Brain and spirit invigorating traditional Chinese medicine composition, preparation method and application thereof

Abstract

This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a brain-strengthening and mind-nourishing traditional Chinese medicine composition, its preparation method, and its application. The brain-nourishing and mind-soothing traditional Chinese medicine composition of the present invention comprises, by weight, the following raw materials: 100-200 parts of Rehmannia glutinosa, 60-120 parts of Cornus officinalis, 50-70 parts of Dioscorea opposita, 100-200 parts of Morinda officinalis, 50-70 parts of Epimedium brevicornu, 50-70 parts of Cuscuta chinensis, 50-70 parts of Codonopsis pilosula, 60-120 parts of Poria cocos, 60-120 parts of Angelica sinensis, 60-120 parts of Ligusticum chuanxiong, 10-20 parts of deer antler glue, 10-20 parts of tortoise shell glue, 60-120 parts of Lycium barbarum, 50-70 parts of Ligustrum lucidum, 50-70 parts of Alisma plantago-aquatica, 60-120 parts of Salvia miltiorrhiza, 50-70 parts of Alpinia oxyphylla, 35-55 parts of Acorus tatarinowii, 20-40 parts of Aquilaria sinensis, and 60-120 parts of Achyranthes bidentata. This invention demonstrates through nematode model organism experiments that Jiannao Yishen Ointment exhibits multidimensional anti-aging effects in the Caenorhabditis elegans model, suggesting that the Jiannao Yishen traditional Chinese medicine composition of this invention has excellent anti-aging effects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a brain-strengthening and mind-nourishing traditional Chinese medicine composition, its preparation method, and its application. Background Technology

[0002] Aging is a systemic and progressive decline in the body's physiological functions with age, and it is closely related to the occurrence and development of various chronic diseases. Delaying aging and achieving healthy aging has become a major global scientific and social issue. Currently, anti-aging strategies mainly include lifestyle interventions, hormone replacement therapy, and drug development targeting specific aging markers (such as oxidative stress, telomere shortening, and cellular senescence). However, many synthetic drugs suffer from problems such as single-target effects, potential side effects, or uncertain long-term safety.

[0003] Aging is a complex process involving the gradual decline of multiple system functions, and effectively delaying aging has always been an important direction in the modernization of traditional Chinese medicine (TCM). TCM compound formulas, with their multi-target effects and holistic regulatory advantages, have shown unique application potential in the field of anti-aging. However, existing TCM anti-aging compound formulas still have many problems: First, the scientific compatibility of the formulations is insufficient; some TCM compositions simply combine single herbs with antioxidant, kidney-tonifying, and yin-nourishing effects, resulting in insignificant anti-aging efficacy. Second, the efficacy of the formulations is singular; existing TCM compositions often focus on single pathogenic mechanisms such as "kidney tonification" or "qi tonification," neglecting the correlation of organ dysfunction during aging and failing to comprehensively improve the aging state of the body. Therefore, it is necessary to develop a scientifically formulated, effective, and safe anti-aging TCM composition to meet clinical needs. Summary of the Invention

[0004] The first objective of this invention is to provide a brain-nourishing and spirit-enhancing traditional Chinese medicine composition; the second objective of this invention is to provide a brain-nourishing and spirit-enhancing ointment; the third objective of this invention is to provide a method for preparing the brain-nourishing and spirit-enhancing ointment; and the fourth objective of this invention is to provide the application of the brain-nourishing and spirit-enhancing traditional Chinese medicine composition.

[0005] According to a first aspect of the present invention, a brain-strengthening and mind-nourishing traditional Chinese medicine composition is provided, comprising, by weight, the following raw materials: 100-200 parts of Rehmannia glutinosa (processed), 60-120 parts of Cornus officinalis (salted), 50-70 parts of Dioscorea opposita (Chinese yam), 100-200 parts of Morinda officinalis (bajitian), 50-70 parts of Epimedium brevicornu (xianpi), 50-70 parts of Cuscuta chinensis (tusizi), 50-70 parts of Codonopsis pilosula (dangshen), 60-120 parts of Poria cocos (fuling), 60-120 parts of Angelica sinensis (danggui), 60-120 parts of Ligusticum chuanxiong (chuanxiong), 10-20 parts of deer antler glue (lujiaojiao), 10-20 parts of tortoise shell glue (guibanjiao), 60-120 parts of Lycium barbarum (gouqizi), 50-70 parts of Ligustrum lucidum (nulizi), 50-70 parts of Alisma plantago-aquatica (zexai), 60-120 parts of Salvia miltiorrhiza (danshen), 50-70 parts of Alpinia oxyphylla (yizhiren), 35-55 parts of Acorus tatarinowii (shichangpu), 20-40 parts of Aquilaria sinensis (chenxiang), and 60-120 parts of Achyranthes bidentata (niuxi).

[0006] This invention posits that aging is not merely the decline of a single organ, but a holistic degenerative process involving all the internal organs, qi and blood, and yin and yang. The cause is not a single factor, but rather the result of the combined effects of congenital heredity and acquired environment, physiological decline and pathological damage. Aging is a process of "deficiency in the root and excess in the branch": deficiency of the kidneys and spleen, and insufficiency of essence and qi are the root causes (internal causes), while phlegm turbidity, blood stasis, and unhealthy lifestyles are the inducing factors that accelerate aging (external causes / excess in the branch). Its core pathogenesis can be summarized as "deficiency" and "stasis," and is most closely related to the kidneys, spleen, and heart. Human aging is the result of the gradual decline of the functions of the five internal organs, with the kidneys and spleen playing a decisive role, and the heart governing vitality. Abundant kidney qi leads to longevity, while deficient kidney qi leads to premature death; the spleen and kidneys are mutually causal. Congenital kidney qi deficiency affects the spleen and stomach's digestive function, while acquired spleen and stomach weakness cannot replenish congenital kidney essence; both accelerate aging. Therefore, aging is not a problem of a single organ, but a holistic decline of the heart, liver, spleen, lungs, and kidneys. According to the theory of "treating the root cause of the disease and addressing both the symptoms and the root cause", the core treatment principle should be "tonifying the kidney and strengthening the spleen, replenishing qi and nourishing blood, and resolving phlegm and removing blood stasis".

[0007] In this formula, Rehmannia glutinosa (processed), Cornus officinalis (salted mountain corn), Dioscorea opposita (Chinese yam), Morinda officinalis (ground orange), Epimedium brevicornu (spleen), and Cuscuta chinensis (dodder seed) are the principal herbs. Rehmannia glutinosa (processed), Cornus officinalis (salted mountain corn), and Dioscorea opposita (Chinese yam) form the core of the "three tonifying" principle. Rehmannia glutinosa (processed) nourishes the kidneys and replenishes essence, greatly tonifying true yin; Cornus officinalis (salted mountain corn) tonifies the liver and kidneys, astringes essence and prevents leakage; Dioscorea opposita (Chinese yam) tonifies spleen yin and also strengthens the kidneys. The three herbs combined tonify the yin of the liver, spleen, and kidneys, strengthening the foundation and nourishing the source. Morinda officinalis (ground orange), Epimedium brevicornu (spleen), and Cuscuta chinensis (dodder seed) warm and tonify kidney yang, enabling "yang to receive the assistance of yin and generate endless transformations," achieving the goal of tonifying both yin and yang, maintaining yin-yang balance, and resisting aging.

[0008] The formula uses Codonopsis pilosula, Poria cocos, Angelica sinensis, Ligusticum chuanxiong, deer antler glue, tortoise shell glue, Lycium barbarum, and Ligustrum lucidum as assistant herbs. Codonopsis pilosula, Poria cocos, Angelica sinensis, and Ligusticum chuanxiong invigorate the spleen, replenish qi, and nourish blood to replenish the innate essence. Deer antler glue is more yang-tonifying, while tortoise shell glue is more yin-nourishing; together, they can "strongly replenish the essence and marrow, and connect the Ren and Du meridians." Lycium barbarum and Ligustrum lucidum nourish the liver and kidney yin and blood, enhancing the effects of nourishing the liver and kidneys, replenishing essence, and improving eyesight.

