0.5-1% of the mass of the Cistanche deserticola mixture is added, with cellulase having an activity of 20,000 u / g and pectinase having an activity of 30,000 u / g; the mass ratio of the cellulase to the pectinase is 1:0.8-1.2.

[0022] In some embodiments, the enzymatic hydrolysis is performed at 45–55°C for 100–140 min.

[0023] In some embodiments, the carbon source in step (4) is any one of white sugar, fructooligosaccharide, isomaltooligosaccharide, and inulin, and its addition amount is 2-5 wt% of the Cistanche deserticola enzymatic hydrolysate system.

[0024] This invention provides a Cistanche deserticola fermentation liquid that enhances energy and improves cell aging, prepared by the method described above.

[0025] This invention provides a functional product containing the aforementioned Cistanche deserticola fermentation liquid. The functional product is a pharmaceutical product and has at least one of the following functions: boosting energy, enhancing immunity, anti-oxidation, and increasing testosterone levels.

[0026] The beneficial effects of this invention are:

[0027] This invention utilizes a specific mixture of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 in a designated ratio for fermentation, significantly increasing the content of echinacoside and verbascoside in the fermentation broth of *Cistanche deserticola*. Experiments show that the echinacoside content can reach over 68.42 mg / 100mL, and the verbascoside content can reach over 8.09 mg / 100mL.

[0028] The Cistanche deserticola fermentation broth prepared in this invention exhibits significant immunomodulatory activity. Experimental data show that this fermentation broth can effectively increase the secretion of NO and TNF-α and the phagocytic rate of neutral red in RAW264.7 cells, which are important indicators of immune function activation.

[0029] The fermentation broth of Cistanche deserticola prepared by this invention has strong antioxidant capacity. In vitro antioxidant experiments showed that it has a strong scavenging ability against DPPH, ABTS, and hydroxyl radicals. Cell experiments demonstrated that this fermentation broth can reduce the ROS (reactive oxygen species) level in D-gal-induced TM3 testicular interstitial cells and increase the activity of the antioxidant enzyme GSH-Px, thereby protecting cells from oxidative damage.

[0030] The Cistanche deserticola fermentation broth prepared in this invention can significantly enhance testosterone synthesis and release in TM3 cells in a D-gal injury model. Its mechanism of action is related to the upregulation of gene expression of key enzymes in testosterone synthesis, such as StAR, 3β-HSD, 17β-HSD, and CYP17a1.

[0031] This invention utilizes conventional steps such as extraction, homogenization, enzymatic hydrolysis, fermentation, and filtration. The process is simple, highly operable, and requires minimal equipment. The entire process does not use organic solvents, complies with environmental and safety standards, and has promising market application prospects. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 The content of echinacoside and verbascoside in the Cistanche deserticola extract prepared in the embodiments and comparative examples of this invention was determined.

[0034] Figure 2 To assess the antioxidant activity of Cistanche deserticola extract under different processing methods;

[0035] Figure 3 The effects of Cistanche deserticola extract on NO and TNF-α secretion and neutral red phagocytosis rate in RAW264.7 cells were investigated in the examples and comparative examples.

[0036] Figure 4 The effects of Cistanche deserticola extract on D-gal-induced intracellular ROS and GSH-PX activities in TM3 cells were studied in the examples and comparative examples.

[0037] Figure 5 The testosterone concentration and synthase-related gene expression levels of Cistanche deserticola extract were measured in the examples and comparative examples. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0039] Example 1

[0040] (1) Weigh 100 g of Cistanche deserticola strips (Cistanche deserticola, Hotan City, Xinjiang, echinacoside 980 mg / 100g, verbascoside 95 mg / 100g) and add them to 2000 mL of deionized water at 95 ℃. The mass ratio of Cistanche deserticola to deionized water is 1:20. Stir and extract for 1 h, then soak in hot water to prepare Cistanche deserticola water extract.

[0041] (2) Add the water extract of Cistanche deserticola to a colloid mill and grind for 5 minutes to effectively crush the Cistanche deserticola and mix it evenly with the liquid to obtain a Cistanche deserticola mixture.

