WO2024021876A1 - 杜仲雄花提取物在制备抗肌肉衰老的食品或药物中的应用 - Google Patents

杜仲雄花提取物在制备抗肌肉衰老的食品或药物中的应用 Download PDF

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WO2024021876A1
WO2024021876A1 PCT/CN2023/098531 CN2023098531W WO2024021876A1 WO 2024021876 A1 WO2024021876 A1 WO 2024021876A1 CN 2023098531 W CN2023098531 W CN 2023098531W WO 2024021876 A1 WO2024021876 A1 WO 2024021876A1
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male flower
extract
flower extract
eucommia
aging
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陆柏益
陈祁
黄伟素
吴礼鹏
朱宇航
汪怿璇
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Zhejiang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/46—Eucommiaceae (Eucommia family), e.g. hardy rubber tree
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to the technical field of plant extracts, and specifically relates to the application of Eucommia ulmoides male flower extract containing iridoid components in the preparation of anti-muscle aging foods (including pet foods) or medicines.
  • Skeletal muscle is the driving force of the human body's movement system, and its aging is an important sign of the aging of the body. As the most abundant plastic organ among vertebrates, it plays an important role in metabolism, movement, respiration, protection, daily physical activities, and maintenance of posture and balance. Sarcopenia (characterized by progressive and systemic loss of skeletal muscle mass and function) was officially recognized as a disease in 2016 with the code ICD-10-CM (M62.84), raising concerns about this age-related disorder. of extra attention. Among them, aging-related decline in muscle function is an important feature of the occurrence and development of sarcopenia.
  • Sarcopenia especially the aging-related decline in muscle function, is closely related to mitochondrial dysfunction in skeletal muscle. Clearance of dysfunctional mitochondria to maintain mitochondrial homeostasis during aging is mainly carried out through a mitochondria-specific autophagy mode, namely mitophagy. However, mitophagy activity decreases during muscle aging. These indicate the importance of mitophagy in maintaining muscle function during aging.
  • Natural products are potential sources of mitophagy-modulating and anti-muscle aging active compounds.
  • edible flowers have become one of the new trends in the consumption of food resources and the development of plant-derived natural products. What is outstanding is that edible flower resources have potential value in delaying aging and related diseases.
  • male flowers of Eucommia ulmoides Oliver have physiological activities such as lowering blood lipids, anti-obesity, antioxidant, and anti-fatigue.
  • iridoids are widely distributed in traditional Chinese medicine, including Eucommiaceae, Rubiaceae and other plants.
  • the purpose of the present invention is to discover new functions of Eucommia male flowers in preventing and alleviating skeletal muscle aging, and to extract active ingredients from Eucommia male flowers to fill the current research gaps in the above-mentioned effects and functional ingredients of Eucommia male flower extracts.
  • the present invention uses Caenorhabditis elegans to screen 30 kinds of common domestic edible flowers and finds that the extract of Eucommia male flowers effectively extends the healthy lifespan of nematodes, and especially improves the age-related decline in muscle function.
  • Qualitative and quantitative analysis using high-resolution mass spectrometry showed that the main anti-aging active ingredients in the above-mentioned Eucommia male flower extract are iridoids, including trifoliolin, aucubin, genipin and genipinic acid.
  • iridoids including trifoliolin, aucubin, genipin and genipinic acid.
  • human mt-Keima HEK 293T cells in vitro, it was found that these iridoids have mitophagy activating activity.
  • the preventive and alleviating effects of Eucommia male flower extract on aging-related muscle dysfunction were clarified in mammalian models.
  • the present invention provides the use of Eucommia male flower extract in the preparation of food or medicine for preventing or alleviating aging-related muscle dysfunction, and the active ingredients in the Eucommia male flower extract include iridoids.
  • the food products include human food and pet food.
  • the Eucommia male flower extract is an extract obtained by extracting the Eucommia male flower using an organic solvent aqueous solution or a pure aqueous solution, and the main active component is an iridoid extract. Specifically, the extraction temperature is 20 to 65°C.
  • the iridoid compounds include one or more of aucubin, geniposide, geniposide, and trifoliolin.
  • the prevention or alleviation of aging-related muscle dysfunction includes improving skeletal muscle mass, skeletal muscle strength, mitochondrial function and body exercise capacity.