[0009] The formula uses Alisma plantago-aquatica, Salvia miltiorrhiza, Alpinia oxyphylla, and Acorus tatarinowii as adjuvant herbs. Alisma plantago-aquatica promotes diuresis and eliminates turbidity, preventing the cloying effect of Rehmannia glutinosa. Salvia miltiorrhiza clears the heart and relieves irritability, while also clearing heat stagnation in the blood. The combination of these two herbs makes the entire formula nourishing without being cloying, achieving a balance between activity and rest, and complementing each other effectively. Alpinia oxyphylla and Acorus tatarinowii, when used together, can calm the mind, improve intelligence, and enhance memory.

[0010] Agarwood and Achyranthes bidentata are used as guiding herbs. Agarwood is pungent, fragrant, and warm, guiding the medicine to the kidneys, promoting qi circulation and resolving stagnation, making the whole formula "nourishing without causing stagnation, and nourishing without being greasy." Achyranthes bidentata can nourish the liver and kidneys, strengthen tendons and bones, and guide blood downwards, allowing the medicinal power to directly reach the lower jiao (lower burner) where the liver and kidneys are located.

[0011] When used together, these herbs nourish both Yin and Yang, regulate the spleen and kidneys, and balance Qi and blood. By combining tonification and unblocking, they work together to strengthen the body's foundation, nourish both Yin and Yang, replenish essence and marrow, invigorate the brain, calm the mind, and combat aging. They are suitable for people under high workplace pressure, those suffering from chronic mental fatigue, age-related forgetfulness, or those with memory loss, who often experience symptoms such as blurred vision, poor sleep, and fatigue.

[0012] The specific ingredients and their effects in this invention are as follows: Prepared Rehmannia: A traditional Chinese medicine made from the fresh or dried tuberous roots of Rehmannia glutinosa Libosch., a plant belonging to the genus Rehmannia in the family Scrophulariaceae, through processing.

[0013] Salted Cornus officinalis pulp: The dried, mature pulp of Cornus officinalis Sieb. et Zucc., a plant in the Cornaceae family, is processed with salt.

[0014] Chinese yam: The dried rhizome of Dioscorea opposita Thunb., a plant in the Dioscoreaceae family.

[0015] Morinda officinalis How, a plant in the Rubiaceae family, is the dried root of the plant.

[0016] Epimedium: The dried leaves of Epimedium, Epimedium sagittatum, Epimedium pubescens, or Epimedium koreanum, all belonging to the Berberidaceae family.

[0017] Dodder: The dried, mature seeds of Cuscuta australis R. Br. or Cuscutachinensis Lam., plants of the Convolvulaceae family.

[0018] Codonopsis: The dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen, or Codonopsis tangshen Oliv., all belonging to the Campanulaceae family.

[0019] Poria: The dried sclerotium of the fungus Poriacocos (Schw.) Wolf, belonging to the genus Poria in the family Polyporaceae.

[0020] Angelica sinensis (Oliv) Diels., a plant belonging to the genus Angelica in the family Apiaceae.

[0021] Chuanxiong: The dried rhizome of Ligusticum chuanxiong Hort., a plant belonging to the genus Ligusticum in the family Apiaceae.

[0022] Deer antler glue: A gelatinous substance made by boiling the antlers of sika deer or red deer.

[0023] Tortoise shell glue: A solid glue made by boiling and concentrating tortoise shells in water.

[0024] Goji berries: the dried, ripe fruit of Lycium barbarum L., a plant belonging to the genus Lycium in the Solanaceae family.

[0025] Privet fruit: The mature fruit of Ligustrum lucidum Ait., a plant belonging to the genus Ligustrum in the family Oleaceae.

[0026] Alisma: The dried tuber of Alisma orientale (Sam.) Juzep. or Alismaplantago-aquatica Linn., belonging to the Alismataceae family.

[0027] Danshen: The dried root and rhizome of Salvia miltiorrhiza Bge., a plant belonging to the genus Salvia in the family Lamiaceae.

[0028] Alpinia oxyphylla Miq., the fruit of Alpinia oxyphylla, a plant belonging to the genus Alpinia in the family Zingiberaceae.

[0029] Sweet flag (Acorus tatarinowii): The dried rhizome of Acorus tatarinowii Schott, a plant belonging to the genus Acorus in the family Araceae.

[0030] Agarwood: The resinous wood of Aquilaria agallocha Roxb. and Aquilaria sinensis (Lour.) Gilg, plants of the genus Aquilaria in the family Thymelaeaceae.

[0031] Achyranthes bidentata BI. (family Amaranthaceae) and Cyathula officinalis Kuan (family Cyathulae) are the roots of plants in the genus Achyranthes.

[0032] In some embodiments, the brain-nourishing and mind-soothing traditional Chinese medicine composition of the present invention comprises, by weight, the following raw materials: 150 parts of Rehmannia glutinosa, 90 parts of Cornus officinalis, 60 parts of Dioscorea opposita, 150 parts of Morinda officinalis, 60 parts of Epimedium brevicornu, 60 parts of Cuscuta chinensis, 60 parts of Codonopsis pilosula, 90 parts of Poria cocos, 90 parts of Angelica sinensis, 90 parts of Ligusticum chuanxiong, 15 parts of deer antler glue, 15 parts of tortoise shell glue, 90 parts of Lycium barbarum, 60 parts of Ligustrum lucidum, 60 parts of Alisma plantago-aquatica, 90 parts of Salvia miltiorrhiza, 60 parts of Alpinia oxyphylla, 45 parts of Acorus tatarinowii, 30 parts of Aquilaria sinensis, and 90 parts of Achyranthes bidentata.

[0033] According to a second aspect of the present invention, a brain-nourishing and mind-enhancing ointment is provided, which is composed of the above-mentioned brain-nourishing and mind-enhancing traditional Chinese medicine composition and pharmaceutically acceptable excipients.

[0034] According to a third aspect of the present invention, a method for preparing the above-mentioned brain-boosting and mind-nourishing ointment is provided, comprising the following steps: (1) Add water to deer antler glue and tortoise shell glue and simmer until dissolved, then filter to obtain glue liquid; (2) Prepare Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Morinda officinalis, Epimedium, Cuscuta chinensis, Codonopsis pilosula, Poria cocos, Angelica sinensis, Ligusticum chuanxiong, Lycium barbarum, Ligustrum lucidum, Alisma plantago-aquatica, Salvia miltiorrhiza, Alpinia oxyphylla, Acorus tatarinowii, Aquilaria sinensis, and Achyranthes bidentata in proportion, add water and decoct, filter to obtain decoction, and then concentrate the decoction to obtain clear extract; (3) Mix the adhesive solution and the clear paste, and add the excipients to obtain a mixture; (4) Concentrate the mixture obtained in step (3) and remove the water to obtain the finished paste.

[0035] In some embodiments, the relative density of the paste is 1.0-1.25, the viscosity is 1100-1400 mPa·s, and the water content of the finished paste is 45-60%.

[0036] According to a fourth aspect of the present invention, the use of the above-described brain-nourishing and mind-soothing traditional Chinese medicine composition in the preparation of a medicament for delaying aging is provided.

[0037] According to a fifth aspect of the present invention, the above-described brain-nourishing and mind-strengthening traditional Chinese medicine composition is provided for use in the preparation of a medicament for prolonging life and / or promoting growth and development and / or enhancing motor function and / or enhancing antioxidant capacity and / or enhancing stress resistance.