[0042] (3) When the Cistanche deserticola mixture cools down to 50±2 ℃, add 0.5% of cellulase and pectinase by mass of the mixture respectively. After 2 h of enzymatic hydrolysis, the Cistanche deserticola hydrolysate is obtained. The cellulase activity is 20000 u / g and the pectinase activity is 30000 u / g.

[0043] (4) Weigh 4% of the oligofructose in the liquid system and add it to the Cistanche deserticola hydrolysate. Then boil the Cistanche deserticola hydrolysate and keep it for 20 minutes to sterilize it.

[0044] (5) After the Cistanche deserticola enzymatic hydrolysate has cooled to 37±2 ℃, weigh out 0.01% of Lactobacillus paracasei B20 bacterial powder and inoculate it into the cooled Cistanche deserticola enzymatic hydrolysate. The viable count of Lactobacillus paracasei B20 bacterial powder is 1~5×10⁻⁶. 12 CFU / g was fermented at 36–38 °C for 18 h to obtain Cistanche deserticola fermentation broth.

[0045] (6) The fermentation liquid of Cistanche deserticola after fermentation is completed is separated into solid and liquid. First, it is coarsely filtered using a 300-mesh filter cloth. Then, the coarsely filtered fermentation liquid is centrifuged at 5000 r / min for 5 min. The supernatant of Cistanche deserticola fermentation after centrifugation is collected.

[0046] (7) The collected Cistanche fermentation supernatant was sterilized at 105 °C for 25 min to obtain sterilized Cistanche fermentation liquid.

[0047] Example 2

[0048] The preparation method in this embodiment is basically the same as that in Example 1, except that the fermentation strain used is an equal amount of *Lactobacillus plantarum* MPs-68 bacterial powder (live count 1-5 × 10⁻⁶). 11 Replace Lactobacillus paracasei B20 with CFU / g.

[0049] Example 3

[0050] The preparation method of this embodiment is basically the same as that of Example 1, except that the fermentation strain uses an equal amount of mixed strain 1, which is a mixture of Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 1:1.

[0051] Example 4

[0052] The preparation method of this embodiment is basically the same as that of Example 1, except that the fermentation strain uses an equal amount of mixed strain 2, which is a mixture of Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 0.9:1.2.

[0053] Example 5

[0054] The preparation method of this embodiment is basically the same as that of Example 1, except that the fermentation strain uses an equal amount of mixed strain 3, which is a mixture of Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 1.1:1.

[0055] Example 6

[0056] The preparation method of this embodiment is basically the same as that of Example 1, except that the fermentation strain uses an equal amount of mixed strain 4, which is a mixture of Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 0.8:1.1.

[0057] Example 7

[0058] The preparation method in this embodiment is basically the same as that in Example 4, except that granulated sugar is used to replace fructooligosaccharides in equal amounts. Equal amounts of mixed bacterial strain 2 are used, which is a mixture of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 at a mass ratio of 0.9:1.2.

[0059] Example 8

[0060] The preparation method in this embodiment is basically the same as that in Example 4, except that isomaltooligosaccharide is used to replace fructooligosaccharide in equal amounts. Equal amounts of mixed bacterial strain 2 are used, which is a mixture of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 at a mass ratio of 0.9:1.2.

[0061] Example 9

[0062] The preparation method in this embodiment is basically the same as that in Example 4, except that inulin is used to replace fructooligosaccharides in an equal amount. An equal amount of mixed bacterial strain 2 is used, which is a mixture of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 at a mass ratio of 0.9:1.2.

[0063] Comparative Example 1: Unfermented and unenzymatically hydrolyzed

[0064] 100 g of Cistanche deserticola strips were weighed and extracted with hot water and homogenized. Then, sterilization, cooling, solid-liquid separation, and final sterilization were performed directly, following the same steps as in Example 1. Enzymatic hydrolysis and fermentation were not performed.

[0065] Comparative Example 2: Unfermented but enzymatically hydrolyzed

[0066] 100 g of Cistanche deserticola strips were weighed and subjected to hot water extraction, homogenization, and enzymatic hydrolysis. After enzymatic hydrolysis, the enzymes were inactivated by heating. Then, solid-liquid separation and final sterilization were performed, following the same steps as in Example 1. Fermentation was not performed.

[0067] Comparative Example 3

[0068] The preparation method of this comparative example is basically the same as that of Example 1, except that the fermentation strain uses an equal amount of commercially available Lactobacillus paracasei F-19 to replace Lactobacillus paracasei B20.