  • the present invention conducts efficacy research on the extract of Eucommia male flowers through in vivo experiments on nematodes and mice.
  • the extract can improve skeletal muscle quality, strength, mitochondrial function and body exercise capacity, etc., indicating that it has the ability to prevent or alleviate aging-related muscle dysfunction and other anti-inflammatory properties. Muscle aging effect.
  • the aging-related muscle dysfunction is closely related to mitochondrial dysfunction of skeletal muscle, and related diseases include sarcopenia.
  • Eucommia male flower extract activates muscle cell mitophagy activity and promotes ATP synthesis and/or maintaining normal mitochondrial morphology to improve mitochondrial function and delay muscle dysfunction.
  • Eucommia male flower extract delays muscle aging by improving the mitochondrial function of skeletal muscles.
  • the present invention identifies active mitophagy components in the male flowers of Eucommia ulmoides and clarifies that the main mitophagy-enhancing active components in the male flower extracts of Eucommia ulmoides are iridoids, including trifoliolin, aucubin, and genipin. and genipinic acid.
  • the present invention specifically stipulates the extraction method based on the water-soluble properties of the Eucommia ulmoides androidene components.
  • the preparation method of the Eucommia ulmoides male flower extract includes the following steps:
  • the male flower of Eucommia ulmoides refers to the stamens of the male tree of Eucommia ulmoides Oliver.
  • the raw material used in the present invention is a dried Eucommia male flower product, which refers to a type of raw material in which fresh Eucommia male flowers or ordinary sun-dried Eucommia male flowers have been dried until the moisture content does not exceed 10%.
  • the raw materials may or may not undergo crushing pretreatment.
  • the dried Eucommia male flower products are crushed to facilitate the dissolution of the active substances.
  • step (1) a higher extraction rate is achieved by controlling the material-to-liquid ratio, extraction temperature, and raw material moisture content.
  • the moisture content of the dried Eucommia male flower product is ⁇ 10%, and the material-to-liquid ratio is 1g:40mL.
  • the extractant used in the present invention can be an aqueous solution containing a solvent or pure water.
  • the organic solvent is ethanol, methanol or n-butanol, and the volume ratio concentration of the aqueous organic solvent solution is 65% to 80%.
  • the leaching temperature is 45-60°C, and the leaching time is 20-30 hours.
  • the extraction process is combined with ultrasonic treatment, and the ultrasonic conditions are: ultrasonic frequency 200-500 kHz, ultrasonic intensity 5-15 W/cm 2 , time 1.5-3.0 h, and let stand after the ultrasonic is completed.
  • step (2) the organic solvent is removed by evaporation under reduced pressure to obtain a concentrated liquid. After adding 5 to 15 times the volume of water of the concentrated extract liquid to fully reconstitute it, filter and separate to obtain the extract liquid, which is then freeze-dried to obtain Eucommia male flower extract. .
  • the active ingredients of the male flower extract of Eucommia ulmoides prepared by the above method are mainly trifoliolin, aucubin, genipin and genipinic acid.
  • the weight content of trifoliolin is 0.05% to 5.0%; the weight content of aucubin is 0.05% to 10.0%; the weight content of geniposide is 0.1% to 5.0%; the weight of geniposide The content is 0.1% to 10.0%.
  • Eucommia male flower extract and its main active ingredient iridoid compounds have anti-muscle aging effects such as preventing and alleviating aging-related muscle dysfunction, it can be used to prepare related foods and medicines.
  • the food is a functional food or drink that has the effect of preventing or alleviating aging-related muscle dysfunction.
  • the Eucommia ulmoides male flower extract or its main active ingredient is mixed with food-acceptable auxiliary materials or auxiliary ingredients to prepare Functional food or drink.
  • the medicine is a pharmaceutical preparation that has the effect of preventing or alleviating aging-related muscle dysfunction.
  • the Eucommia male flower extract or its main active ingredient is mixed with pharmaceutically acceptable auxiliary materials or auxiliary ingredients to prepare a pharmaceutical preparation.
  • the preparation may be tablets, powders, granules, capsules, oral liquids or sustained-release preparations, etc.
  • the present invention reports for the first time that Eucommia male flower extract and the trifoliolin, aucubin, geniposide and geniposide it contains have mitophagy activating activity. These components can effectively stimulate mitophagy. And maintain normal mitochondrial function.