[0038] The beneficial effects of this invention include: This invention demonstrates, through nematode model organism experiments, that the Jiannao Yishen Ointment exhibits multidimensional anti-aging effects in the *C. elegans* model. It extends lifespan in a dose-dependent manner while promoting growth, development, and maintenance of motor function, without interfering with reproductive capacity. This suggests that the Jiannao Yishen herbal composition of this invention possesses excellent anti-aging effects. Attached Figure Description

[0039] Figure 1 This presents the results of the effects of various drugs on the lifespan of wild-type N2 nematodes in the nematode model organism experiment of this invention.

[0040] Figure 2 This is the result of the effect of Jiannao Yishen Ointment on the growth of nematodes in the nematode model organism experiment of the present invention.

[0041] Figure 3This is the result of the effect of Jiannao Yishen Ointment on the nematode's swinging ability in the nematode model organism experiment of the present invention.

[0042] Figure 4 This is the result of the effect of the brain-nourishing and mind-nourishing ointment on the pharyngeal pump function of nematodes in the nematode model organism experiment of the present invention.

[0043] Figure 5 This is the result of the effect of Jiannao Yishen Ointment on the total oviposition of nematodes in the nematode model organism experiment of the present invention.

[0044] Figure 6 This is the result of the effect of Jiannao Yishen Ointment on lipofuscin in the nematode model organism experiment of the present invention.

[0045] Figure 7 This is the result of the effect of Jiannao Yishen Ointment on the survival rate of nematodes under stress conditions in the nematode model organism experiment of the present invention.

[0046] Figure 8 These are images of ROS fluorescence intensity in the intestines of nematodes in each group during the nematode model organism experiment of this invention, along with the results of relative quantitative detection. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. Process steps not disclosed in the embodiments are prior art. Unless otherwise specified, all raw materials are commercially available.

[0048] Example 1 The Brain-Nourishing and Spirit-Enhancing Ointment of this embodiment is made from the following raw materials: 150g Rehmannia glutinosa, 90g Cornus officinalis, 60g Dioscorea opposita, 150g Morinda officinalis, 60g Epimedium brevicornu, 60g Cuscuta chinensis, 60g Codonopsis pilosula, 90g Poria cocos, 90g Angelica sinensis, 90g Ligusticum chuanxiong, 15g Cervi cornu gelatin, 15g Tortoise shell gelatin, 90g Lycium barbarum, 60g Ligustrum lucidum, 60g Alisma plantago-aquatica, 90g Salvia miltiorrhiza, 60g Alpinia oxyphylla, 45g Acorus tatarinowii, 30g Aquilaria sinensis, and 90g Achyranthes bidentata.

[0049] Its preparation method includes the following steps: (1) Add water to deer antler glue and tortoise shell glue and simmer until dissolved, then filter to obtain glue liquid; (2) Mix Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Morinda officinalis, Epimedium, Cuscuta chinensis, Codonopsis pilosula, Poria cocos, Angelica sinensis, Ligusticum chuanxiong, Lycium barbarum, Ligustrum lucidum, Alisma plantago-aquatica, Salvia miltiorrhiza, Alpinia oxyphylla, Acorus tatarinowii, Aquilaria sinensis, and Achyranthes bidentata, add 10 times the weight of water and decoct. After boiling, keep it simmering for 1 hour. Filter to obtain the decoction. Then, repeat the decoction twice on the residue. Mix the decoction obtained each time and concentrate to obtain the clear extract. (3) Mix the adhesive solution and the clear paste, and add the excipients to obtain a mixture; (4) Concentrate the mixture obtained in step (3) and remove the water to obtain the finished paste.

[0050] Example 2 The Brain-Nourishing and Spirit-Enhancing Ointment of this embodiment is prepared from the following raw materials: 200g Rehmannia glutinosa, 120g Cornus officinalis, 70g Dioscorea opposita, 200g Morinda officinalis, 70g Epimedium brevicornu, 70g Cuscuta chinensis, 70g Codonopsis pilosula, 120g Poria cocos, 120g Angelica sinensis, 120g Ligusticum chuanxiong, 20g Cervi cornu gelatin, 20g Tortoise shell gelatin, 120g Lycium barbarum, 70g Ligustrum lucidum, 70g Alisma plantago-aquatica, 120g Salvia miltiorrhiza, 70g Alpinia oxyphylla, 55g Acorus tatarinowii, 40g Aquilaria sinensis, and 120g Achyranthes bidentata.

[0051] Its preparation method is the same as that of Example 1.

[0052] Example 3 The Brain-Nourishing and Spirit-Enhancing Ointment of this embodiment is made from the following raw materials: 100g Rehmannia glutinosa, 60g Cornus officinalis, 50g Dioscorea opposita, 100g Morinda officinalis, 50g Epimedium brevicornu, 50g Cuscuta chinensis, 50g Codonopsis pilosula, 60g Poria cocos, 60g Angelica sinensis, 60g Ligusticum chuanxiong, 10g Cervi cornu gelatin, 10g Tortoise shell gelatin, 60g Lycium barbarum, 50g Ligustrum lucidum, 50g Alisma plantago-aquatica, 60g Salvia miltiorrhiza, 50g Alpinia oxyphylla, 35g Acorus tatarinowii, 20g Aquilaria sinensis, and 60g Achyranthes bidentata.

[0053] Its preparation method is the same as that of Example 1.

[0054] *C. elegans* is an important model organism for aging research, exhibiting high genetic conservation and sharing 60%-80% gene similarity with humans, making it widely used in aging studies. The following study uses *C. elegans* as a model to explore the anti-aging effects of a traditional Chinese medicine composition for nourishing the brain and calming the mind.

[0055] I. Experimental Materials 1. Experimental reagents The experimental reagents are shown in Table 1: Table 1 Experimental Reagents

[0056] 2. Experimental apparatus The experimental apparatus is shown in Table 2: Table 2 Experimental Instruments

[0057] 3. Experimental Biology Both Escherichia coli OP50 and wild-type C. elegans Bristol N2 were provided by the Department of Neurology Laboratory of the First Affiliated Hospital of Sun Yat-sen University.

[0058] 4. Preparation of main reagents (1) Preparation of experimental drug solutions Brain-Boosting and Nourishing Ointment Solution: Weigh 9 g of the Brain-Boosting and Nourishing Ointment prepared in Example 1 and dissolve it in 30 mL of ultrapure water. After vortexing to fully dissolve, centrifuge at 2000 rpm for 2 min. Take the supernatant and filter it through a 0.22 μm microporous membrane to obtain a stock solution with a concentration of 300 mg / mL. Dilute sequentially at volume ratios of 1:1, 1:2, and 1:9 to obtain working concentrations of 150 mg / mL, 50 mg / mL, and 5 mg / mL. All solutions are aliquoted into sterile 1.5 mL centrifuge tubes, labeled with the concentration and preparation date, and stored at -20℃. Before the experiment, thaw the required solutions at room temperature before use.

[0059] Metformin solution: Accurately weigh 0.2484 g of metformin hydrochloride and dissolve it in 30 mL of ultrapure water. After bacterial filtration, a metformin solution with a concentration of 50 mM is obtained.

[0060] (2) Preparation of experimental stock solutions and culture media 1 M MgSO4 solution: Weigh 12.325 g of MgSO4, add 50 mL of dH2O to dissolve, filter to remove bacteria and set aside for later use.

[0061] 1 M CaCl2 solution: Weigh 2.775 g of CaCl2, add 50 mL of dH2O to dissolve, filter to remove bacteria and set aside for later use.

[0062] 5 mg / mL cholesterol solution: Weigh 0.125 g of cholesterol, add 50 mL of anhydrous ethanol, sonicate to dissolve, filter to sterilize, and set aside for later use.