[0069] Comparative Example 4

[0070] The preparation method of this comparative example is basically the same as that of Example 1, except that commercially available Lp90 Lactobacillus plantarum is used to replace Lactobacillus plantarum MPs-68 in an equal amount.

[0071] Comparative Example 5

[0072] The preparation method of this comparative example is basically the same as that of Example 4, except that commercially available Lactobacillus paracasei B20 is replaced with an equal amount of commercially available Lactobacillus paracasei F-19.

[0073] Comparative Example 6

[0074] The preparation method of this comparative example is basically the same as that of Example 4, except that commercially available Lp90 Lactobacillus plantarum is used to replace Lactobacillus plantarum MPs-68 in an equal amount.

[0075] Performance Testing and Results Analysis

[0076] Result Example 1: Determination of echinacoside and verbascoside content

[0077] The contents of echinacoside and verbascoside in Cistanche deserticola extract were determined according to the detection methods specified in Part I of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 1.

[0078] Table 1. Content of echinacoside and verbascoside in the Examples and Comparative Examples

[0079]

[0080] Note: Compared with Comparative Example 1, *p<0.05; compared with Example 4, #p<0.05, n=3.

[0081] As shown in Table 1, after fermentation, the contents of echinacoside and verbascoside in the embodiments of the present invention are much higher than those in Comparative Examples 1 and 2 without fermentation; among them, compared with Comparative Example 1, the echinacoside content in Example 4 increased by 38.08%, and the verbascoside content increased by 33.94%. This indicates that the preparation method of the present invention can effectively increase the contents of echinacoside and verbascoside in Cistanche deserticola extract.

[0082] Comparative Examples 3 and 4 used commercially available *Lactobacillus paracasei* F-19 and *Lactobacillus plantarum* Lp90 for fermentation, respectively. In contrast, the echinacoside and verbascoside contents of the *Cistanche deserticola* fermentation broth in this embodiment were significantly higher than those in Comparative Examples 3 and 4. The dual-strain co-fermentation optimized the fermentation microenvironment through metabolic complementarity. B20 exhibits strong glycoside hydrolase activity, specifically cleaving the glycosyl bonds of phenylethyl glycosides, while MPs-68 may lower the system pH by producing organic acids (such as lactic acid and acetic acid), promoting the dissolution and stabilization of target components. Simultaneously, its extracellular enzyme system can further degrade cell wall polysaccharides and protein impurities, reducing substrate inhibition effects, and collectively promoting the enrichment of echinacoside and verbascoside. This demonstrates that the strains selected in this invention have a significant effect on increasing the echinacoside and verbascoside content in the *Cistanche deserticola* broth.

[0083] The effect of strain ratio:

[0084] Comparing Examples 3-6 with Examples 1 and 2, it can be seen that when two strains are combined in different proportions and fermented with Cistanche deserticola, the contents of echinacoside and verbascoside in the fermentation broth are higher than those of single-strain fermentation.

[0085] Comparing Examples 3 to 6, it is evident that Example 4, which used a mixed strain 2 (a mixture of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 at a mass ratio of 0.9:1.2) to ferment the *Cistanche deserticola* enzymatic hydrolysate, yielded the highest content of echinacoside and verbascoside in the fermentation broth. This is because the mixed strains at this ratio can form an optimal synergistic effect during fermentation. The dual-strain synergistic fermentation can activate the biotransformation pathways of precursor substances in the *Cistanche deserticola* matrix. Through cross-talk between signaling molecules between strains, it regulates secondary metabolic processes, potentially generating new active derivatives or improving the bioavailability of existing components. This achieves optimization of the synergistic effect of the strains, optimization of the fermentation environment, and more full utilization of nutrients, all working together to promote the release and transformation of effective components such as echinacoside and verbascoside compared to other mixed strain ratios.