[0063] 5 M NaOH solution: Weigh 20 g of NaOH and dissolve it in 100 mL of dH2O.

[0064] 1 M potassium phosphate buffer (pH=6.0): Weigh 108.3 g KH2PO4, 35.6 g K2HPO4, and 15.12 g Na2HPO4, add dH2O to bring the volume to 1 L, adjust the pH to 6.0, autoclave, and store at room temperature.

[0065] M9 buffer: Weigh 3 g KH2PO4, 6 g Na2HPO4, and 5 g NaCl, add dH2O to bring the volume to 1 L, autoclave, and after cooling, add 1 mL 1 M MgSO4.

[0066] Nematode growth medium (NGM): Weigh 3 g NaCl, 2.5 g tryptone, and 17 g agar powder, add dH2O to a final volume of 975 mL, and autoclave at 121℃ for 15 min. Cool to approximately 60℃, and in a clean bench, add 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 25 mL of 1 M potassium phosphate buffer (pH=6.0) sequentially. Mix well, pour the mixture onto a plate, allow it to solidify, and then sterilize by UV irradiation for 2 h. Invert and seal the plate, and store at 4℃ for later use.

[0067] Cryopreservation solution for nematodes: Weigh 1.17 g NaCl and 1.36 g K2HPO4, add 60 mL of 30% glycerol and 140 mL dH2O, autoclave, add 0.6 mL of 0.1 M MgSO4, and store at 4℃.

[0068] II. Experimental Methods 1. Culture of Escherichia coli OP50 In a clean bench, aseptically take a cryopreservation tube of *E. coli* OP50 or the original bacterial culture, dip a small amount of bacterial solution into an inoculation loop, and streak it in four zones on a blood agar plate to obtain single colonies. Invert the streaked plate and incubate it at 37°C for 48 hours until clearly visible single colonies appear. Pick a single colony with neat edges and inoculate it into an Erlenmeyer flask containing 100 mL of LB liquid medium (the medium must be sterilized and cooled to room temperature beforehand). Place the Erlenmeyer flask in a shaker at 37°C and 220 rpm, and incubate overnight (approximately 12-16 hours) with continuous shaking until the bacterial solution becomes uniformly turbid. The cultured bacterial solution can be used as a working solution and stored at 4°C for up to two weeks.

[0069] 2. Making a nematode food board Before spreading, remove the preserved bacterial suspension and gently shake to mix. In a laminar flow hood, accurately pipette 200 μL of the bacterial suspension and vertically drop it into the center of a solidified nematode growth medium (NGM) agar plate. Sterilize a glass rod by flaming it with an alcohol lamp. After cooling, gently spread the bacterial suspension from the center of the plate outwards with even pressure, covering the entire surface of the plate as much as possible, but keeping the suspension about 0.5 cm from the edge of the plate to prevent bacterial growth and spread along the plate walls. Apply even pressure during spreading to avoid scratching the agar surface. After spreading, slightly open the plate lid and allow it to air dry in the laminar flow hood. Incubate the plate overnight at 37°C before using it for nematode culture. After preparation, immediately invert the plate and seal it in a 4°C refrigerator for 1-2 weeks. Before use, check the plate for contamination and ensure the bacterial growth is uniform.

[0070] 3. Preparation of agarose mats Weigh 0.5 g of agarose powder and place it in a clean glass container. Add 25 mL of ultrapure water and shake well to prevent clumping. Place in a microwave oven and heat on medium-high for about 60 seconds, observing the solution's state. During heating, be careful to prevent bumping. Continue heating until the solution becomes completely transparent. After slightly cooling, use a dropper to quickly add an appropriate amount of agarose solution to the center of a clean, dry glass slide. Take a coverslip and gently cover the droplet in a crisscross pattern. If air bubbles appear during this process, gently press the coverslip to remove them. Let the prepared slide stand at room temperature for 5-10 minutes until the agarose solution has completely cooled and solidified. Slowly and evenly peel the coverslip off the slide to obtain a complete agarose mat on the coverslip. The mat should be of uniform thickness, with a smooth, flat surface, free of air bubbles and damage. Use immediately after preparation; store at 4°C for no more than 2 hours. 4. General Culture of Caenorhabditis elegans (1) Cultivation of nematodes All nematodes used in this experiment were cultured in a stable environment with a temperature of 20℃ and humidity. The nematodes fed on Escherichia coli OP50, and nematodes at all developmental stages were cultured on solid culture plates coated with OP50 bacterial solution.

[0071] (2) Nematode propagation Nematodes in their oviposition stage were selected for subculturing. Before subculturing, the blade was sterilized by flaming with an alcohol lamp. After cooling, an appropriately sized piece of agar was cut from the old culture dish, and the side containing the nematodes was gently placed on the surface of the agar in a new culture dish pre-added with *E. coli* OP50. The dish was sealed with sealing film and labeled with the strain name and subculturing date. The culture dish was then placed in a 20°C incubator for further cultivation. The first generation of nematodes needed to be cultured for at least three generations before being used as experimental material for subsequent observation and experiments.

[0072] (3) Nematode synchronization Nematodes in their oviposition stage were selected for synchronization treatment. The surface of the culture dish was repeatedly rinsed with 1 mL ddH2O. Nematodes and eggs were collected into 15 mL centrifuge tubes, and this process was repeated three times, with ddH2O added to bring the total volume to 3.5 mL. Then, 0.5 mL of 5M NaOH solution and 1 mL of 10% NaOCl solution were added sequentially. The tubes were immediately capped, and the tubes were vigorously vortexed for 30 seconds, then allowed to stand for 2 minutes. This vortex-stand cycle was repeated 5 times until no visible flocculent matter was observed in the tubes. The tubes were then centrifuged at 1300 rpm for 1 minute, and the supernatant was discarded. The precipitate was resuspended in 5 mL ddH2O, centrifuged again, and the supernatant was discarded. The washing process was repeated once more. The resuspended egg precipitate was transferred to a new culture dish pre-coated with E. coli OP50. At this point, the eggs in the dish were all at the same developmental stage. After incubating at 20℃ for 48 hours, most fertilized eggs had developed to the L4 stage. Synchronization was then complete and the tubes were ready for subsequent experiments.

[0073] (4) Cryopreservation and thawing of nematodes Inoculate an appropriate amount of nematodes onto 3-5 60 mm NGM plates coated with *E. coli* OP50. Incubate at 20°C until more than 60% of the nematodes enter the Dauer phase, the bacterial colony is largely consumed, and the plates are free of contamination. In a clean bench, add an appropriate amount of M9 buffer to each plate, agitate the agar surface to collect the nematodes, and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 1500 rpm for 30 s, discard the supernatant, and repeat the centrifugation and washing process 2-3 times. Resuspend the nematodes in 0.6 mL of M9 buffer and transfer them to cryovials. Add 0.6 mL of cryopreservation solution to each cryovial, invert to mix, and label with the strain, date, and operator. Place the cryovials in a gradient cooling box and freeze at -80°C for 24 h, then transfer to a cryopreservation box for long-term storage. After one month, collect 1-2 tubes of this batch of frozen samples to test the success rate of resuscitation. After thawing the liquid in the cryovials in a 37°C incubator, pour the liquid into NGM plates and incubate at 20°C for one to two days, observing the nematode recovery. Those that successfully recover can be cultured for subsequent experiments.

[0074] 5. Lifespan test of Caenorhabditis elegans In the screening of anti-aging drugs, whether it can effectively extend the survival time of biological individuals is the most intuitive and crucial criterion. By observing the changes in the average lifespan and median survival time of model animals, the overall regulatory effect of drugs on the aging process can be objectively reflected, which is also a widely used evaluation method in current research in this field. To explore whether the Jinnian Yishen Ointment has the effect of delaying aging, this study used wild-type N2 nematodes as a model, and set up a blank control group (referred to as the Control group, administered 150 μL of ultrapure water), a positive drug metformin group (referred to as the Metformin group, administered 150 μL of 50 mM metformin solution), and a Jinnian Yishen Ointment treatment group (referred to as the JNYS group, administered 150 μL of Jinnian Yishen Ointment solution at different concentrations (5, 50, 150, 300 mg / mL)), and systematically observed and recorded the changes in the survival time of nematodes in each group.

[0075] After synchronizing the passaged nematodes and culturing them for 48 h until they developed to the L4 stage, the synchronized L4-stage nematodes were transferred to NGM plates containing different drugs according to the above grouping, and 60 were randomly selected for each plate. During the experiment, the nematodes were cultured at 20 °C, and transferred to new NGM plates at the same time every day to ensure food supply and drug concentration, and to exclude the influence of metabolite accumulation. The number of dead nematodes was observed and recorded daily. If the nematodes had no response to the platinum wire stimulation and their bodies were rigid in an "S" shape or a straight line, they were judged to be dead. During the experiment, those that accidentally crawled out of the culture dish, hatched inside the body, or were deformed were excluded. The experiment was repeated three times.

[0076] 6. Measurement of the body length of Caenorhabditis elegans The body length is an intuitive indicator reflecting the growth and development of nematodes. To explore the maintenance of the quality of life by the Jinnian Yishen Ointment, this study used wild-type N2 nematodes as a model, and set up a blank control group (referred to as the Control group, administered 150 μL of ultrapure water), a positive drug metformin group (referred to as the Metformin group, administered 150 μL of 50 mM metformin solution), and a Jinnian Yishen Ointment treatment group (referred to as the JNYS group, administered 150 μL of 300 mg / mL Jinnian Yishen Ointment solution), and systematically observed and recorded the body lengths of nematodes in each group.

[0077] Synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs, with 60 nematodes per group, and cultured at 20°C. Drug administration continued until day 3 and day 10 of adulthood. To prevent interference from progeny nematodes, they were transferred to fresh NGM plates every 24 hours. On day 3 of drug administration, 10 nematodes were randomly selected from the 60 nematodes to measure their body length. On day 10 of drug administration, 10 nematodes were randomly selected from the remaining 50 nematodes to measure their body length. For body length measurement, a drop of 15 M levamisole hydrochloride solution was placed on a glass slide, and the selected nematodes were anesthetized by placing them in the drop for 15 minutes. The nematodes were photographed under a microscope with a suitable field of view that exposed their full body length, and their body length was measured using ImageJ software. The experiment was repeated three times.

[0078] 7. Detection of the wiggling ability of Caenorhabditis elegans Swinging is one of the important indicators for assessing motor ability. To investigate the effect of Jiannao Yishen Ointment on motor ability, this study used wild-type N2 nematodes as a model and set up a blank control group (Control group, administered 150 μL of ultrapure water), a positive control group (Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution). The swinging behavior of nematodes in each group was systematically observed and recorded.

[0079] Synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping method, with 60 nematodes per group. They were cultured at 20°C and continuously administered the drugs until day 3 and day 10 of adulthood. To prevent interference from progeny nematodes, they were transferred to fresh NGM plates every 24 hours. On day 3 of drug administration, 10 nematodes were randomly selected from the 60 nematodes to measure their motility. On day 10 of drug administration, 10 nematodes were randomly selected from the remaining 50 nematodes to measure their motility. For motility measurement, a drop of M9 buffer was placed on a glass slide, and each nematode was picked up and placed into the M9 droplet, allowing it to adapt for 5 seconds. Then, under a stereomicroscope, the number of body movements within 15 seconds was counted. Each movement of the nematode's head from one side to the other and back to the original side was counted as one movement. The experiment was repeated three times.

[0080] 8. Detection of pharyngeal pump capacity in Caenorhabditis elegans The pharyngeal pumping frequency of nematodes directly reflects the rhythmic contraction ability of their pharyngeal muscles, which is closely related to feeding efficiency and is one of the important indicators for evaluating the physiological function and health status of nematodes. To explore the effects of Jiannao Yishen Ointment on physiological function and health status, in this study, wild-type N2 nematodes were used as a model, and a blank control group (abbreviated as Control group, administered 150 μL of ultrapure water), a positive drug metformin group (abbreviated as Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (abbreviated as JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution) were set up. The pharyngeal pumping frequencies of nematodes in each group were systematically observed and recorded.

[0081] The synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 60 nematodes in each group, cultured at 20 °C, and continuously administered until the 3rd and 10th days after adulthood. To prevent interference from offspring nematodes, they were transferred to new NGM plates every 24 h. On the 3rd day of administration, randomly select 10 nematodes from 60 nematodes to measure their pharyngeal pumping times, and on the 10th day of administration, randomly select 10 nematodes from the remaining 50 nematodes to measure their pharyngeal pumping times. When measuring the motile pharyngeal pumping times, transfer each nematode to a new NGM plate, place it under a microscope, and continuously photograph for 10 s through an imaging system connected to a camera. Use video playback software to play back the images frame by frame and record the number of pharyngeal contractions of the nematodes. The experiment was repeated three times.

[0082] 9. Detection of the reproductive ability of Caenorhabditis elegans The reproductive ability of nematodes is closely related to lifespan and the aging process. There is a classic trade-off relationship between reproductive investment and somatic maintenance. The intensity of reproductive activity is one of the key physiological factors affecting individual lifespan. Excessive reproduction often accompanies a shortened lifespan, and intervention measures to extend lifespan often accompany reproductive inhibition. To explore whether the mechanism of action of Jiannao Yishen Ointment in delaying aging is related to reproduction, in this study, wild-type N2 nematodes were used as a model, and a blank control group (abbreviated as Control group, administered 150 μL of ultrapure water), a positive drug metformin group (abbreviated as Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (abbreviated as JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution) were set up. The total egg production of nematodes in each group was systematically observed and recorded.

[0083] The synchronized hermaphrodite nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 1 nematode per plate and 10 nematodes in each group. They were transferred to new NGM plates every 24 h until the end of the egg-laying period. The original plates were continued to be cultured in a 20 °C incubator for 48 h, and the number of offspring hatched by nematodes in each NGM plate was counted. The experiment was repeated three times.

[0084] 10. Lipofuscin fluorescence detection in Caenorhabditis elegans As an important marker of cellular senescence, the accumulation level of lipofuscin directly reflects the level of oxidative damage and the aging process of the body. To explore the anti-aging ability of Jiannao Yishen Ointment, in this study, wild-type N2 nematodes were used as a model, and a blank control group (referred to as the Control group, administered 150 μL of ultrapure water), a positive drug metformin group (referred to as the Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (referred to as the JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution) were set up. The fluorescence intensity in nematodes of each group was systematically observed and recorded.

[0085] The synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 60 nematodes in each group, cultured at 20 °C, and continuously administered drugs until the 5th day after adulthood. To prevent interference from offspring nematodes, they were transferred to new NGM plates every 24 h. On the 5th day of drug administration, 10 nematodes were randomly selected from 60 nematodes to measure their lipofuscin fluorescence intensity. When measuring the lipofuscin fluorescence intensity, the nematodes were anesthetized with 15 M levamisole hydrochloride for 15 min. After the nematodes stopped moving, they were transferred to a 2% agarose pad. The autofluorescence of lipofuscin in the nematode intestine was observed and photographed under a fluorescence microscope. Green excitation was used to excite red fluorescence, and the pictures were taken and saved. The fluorescence intensity in the nematodes was quantitatively analyzed using Image J software. The experiment was repeated three times.