[0086] Comparing Example 4 with Comparative Examples 5 and 6, different mixed bacterial strains were used for fermentation. In Example 4, the fermentation broth of *Cistanche deserticola* obtained using a mixed strain of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68 showed significantly higher contents of echinacoside and verbascoside than Comparative Example 5 (using a mixed strain of *Lactobacillus paracasei* F-19 and *Lactobacillus plantarum* MPs-68) and Comparative Example 6 (using a mixed strain of *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* Lp90). This indicates a specific synergistic effect between *Lactobacillus paracasei* B20 and *Lactobacillus plantarum* MPs-68, which allows them to mutually promote each other during mixed fermentation, thereby increasing the contents of echinacoside and verbascoside. Furthermore, compared to Comparative Examples 5 and 6, B20, MPs-68 may have a less synergistic promoting effect with F-19 and Lp90, thus reducing nutrient absorption and metabolite conversion, resulting in a smaller increase in the contents of echinacoside and verbascoside.

[0087] The effects of different carbon sources:

[0088] As shown in Examples 4 and 7-9, under the same carbon source addition, the 4% addition of fructooligosaccharides in Example 4 significantly increased the content of echinacoside and verbascoside in the Cistanche deserticola fermentation broth. Compared with white sugar, isomaltooligosaccharides, and inulin, the 4% addition of fructooligosaccharides can achieve a better microbial community balance, optimize the fermentation microbial community structure, inhibit competition from other microorganisms, and thus improve the decomposition efficiency of cellulase and pectinase on the Cistanche deserticola cell wall, thereby being more conducive to the synthesis of effective components such as echinacoside.

[0089] Therefore, it can be shown that the strains selected in this invention have a significant effect on increasing the release rate of echinacoside and verbascoside in Cistanche deserticola extract.

[0090] Result Example 2: Antioxidant activity of Cistanche deserticola extract prepared in the examples and comparative examples

[0091] Take 20 ml of the Cistanche deserticola extract from Examples 1, 2, 4 and the comparative example as a sample solution, and test its hydroxyl radical scavenging rate, DPPH radical scavenging rate and ABTS radical scavenging rate. See Table 2 for specific data.

[0092] The detection methods for DPPH and ABTS free radical scavenging rates of Cistanche deserticola extract were based on the DPPH and ABTS methods for antioxidant determination in GB / T39100-2020, and the detection method for hydroxyl free radical scavenging rate was based on the study of antioxidant activity of Panax notoginseng polysaccharides.

[0093] Table 2. Antioxidant activity of Cistanche deserticola extract under different processing methods

[0094]

[0095] Note: Compared with Comparative Example 1, *p<0.05; compared with Example 4, #p<0.05, n=3.

[0096] As shown in Table 2, in Example 4, the fermentation broth of Cistanche deserticola fermented with mixed strain 2 (Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 mixed at a mass ratio of 0.9:1.2) had a DPPH free radical scavenging rate that was 49.32% higher than that of the unfermented Cistanche deserticola water extract in Comparative Example 1, an ABTS free radical scavenging rate that was 5.14% higher than that of the unfermented Example 1, and a hydroxyl free radical scavenging rate that was 20.3% higher than that of the unfermented Example 1.

[0097] Compared with Comparative Examples 1 and 2, the antioxidant activity of Cistanche deserticola in Examples 1, 2, and 4 of the present invention and Comparative Examples 3 to 6 was improved after fermentation, indicating that Cistanche deserticola has stable antioxidant properties in various environments after fermentation, which can effectively prevent the immune system from being damaged by excessive free radicals and alleviate cell aging.

[0098] Comparing Examples 1 and 2 with Comparative Examples 3 and 4, it can be found that, only under the condition of different selected bacterial strains, the free radical scavenging rate of the Cistanche deserticola fermentation broth in Examples 1 and 2 is higher than that in Comparative Examples 3 and 4. Combined with Comparative Examples 3 and 4, it can be seen that the bacterial strains selected in this invention, while increasing the release rate of echinacoside and verbascoside in Cistanche deserticola, also have a better effect on improving the free radical scavenging rate than Lactobacillus paracasei F-19 and Lp90 Lactobacillus plantarum.

[0099] Comparing Example 4 with Comparative Examples 5 and 6, under the condition of using different mixed microbial strains in the same proportion, the free radical scavenging rate of the Cistanche deserticola fermentation broth in Example 4 was higher than that in Comparative Examples 5 and 6. This indicates that, compared with the mixed microbial strains used in Comparative Examples 5 and 6, the mixed microbial strains selected in Example 4 of this invention have more advantages in promoting the release of effective components of Cistanche deserticola and enhancing its antioxidant properties.