[0086] 11. Stress resistance experiment in Caenorhabditis elegans The stress resistance of organisms gradually decreases with age, and the response degree to external environmental stress can directly reflect the physiological state of the body. In this study, wild-type N2 nematodes were used as a model, and a blank control group (referred to as the Control group, administered 150 μL of ultrapure water), a positive drug metformin group (referred to as the Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (referred to as the JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution) were set up. By detecting the survival rate of Caenorhabditis elegans under acute stress conditions, the potential effect of the drug on delaying aging can be measured to a certain extent.

[0087] Heat stress experiment: The synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 30 nematodes in each group. After culturing at 20 °C for 5 days, the nematodes were transferred to blank NGM plates without food and drugs, sealed with parafilm, and cultured in a 35 °C incubator for 4 h, and then transferred to a 20 °C incubator to recover for 12 h. Note that the culture dishes should not be stacked together. After 12 h, the survival and death of the nematodes were observed under a microscope. The nematodes were judged dead if they did not move when gently touched with a platinum wire. The experiment was repeated three times.

[0088] Oxidative stress experiment: In the dark, 6 μL of 30% hydrogen peroxide solution was added to 20 mL of M9 buffer, and mixed well to obtain 3 mM hydrogen peroxide solution. It was wrapped with tin foil and stored at 4 °C for later use. The synchronized nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 30 nematodes in each group. After culturing at 20 °C for 5 days, the nematodes were collected with M9 buffer and washed 3 times repeatedly to wash away the Escherichia coli adhering to the nematodes to prevent affecting the experimental results. Add 2 mL of the prepared hydrogen peroxide solution to a 3.5 cm culture dish, transfer the nematodes of each group to the culture dish containing hydrogen peroxide solution, mark it, and place it in a 25 °C incubator for dark culture for 4 h. The survival of the nematodes was observed under a stereomicroscope. Note that the experiment process should be strictly in the dark. The experiment was repeated three times.

[0089] 12. Determination of ROS level in Caenorhabditis elegans Reactive oxygen species (ROS) are the products of mitochondrial oxidative metabolism. Their excessive accumulation can induce oxidative stress damage and then accelerate cell senescence and tissue function decline. Therefore, detecting the ROS level in vivo is often used to evaluate the antioxidant capacity of drugs. To explore the antioxidant capacity of Jiannao Yishen Ointment, in this study, wild-type N2 nematodes were used as a model, and a blank control group (abbreviated as Control group, administered 150 μL of ultrapure water), a positive drug metformin group (abbreviated as Metformin group, administered 150 μL of 50 mM metformin solution), and a Jiannao Yishen Ointment treatment group (abbreviated as JNYS group, administered 150 μL of 300 mg / mL Jiannao Yishen Ointment solution) were set up, and the fluorescence intensity in the nematodes of each group was systematically observed and recorded.

[0090] The ROS-sensitive probe DCFH-DA was used to detect the ROS level in nematodes. DCFH-DA can freely penetrate the nematode cell membrane and enter the cell, and is hydrolyzed by intracellular esterase to generate DCFH. Since DCFH cannot cross the membrane and leak out, it remains in the cell and is oxidized to generate fluorescent dichlorofluorescein (DCF) under the action of ROS. By measuring the fluorescence intensity of DCF, the content of ROS in the cell can be indirectly reflected.

[0091] After synchronization, nematodes cultured to the L4 stage were transferred to NGM plates containing different drugs according to the above grouping, with 60 nematodes per group. They were cultured at 20°C and continuously administered the drugs until day 5 of adulthood. To prevent interference from progeny nematodes, they were transferred to a new NGM plate every 24 hours. On day 5 of drug administration, 10 nematodes were randomly selected from the 60 nematodes to measure their ROS fluorescence intensity. When measuring ROS fluorescence intensity, nematodes were collected with M9 buffer and washed three times to remove any adhering E. coli to prevent interference with the experimental results. After centrifugation at 1500 rpm for 1 min, the nematode pellet was collected and 100 μL of 5 mM DCFH-DA solution was added. The pellet was incubated at 37°C on a shaker at 50 rpm for 30 min, and repeatedly washed with M9 buffer until no excess dye remained on the nematode. Nematodes were anesthetized with 15 M levamisole hydrochloride for 15 min. After the nematodes became immobile, they were transferred to a 2% agarose mat. Fluorescence microscopy was used to observe the fluorescence under blue light, which excited the green fluorescence. Images were taken and saved, and the fluorescence intensity within the nematodes was quantitatively analyzed using ImageJ software. The experiment was repeated three times. III. Statistical Methods All experimental data were processed and analyzed using GraphPad Prism 10.0 software. Lifespan data were expressed as mean lifespan and median lifespan. Survival curves were plotted using the Kaplan-Meier method, and the log-rank test was used to compare the survival curves. One-way ANOVA was used to compare means among multiple groups that met the requirements of normality and homogeneity of variance. The Bonferroni test was used for pairwise comparisons in the ANOVA. Chi-square tests were used for comparisons between groups for categorical data, and the Bonferroni test was used to adjust the significance level for multiple comparisons.

[0092] IV. Experimental Results 1. The effect of Jiannao Yishen Ointment on the lifespan of Caenorhabditis elegans The effects of each drug group on the mean lifespan and median survival of wild-type N2 nematodes are shown in Table 3. The effects of each drug group on the lifespan of wild-type N2 nematodes are also shown in Table 3. Figure 1 As shown.

[0093] Table 3. Effects of each drug group on the mean lifespan and median survival of wild-type N2 nematodes.

[0094] Note: Survival curves were plotted using the Kaplan-Meier method. Overall comparisons between groups were performed using the log-rank test, and multiple pairwise comparisons were corrected using the Bonferroni method (corrected significance level α′=0.01). A p-value < 0.01 was considered statistically significant. In the table, * indicates p < 0.01 compared to the blank control group; *** indicates p < 0.0001 compared to the blank control group.

[0095] From Table 3 and Figure 1 It can be seen that among the various dosage groups of Jiannao Yishen Gao, the average lifespan of the low-dose (5 mg / mL) and medium-dose (50 mg / mL) treatment groups was 15.96 ± 0.40 days and 16.31 ± 0.29 days, respectively, with no statistically significant difference compared with the blank control group (P values ​​were 0.8432 and 0.8453, respectively). The average lifespan of the 150 mg / mL treatment group was 16.85 ± 0.41 days, showing a certain trend of prolongation, but not reaching a significant level of lifespan extension (P=0.2080). It is worth noting that although there was no significant difference in average lifespan between the low- and medium-dose groups, the median survival time showed a dose-dependent increasing trend, at 15.50 days, 17.00 days, and 18.00 days, respectively, suggesting that Jiannao Yishen Gao at low and medium concentrations had a certain positive effect on the survival of nematodes, but its effect was not enough to change the overall average lifespan. The average lifespan of the high-dose Jiannao Yishen Gao (300 mg / mL) treatment group reached 19.14 ± 0.18 days, showing a highly significant life-prolonging effect compared to the blank control group, with a median survival time extended to 20.00 days. Statistical analysis showed a highly significant difference (P < 0.0001). This result indicates that Jiannao Yishen Gao can effectively prolong the lifespan of wild-type N2 nematodes at higher concentrations, and its life-prolonging effect exhibits a clear dose-dependent characteristic.

[0096] Based on the above results, 300 mg / mL of Jiannao Yishen Ointment showed the best life-prolonging effect, and the difference was statistically significant. Therefore, 300 mg / mL was established as the dosage for subsequent experiments.

[0097] 2. Effects of Jiannao Yishen Ointment on Physiological Indicators of Caenorhabditis elegans (1) Effect of Jiannao Yishen Ointment on the body length of Caenorhabditis elegans The effects of Jiannao Yishen Ointment on nematode growth are as follows: Figure 2 As shown in Table 4. Figure 2 The results of the effect of Jiannao Yishen Ointment on the growth of nematodes are shown in the figures. (A) represents the body length of nematodes in each group on the 3rd day after administration, and (B) represents the body length of nematodes in each group on the 10th day after administration.

[0098] Table 4. Effects of each drug group on nematode body length

[0099] Note: The 3-day body length was measured using the Kruskal-Wallis test. H =10.69, P =0.0048), and the Dunn test was used for multiple comparisons; the 10-day body length was analyzed using one-way ANOVA (...). F (2,87)=7.655,P =0.0009), multiple comparisons were performed using the Bonferroni test. Data are presented as median (interquartile range). * ,** .

[0100] From Table 4 and Figure 2 As shown in (A), after 3 days of culture, there was no significant difference in the body length of nematodes in the metformin group compared with the blank control group (P>0.05), but the body length of the nematode treated with Jiannao Yishen Gao was significantly longer than that in the blank control group, indicating that Jiannao Yishen Gao can promote the growth and development of nematodes from a young age. (See Table 4 and...) Figure 2 As shown in (B), after 10 days of culture, compared with the blank control group, the body length of nematodes in the metformin group was significantly increased (P<0.05), and the body length in the Jiannao Yishen Gao group was extremely significantly increased (P<0.01). The body length of the Jiannao Yishen Gao treatment group was slightly higher than that in the metformin group. The above results indicate that Jiannao Yishen Gao can promote the growth and development of Caenorhabditis elegans.

[0101] (2) Effect of Jiannao Yishen Ointment on the number of swings of Caenorhabditis elegans The results of the effect of Jiannao Yishen Ointment on the wriggling ability of nematodes are as follows: Figure 3 As shown in Table 5. Figure 3 The results of the effect of Jiannao Yishen Ointment on the nematode motility were as follows: (A) shows the motility of nematodes in each group on the 3rd day after administration, and (B) shows the motility of nematodes in each group on the 10th day after administration.

[0102] Table 5. Effects of each drug group on the number of nematode swings.

[0103] Note: The number of oscillations over 3 days was determined using the Kruskal-Wallis test. H =16.02, P =0.0003), and the Dunn test was used for multiple comparisons; the number of oscillations over 10 days was analyzed using one-way ANOVA (). F (2,87)=11.28, P <0.0001), multiple comparisons were performed using the Bonferroni test. Data are presented as median (interquartile range). *** .

[0104] From Table 5 and Figure 3As shown in (A), compared with the blank control group, the number of body swings of nematodes treated with Jiannao Yishen Gao was significantly increased after 3 days of culture (P<0.001), and compared with the metformin group, the number of body swings of nematodes treated with Jiannao Yishen Gao was significantly increased after 3 days of culture (P<0.01). However, there was no significant difference between the metformin group and the blank control group, indicating that Jiannao Yishen Gao has a significant promoting effect on the motility of juvenile Caenorhabditis elegans. (See Table 5 and...) Figure 3 As shown in (B), after 10 days of culture, compared with the blank control group, the number of nematode movements in both the metformin group and the Jiannao Yishen Gao treatment group was significantly increased (P<0.001), and there was no significant difference between the two groups. This suggests that both Jiannao Yishen Gao and the positive control drug can effectively improve the reduced motility of nematodes due to aging. The above results indicate that Jiannao Yishen Gao can enhance the motility of Caenorhabditis elegans in both youth and old age.

[0105] (3) Effects of Jiannao Yishen Ointment on Pharyngeal Pump Function of Caenorhabditis elegans The results of the effect of Jiannao Yishen Ointment on the pharyngeal pump function of nematodes are as follows: Figure 4 As shown in Table 6. Figure 4 The results of the effect of Jiannao Yishen Ointment on the pharyngeal pump function of nematodes are as follows: (A) is the pharyngeal pump frequency of each group of nematodes on the 3rd day after administration, and (B) is the pharyngeal pump frequency of each group of nematodes on the 10th day after administration.

[0106] Table 6. Effects of Jiannao Yishen Ointment on the Pharyngeal Pump Frequency of Nematodes

[0107] Note: The number of pharyngeal pumps over 3 days was analyzed using the Kruskal-Wallis test (H=1.214, P=0.5450), and the Dunn test was used for multiple comparisons; the number of pharyngeal pumps over 10 days was analyzed using one-way ANOVA (F(2,87)=9.204, P=0.0002), and the Bonferroni test was used for multiple comparisons. Data are expressed as median (interquartile range). * P<0.05, *** P<0.001.

[0108] From Table 6 and Figure 4 As shown in (A), on the 3rd day after administration, the pharyngeal pump frequency of nematodes in each group was at a similar level. There was no statistically significant difference between the Jiannao Yishen Ointment treatment group and the blank control group and the metformin group (P>0.05), indicating that Jiannao Yishen Ointment had no significant effect on the pharyngeal muscles of juvenile nematodes. (See Table 6 and...) Figure 4As shown in (B), significant differences began to appear among the groups on day 10 of culture. Compared with the blank control group, the pharyngeal pump frequency in the metformin group was significantly increased (P<0.05), while the increase in pharyngeal pump frequency in the Jiannao Yishen Gao treatment group was even more significant, reaching a highly significant level (P<0.001). These results indicate that Jiannao Yishen Gao can specifically enhance the pharyngeal muscle motility of adult nematodes. Combined with the association between pharyngeal pump frequency and feeding behavior, this suggests that the anti-aging effect of Jiannao Yishen Gao may be related to its ability to maintain the feeding function of nematodes in their old age.

[0109] (4) Effects of Jiannao Yishen Ointment on the reproductive capacity of Caenorhabditis elegans The effect of Jiannao Yishen Ointment on the total oviposition of nematodes is as follows: Figure 5 As shown in Table 7.

[0110] Table 7. Effects of Jiannao Yishen Ointment on Total Oviposition of Nematodes

[0111] Note: Each group consisted of 10 nematodes, and each nematode was an independent biological replicate. Three replicate experiments were conducted, resulting in a total of 30 nematodes. Therefore, N=30.

[0112] from Figure 5 As shown in Table 7, the average total number of eggs laid was 186.57±14.53 in the blank control group, 188.23±11.78 in the metformin group, and 188.97±14.41 in the Jiannao Yishen Gao treatment group. Statistical analysis showed no significant difference between the Jiannao Yishen Gao treatment group and the blank control group (P=0.7746), and there was also no significant difference between the metformin group and the blank control group (P=0.8839). The results indicate that Jiannao Yishen Gao has no reproductive toxicity to nematodes, suggesting that its effect of prolonging nematode lifespan is not achieved by inhibiting reproduction.

[0113] (5) Effects of Jiannao Yishen Ointment on intestinal lipofuscin in Caenorhabditis elegans The results of the effect of Jiannao Yishen Ointment on lipofuscin in the intestine of nematodes are as follows: Figure 6 As shown in Table 8.

[0114] Table 8. Effects of each drug group on lipofuscin in nematodes

[0115] Note: The relative fluorescence intensity of lipofuscin was measured using the Kruskal-Wallis test. H =47.97, P <0.0001), multiple comparisons were performed using the Dunn test. Data are presented as median (interquartile range). **** On the 5th day after administration, 10 nematodes were randomly selected from 60 nematodes to measure their lipofuscin fluorescence intensity. The experiment was repeated three times, and a total of 30 nematodes were measured for lipofuscin fluorescence intensity, so N=30.