[0100] During strenuous exercise, the mitochondrial respiratory chain produces a large amount of ROS (reactive oxygen species), leading to muscle fatigue and damage. The results and analysis above indicate that the Cistanche deserticola fermentation liquid prepared in this invention has high antioxidant activity, effectively scavenging excess free radicals in the body, thereby reducing oxidative damage after exercise and accelerating recovery.

[0101] Result Example 3: Effects of Cistanche deserticola extract prepared in the examples and comparative examples on NO and TNF-α secretion and neutral red phagocytosis rate in RAW264.7 cells (Zhejiang Nuobo Biotechnology Co., Ltd.)

[0102] After counting the Raw264.7 cells in the logarithmic growth phase, the cell suspension concentration was adjusted to 5 × 10⁻⁶. 4 100 μL of the *Cistanche deserticola* extract from Examples 1, 2, 4, and the comparative examples was seeded into 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. 20 mL of the *Cistanche deserticola* extract from each plate was used as a sample solution and diluted with 10% FBS in DMEM complete medium to a final concentration of 50 μL / mL. The samples were then incubated for 24 h. The blank and model groups were incubated with equal volumes of PBS and 1 μg / mL LPS, respectively, for 24 h. The effects of the reagents on NO and TNF-α secretion and neutral red phagocytosis rate in RAW264.7 cells were detected using the kit. Detailed data are shown in Table 3.

[0103] Table 3. Effects of Cistanche deserticola extract on NO and TNF-α secretion and neutral red phagocytosis rate in RAW264.7 cells in the examples and comparative examples.

[0104]

[0105] Note: Compared with the blank group, *p<0.05; compared with the LPS group, #p<0.05; compared with Comparative Example 1, △p<0.05, n=3.

[0106] RAW264.7 cells can secrete NO upon appropriate stimulation. NO participates in immune regulation by mediating intercellular communication (such as promoting T lymphocyte activation and proliferation), and its secretion level is positively correlated with the enhancement of the body's immune function.

[0107] TNF-α maintains immune homeostasis by positively regulating other cytokines, such as inducing IL-6 secretion. Its synergistic effect with IL-6 manifests in ways such as co-activating natural killer (NK) cells and cytotoxic T cells (CTLs), enhancing the killing ability against virus-infected cells; or forming a cytokine network to dynamically balance pro-inflammatory and anti-inflammatory responses. Neutral red is a dye that can be taken up by living cells (especially immune cells such as macrophages and neutrophils) through endocytosis (phagocytosis or endocytosis). Increased phagocytosis usually indicates immune cell activation and enhanced phagocytic function. Elevated levels of TNF-α secretion and neutral red phagocytosis are hallmark events of immune cell activation, and their synergistic effect can significantly enhance both specific and non-specific immune responses.

[0108] As shown in Table 3, the RAW264.7 cells treated with Cistanche deserticola fermentation broth obtained in Examples 1, 2 and 4 of this invention had higher levels of NO and TNF-α secretion and neutrophil phagocytosis than those treated with Cistanche deserticola broth obtained in Comparative Example 1.

[0109] Comparative Examples 3 and 4 used commercially available Lactobacillus paracasei strain F-19 and Lp90 Lactobacillus plantarum strains, respectively. Only under the condition of different strains, after the Cistanche deserticola fermentation broth in Examples 1 and 2 was used to intervene in RAW264.7 cells, the NO and TNF-α secretion and neutral red phagocytosis rates were higher than those in Comparative Examples 3 and 4.

[0110] As shown in Examples 4 and Comparative Examples 3-6, when 4% fructooligosaccharides were added, the fermentation of Cistanche deserticola by a mixture of two strains, Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68, significantly increased the phagocytic rate of NO, TNF-α, and neutral red produced by RAW264.7 cells. The effect was best when B20 and MPs-68 were mixed and fermented at a mass ratio of 0.9:1.2. This indicates that the mixed strain fermentation selected in this invention is beneficial to the enhancement of NO and TNF-α secretion and neutral red phagocytic rate of Cistanche deserticola cells, and is superior to commercially available Lactobacillus paracasei F-19 and Lp90 Lactobacillus plantarum.

[0111] Result Example 4: Effects of Cistanche deserticola extract prepared in the examples and comparative examples on D-gal-induced intracellular reactive oxygen species (ROS) and antioxidant enzyme activities in TM3 cells.

[0112] Reactive oxygen species (ROS) detection: TM3 cells (Zhejiang Nuobo Biotechnology Co., Ltd.) in logarithmic growth phase were counted, and the cell suspension concentration was adjusted to 10,000 cells / well, then seeded in 96-well plates (black) and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. Cistanche deserticola extract (1% concentration) from Examples 1, 2, 4, and the comparative examples was added to the culture medium for intervention for 24 h. After intervention, the blank group was treated with an equal volume of PBS, while the model and experimental groups were treated with 160 mmol / L D-gal for 24 h. The fluorescence intensity of TM3 cells before and after stimulation was detected using a microplate reader; specific data are shown in Table 4.

[0113] Glutathione peptidase (GSH-PX) detection: After counting TM3 cells in logarithmic growth phase, the cell suspension concentration was adjusted to 50,000 cells / well and seeded in 12-well plates, and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. Cistanche deserticola extract (1% concentration of Examples 1, 2, 4 and the comparative example) was added to the culture medium for intervention for 24 h. After intervention, the blank group was added with an equal volume of PBS, while the model group and experimental group were cultured with 160 mmol / L D-gal for 24 h. Glutathione peptidase in TM3 cells was detected using a kit; specific data are shown in Table 4.

[0114] Table 4. Effects of Cistanche deserticola extract on D-gal-induced intracellular ROS and GSH-PX activities in TM3 cells in the examples and comparative cases.

[0115]

[0116] Note: Compared with the blank group, *p<0.05; compared with the LPS group, #p<0.05; compared with Comparative Example 1, △p<0.05, n=3.

[0117] D-galactose (D-gal) is a reducing sugar commonly used to construct aging-related cell or animal models (by inducing oxidative stress and the accumulation of advanced glycation end products). D-gal increases intracellular ROS levels by mimicking the natural aging process, leading to mitochondrial dysfunction and oxidative damage, affecting the survival and function of TM3 cells (such as decreased testosterone synthesis). It is a classic model for studying anti-aging mechanisms. GSH is a core endogenous antioxidant, while ROS, through oxidative modification, inhibits StAR expression, reduces cholesterol transport to mitochondria, and decreases testosterone synthesis substrates.

[0118] Table 4 shows that after D-gal intervention in testicular interstitial cells TM3, the ROS content significantly increased and the GSH-PX content significantly decreased. Compared with the D-gal group, the Cistanche deserticola extracts in both the examples and comparative examples reduced ROS content and increased the enzyme activities of ROS and GSH-PX. Compared with unfermented Cistanche deserticola extract (Comparative Examples 1 and 2), the ROS content of Cistanche deserticola fermented with different single or mixed strains in Examples 1, 2, 4 and Comparative Examples 3 to 6 was significantly reduced, while the GSH-PX content was significantly increased.

[0119] As shown in Example 4, when B20 and MPs-68 were mixed and fermented in a mass ratio of 0.9:1.2, the ROS content was the lowest and the GSH-PX enzyme activity was the strongest, which was superior to the commercially available Lactobacillus paracasei F-19 and Lp90 Lactobacillus plantarum in Comparative Examples 3 to 6.

[0120] Therefore, it can be explained that the fermentation broth of Cistanche deserticola can alleviate the effects of D-gal on the reduction of ROS and GSH-PX enzyme activity in cells. This indicates that the fermentation broth of Cistanche deserticola can protect testicular interstitial cells damaged by D-gal by targeting and inhibiting ROS accumulation and oxidative damage, thereby delaying the aging process at the cellular level. It also suggests that the decrease in testosterone secretion may be related to the decrease in antioxidant enzyme activity.

[0121] Result Example 5: Effects of Cistanche deserticola extract prepared in the examples and comparative examples on D-gal-induced testosterone synthesis and expression of synthase-related genes in TM3 cells.