[0116] From Table 8 and Figure 6 It can be seen that, compared with the blank control group, the fluorescence intensity of lipofuscin in the nematode intestine of the Jiannao Yishen Ointment treatment group and the metformin group was significantly reduced (P<0.0001), and the reduction in lipofuscin level was similar in both groups. This indicates that Jiannao Yishen Ointment can effectively inhibit the abnormal deposition of lipofuscin during the aging process of nematodes, reduce oxidative stress damage, and show good anti-aging potential.

[0117] (6) Effects of Jiannao Yishen Ointment on the survival rate of Caenorhabditis elegans under stress conditions The effects of Jiannao Yishen Ointment on the survival rate of nematodes under stress conditions are as follows: Figure 7 As shown in Table 9-10. Figure 7 The results show the effect of Jiannao Yishen Ointment on the survival rate of nematodes under stress conditions. Among them, (A) represents the survival of nematodes under acute heat stress, and (B) represents the survival of nematodes under acute oxidative stress.

[0118] Table 9. Survival status of nematodes under heat stress conditions

[0119] Note: Chi-square test was used for pairwise comparisons, and Bonferroni correction was applied (corrected significance level α). ’ =0.0167), *P<0.0167, **P<0.01, ***P<0.001.

[0120] From Table 9 and Figure 7 As shown in (A), under heat stress, the survival rates of the blank control group, metformin group, and Jiannaoshen ointment treatment group were 66.7%, 84.4%, and 96.7%, respectively. Compared with the blank control group, the survival rates of the metformin group (P=0.0012) and the Jiannaoshen ointment treatment group (P<0.0003) were significantly increased. Although there was no statistically significant difference between the Jiannaoshen ointment treatment group and the metformin group (P=0.027>0.0167), the survival rate of the Jiannaoshen ointment treatment group showed a numerical trend of being better, indicating that it can more effectively improve the resistance of nematodes under high temperature stress.

[0121] Table 10 Survival status of nematodes under oxidative stress

[0122] Note: Chi-square test was used for pairwise comparisons, and Bonferroni correction was applied (corrected significance level α). ’=0.0167), *P<0.0167, ***P<0.001.

[0123] From Table 10 and Figure 7 As shown in (B), under oxidative stress, the survival rates of the blank control group, metformin group, and Jiannaoshen ointment treatment group were 42.2%, 64.4%, and 84.4%, respectively. Compared with the blank control group, the survival rates of the metformin group (P=0.0132) and the Jiannaoshen ointment treatment group (P<0.0003) were significantly increased, and the survival rate of the Jiannaoshen ointment treatment group was also significantly higher than that of the metformin group (P=0.0102). The results indicate that both Jiannaoshen ointment and metformin can significantly enhance the oxidative stress tolerance of nematodes, and the protective effect of Jiannaoshen ointment is significantly better than that of metformin.

[0124] The above results indicate that the Brain Nourishing and Divine Ointment can delay aging by significantly enhancing the resistance of nematodes to heat and oxidative stress.

[0125] (7) Effect of Jiannao Yishen Ointment on ROS Levels in Caenorhabditis elegans Images of ROS fluorescence intensity in the intestines of each group of nematodes and the results of relative quantification are shown below. Figure 8 As shown in Table 11.

[0126] Table 11 Effects of each drug group on ROS in nematodes

[0127] Note: The relative fluorescence intensity of ROS was measured using the Kruskal-Wallis test. H =71.22, P <0.0001), multiple comparisons were performed using the Dunn test. Data are presented as median (interquartile range). **** On day 5 of drug administration, 10 nematodes were randomly selected from 60 nematodes to measure their ROS fluorescence intensity. The experiment was repeated three times, and a total of 30 nematodes were measured for ROS fluorescence intensity, so N=30.

[0128] From Table 11 and Figure 8 As can be seen, compared with the blank control group, the ROS fluorescence intensity of the Jiannao Yishen Gao treatment group was significantly reduced (P<0.0001), and it was also significantly reduced compared with the metformin group (P<0.01). The results indicate that Jiannao Yishen Gao can significantly inhibit ROS accumulation in nematodes, and it may delay the aging of nematodes by enhancing antioxidant capacity.

[0129] In summary, the Brain-Boosting and Nourishing Ointment demonstrated multidimensional anti-aging effects in the *C. elegans* model. It not only extended lifespan in a dose-dependent manner but also promoted growth, development, and motor function maintenance without interfering with reproductive capacity. These physiological improvements are closely related to its enhanced antioxidant defense, reduced oxidative damage, and improved stress resistance.

[0130] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A brain-boosting and mind-nourishing traditional Chinese medicine composition, characterized in that, The raw materials, by weight, consist of: 100-200 parts Rehmannia glutinosa, 60-120 parts Cornus officinalis, 50-70 parts Dioscorea opposita, 100-200 parts Morinda officinalis, 50-70 parts Epimedium, 50-70 parts Cuscuta chinensis, 50-70 parts Codonopsis pilosula, 60-120 parts Poria cocos, 60-120 parts Angelica sinensis, 60-120 parts Ligusticum chuanxiong, 10-20 parts Cervi cornu colla, 10-20 parts Testudinis plastrum colla, 60-120 parts Lycium barbarum, 50-70 parts Ligustrum lucidum, 50-70 parts Alisma plantago-aquatica, 60-120 parts Salvia miltiorrhiza, 50-70 parts Alpinia oxyphylla, 35-55 parts Acorus tatarinowii, 20-40 parts Aquilaria sinensis, and 60-120 parts Achyranthes bidentata.

2. The brain-nourishing and mind-soothing traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials, by weight, consist of: 150 parts Rehmannia glutinosa, 90 parts Cornus officinalis, 60 parts Dioscorea opposita, 150 parts Morinda officinalis, 60 parts Epimedium, 60 parts Cuscuta chinensis, 60 parts Codonopsis pilosula, 90 parts Poria cocos, 90 parts Angelica sinensis, 90 parts Ligusticum chuanxiong, 15 parts Cervi cornu colla, 15 parts Testudinis plastrum colla, 90 parts Lycium barbarum, 60 parts Ligustrum lucidum, 60 parts Alisma plantago-aquatica, 90 parts Salvia miltiorrhiza, 60 parts Alpinia oxyphylla, 45 parts Acorus tatarinowii, 30 parts Aquilaria sinensis, and 90 parts Achyranthes bidentata.

3. Brain-Boosting and Spirit-Nourishing Ointment, characterized in that, It consists of the brain-nourishing and mind-strengthening traditional Chinese medicine composition as described in claim 1 or 2 and pharmaceutically acceptable excipients.

4. The preparation method of the brain-nourishing and mind-soothing ointment according to claim 3, characterized in that, Includes the following steps: (1) Add water to deer antler glue and tortoise shell glue and simmer until dissolved, then filter to obtain glue liquid; (2) Prepare Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Morinda officinalis, Epimedium, Cuscuta chinensis, Codonopsis pilosula, Poria cocos, Angelica sinensis, Ligusticum chuanxiong, Lycium barbarum, Ligustrum lucidum, Alisma plantago-aquatica, Salvia miltiorrhiza, Alpinia oxyphylla, Acorus tatarinowii, Aquilaria sinensis, and Achyranthes bidentata in proportion, add water and decoct, filter to obtain decoction, and then concentrate the decoction to obtain clear extract; (3) Mix the adhesive solution and the clear paste, and add the excipients to obtain a mixture; (4) Concentrate the mixture obtained in step (3) and remove the water to obtain the finished paste.

5. The method for preparing the Brain-Boosting and Spirit-Nourishing Ointment according to claim 4, characterized in that, The finished paste has a moisture content of 45-60%.

6. The use of the brain-nourishing and mind-soothing traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a drug for delaying aging.

7. The use of the brain-nourishing and mind-strengthening traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for prolonging life and / or promoting growth and development and / or enhancing motor function and / or enhancing antioxidant capacity and / or enhancing stress resistance.

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