[0122] Testosterone content detection: After counting TM3 cells in the logarithmic growth phase, the cell suspension concentration was adjusted to 25,000 cells / well and seeded in 24-well plates, then incubated at 37 ℃ in a 5% CO2 incubator for 24 h. A 1% concentration of Cistanche deserticola extract from Examples 1, 2, 4, and the comparative examples was added to the culture medium for intervention for 24 h. An equal volume of PBS was added to the blank group, while 160 mmol / L D-gal was added to the model and experimental groups for 24 h. Testosterone concentration was detected using the Elabscience® Testosterone (T) Enzyme-Linked Immunosorbent Assay Kit.

[0123] Gene expression level detection: After counting TM3 cells in the logarithmic growth phase, the cell suspension concentration was adjusted to 50,000 cells / well and seeded in 12-well plates, and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. Cistanche deserticola extract (1% concentration of Examples 1, 2, 4 and comparative examples) was added to the culture medium for 24 h. The blank group was treated with an equal volume of PBS, while the model group and experimental group were treated with 160 mmol / L D-gal for 24 h. Real-time qPCR was used to detect the testosterone synthase gene (…). StAR, 3β- HSD, CYP17a1 and 17β-HSD The expression level of ) is detailed in Table 5.

[0124] Table 5. Testosterone concentration and expression levels of synthase-related genes in Cistanche deserticola extract from the examples and comparative cases.

[0125]

[0126] Note: Compared with the blank group, *p<0.05; compared with the LPS group, #p<0.05; compared with Comparative Example 1, △p<0.05, n=3.

[0127] Testosterone is one of the most important androgens secreted into the bloodstream. It is synthesized from pregnenolone, which itself is formed from cholesterol. Testosterone is the main androgen secreted by interstitial cells in the testes, influencing primary and secondary sexual development. Testosterone synthesis occurs in the mitochondria and endoplasmic reticulum, using cholesterol as a raw material. StAR transports cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, which is the rate-limiting step in testosterone synthesis. Decreased StAR expression directly leads to reduced testosterone synthesis. 3β-HSD catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrosterone (DHEA) to androstenedione. CYP17A1 is a branching enzyme that determines the substrate flow pathway to testosterone or cortisol. 17β-HSD reduces androstenedione to testosterone. Therefore, to further investigate the effect of Cistanche deserticola extract on testosterone synthesis in TM3 interstitial cells of the testes, the effects of Cistanche deserticola extract intervention on the mRNA expression levels of StAR, 3β-HSD, CYP17A1, and 17β-HSD were observed by testosterone concentration detection and q-PCR.

[0128] The data in Table 5 show that D-gal intervention significantly reduced testosterone concentration and gene expression levels of related synthetic enzymes in TM3 cells. Compared with the D-gal group, Cistanche deserticola solution intervention significantly increased testosterone concentration and mRNA expression levels of StAR, 3β-HSD, CYP17a1 and 17β-HSD in TM3 cells.

[0129] As shown in Table 5, the concentration of testosterone and the expression level of related genes in fermented Cistanche deserticola were higher than those in Comparative Examples 1 and 2, indicating that the fermented Cistanche deserticola extract was superior to the unfermented Cistanche deserticola extract in alleviating the effect of D-gal on the reduction of cellular testosterone secretion. Among them, Example 4 showed the highest increase in testosterone concentration and related gene expression, with its testosterone concentration increasing by 62.5% compared to Comparative Example 1. This indicates that the fermented Cistanche deserticola broth can significantly increase the testosterone secretion of D-gal-induced stress-induced TM3 cells and has a protective effect on D-gal-damaged testicular interstitial TM3 cells.

[0130] As can be seen from the data in Table 5 for Examples 4, 5, and 6, the testosterone concentration and mRNA expression levels of StAR, 3β-HSD, CYP17a1, and 17β-HSD in Example 4 were higher than those in Comparative Examples 5 and 6. This indicates that when using different mixed bacterial strains to ferment Cistanche deserticola, the fermentation broth obtained by fermenting a mixed bacterial strain of B20 and MPs-68 at a mass ratio of 0.9:1.2 is more effective in alleviating D-gal-induced loss of testicular interstitial TM3 cells.

[0131] In summary, the Cistanche deserticola fermentation liquid of the present invention has the following characteristics:

[0132] 1. Increase the content of echinacoside and verbascoside;

[0133] 2. It has antioxidant activity;

[0134] 3. It can enhance the phagocytic activity of phagocytes;

[0135] 4. It possesses the activity of promoting the secretion of NO and TNF-α by phagocytes;

[0136] 5. It can enhance the activity of ROS and GSH-PX in TM3 cells;

[0137] 6. It has the ability to alleviate D-gal-induced TM3 cell damage and promote testosterone secretion.

[0138] Based on this, according to the functional characteristics mentioned in 1-6 above, Cistanche deserticola fermentation liquid can also be used in any substance that provides preventive and / or other beneficial effects, including but not limited to antioxidant, immune-boosting, and energy-enhancing effects.

[0139] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing Cistanche deserticola fermentation broth that enhances energy and improves cellular aging, characterized in that, Includes the following steps: (1) Extraction: Mix Cistanche deserticola with deionized water and extract with hot water to obtain Cistanche deserticola aqueous extract; (2) Homogenization: The aqueous extract of Cistanche deserticola is homogenized to obtain a Cistanche deserticola mixture; (3) Enzymatic hydrolysis: Cellulase and pectinase are added to the Cistanche deserticola mixture for enzymatic hydrolysis to obtain Cistanche deserticola hydrolysate; (4) Add carbon source: Add carbon source to the Cistanche deserticola enzymatic hydrolysate; (5) Sterilization before fermentation: Sterilize the Cistanche deserticola hydrolysate after adding carbon source, and then cool it to the fermentation temperature; (6) Fermentation: Lactic acid bacteria were inoculated into the cooled Cistanche deserticola enzymatic hydrolysate for fermentation to obtain Cistanche deserticola fermentation broth; the lactic acid bacteria were Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68; the preservation number of Lactobacillus paracasei B20 was CGMCC No. 32434, and the preservation number of Lactobacillus plantarum MPs-68 was CGMCC No. 31976; the lactic acid bacteria were obtained by mixing Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 in a mass ratio of 0.8-1.1:1-1.

2. (7) Solid-liquid separation: The fermentation liquid of Cistanche deserticola is subjected to solid-liquid separation to obtain the fermentation supernatant of Cistanche deserticola; (8) Sterilization after fermentation: Sterilize the supernatant of the fermentation of Cistanche deserticola to obtain the fermentation liquid of Cistanche deserticola.

2. The preparation method according to claim 1, characterized in that: The lactic acid bacteria strain was obtained by mixing Lactobacillus paracasei B20 and Lactobacillus plantarum MPs-68 at a mass ratio of 0.9:1.

2.

3. The preparation method according to claim 2, characterized in that: In step (6), the amount of lactic acid bacteria added is 0.005–0.015 wt% of the Cistanche deserticola enzymatic hydrolysate system; the viable count of the Lactobacillus paracasei B20 bacterial powder is 1–5 × 10⁻⁶. 12 CFU / g; the viable count of the *Lactobacillus plantarum* MPs-68 powder is 1–5 × 10⁻⁶. 11 CFU / g; the fermentation conditions are 36-38℃ for 15-20 h.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of Cistanche deserticola to deionized water is 1:15-25; the hot water extraction conditions are water extraction at 90-100℃ for 60-80 min.

5. The preparation method according to claim 1, characterized in that: In step (3), 0.5-1% of the mass of the Cistanche deserticola mixture is added to cellulase and pectinase respectively, with cellulase activity of 20,000 u / g and pectinase activity of 30,000 u / g; the mass ratio of cellulase to pectinase is 1:0.8-1.

2.

6. The preparation method according to claim 5, characterized in that: The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis at 45–55°C for 100–140 min.

7. The preparation method according to claim 1, characterized in that: In step (4), the carbon source is any one of white sugar, fructooligosaccharide, isomaltooligosaccharide, and inulin.

8. The preparation method according to claim 7, characterized in that: In step (4), the amount of carbon source added is 2-5 wt% of the Cistanche deserticola enzymatic hydrolysate system.

9. A Cistanche deserticola fermentation liquid that enhances energy and improves cellular aging, characterized in that: It is prepared by the preparation method according to any one of claims 1-8.

10. A functional product containing the Cistanche deserticola fermentation liquid according to claim 9, characterized in that: The functional product is a pharmaceutical product that has at least one of the following functions: boosting energy, enhancing immunity, anti-oxidation, and increasing testosterone levels.

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