  • the extract has strong ability to prevent or alleviate aging-related muscle dysfunction, and has great application prospects in the fields of human food, pet food, pharmaceuticals and other fields.
  • Figure 1 is the total ion chromatogram of the Eucommia male flower extract in Example 1, where A is the anion mode and B is the cation mode.
  • Figure 2 shows the secondary mass spectrum of trifoliolin in the extract, where A is the anion mode and B is the cation mode.
  • Figure 3 shows the secondary mass spectrum of aucubin in the extract in anion mode.
  • Figure 4 shows the secondary mass spectrum of geniposide in the extract in cation mode.
  • Figure 5 shows the secondary mass spectrum of genipinic acid in the extract, where A is the anion mode and B is the cation mode.
  • Figure 6 is a quantitative spectrum of iridoid components in the extract, including 1-aucubin, 2-genipin, 3-genipinic acid, and 4-cylinopin. Among them, A is the standard solution and B is the extract sample solution.
  • Figure 7 shows the structural formula of the iridoid components in the extract.
  • Figure 8 shows the changes in movement status of the Eucommia male flower extract group and the vehicle group after feeding nematodes for 1, 5, and 9 days, where Vehicle represents the vehicle group and EUFE represents the Eucommia male flower extract group, the same below.
  • Figure 9 shows the changes in the average movement rate of the Eucommia male flower extract group and the vehicle group after feeding nematodes for 1, 5, and 9 days.
  • Figure 10 shows the changes in body wall muscle fiber morphology of the Eucommia male flower extract group and the vehicle group after feeding nematodes for 1 and 5 days.
  • Figure 11 shows the changes in muscle mitochondrial morphology in the Eucommia male flower extract group and the vehicle group after feeding nematodes for 9 days.
  • Figure 12 shows the changes in muscle mitophagy activity of the Eucommia male flower extract group, vehicle group, and positive control group after feeding nematodes for 1 day.
  • Figure 13 shows the activation of mitophagy by trifoliolin in vitro.
  • Figure 14 is a comparison chart of the mitophagy activation activities of the cylinoid components of Eucommia ulmoides, including Asp-trifoliate, Auc-aucubin, Gen-geniposide, and Gen a-geniposide. .
  • test methods used in the following examples are conventional methods unless otherwise stated; the materials and reagents used, unless otherwise stated, are commercially available reagents and materials.
  • the raw material Eucommia male flowers used in the examples were purchased from Zhangjiajie, Hunan, and dried at 40°C for more than 24 hours.
  • the monomeric components of Eucommia ulmoides androidene used in quantitative analysis and activity examples are trifoliolin (CAS: 14259-45-1), aucubin (CAS: 479-98-1), genipin ( CAS: 24512-63-8) and genipinic acid (CAS: 27741-01-1) were purchased from Chengdu Phytochemical Pure Biotechnology Co., Ltd., with purity ⁇ 98%.
  • Preparation method 1kg of dried Eucommia male flower stamens with a moisture content of ⁇ 10% (measured according to GB 5009.3-2016 direct drying method, if the moisture content requirements are not met, continue to be baked and dried at 40°C) according to a 1g:40mL material-liquid ratio, Add 80% (v/v) ethanol aqueous solution, control the temperature to 45°C, perform leaching for 2 hours at an ultrasonic frequency of 400 kHz and an ultrasonic intensity of 10 W/cm 2 to fully extract and let stand for 24 hours.
  • the extract was separated from the precipitate by centrifugation. Ethanol is recovered by evaporation under reduced pressure to obtain a concentrated solution. After adding 10 times the volume of water of the concentrated solution, stir thoroughly to redissolve, separate the extract from the insoluble matter by centrifugation, and freeze-dry the extract to obtain Eucommia male flower extract.
  • the operating parameters are set as: cone voltage 30v, capillary voltage 2kv, source temperature 100°C. Data were recorded in the mass-to-charge ratio (m/z) range of 95 to 1400, with a scan time of 0.25 s, a scan interval of 0.02 s, and a duration of 10 min. On this basis, the possible components were determined by combining the mass spectrometry database Compound Discoverer TM search matching and literature query.
  • the iridoid components of the extract were quantitatively analyzed by a second-class HPLC chromatograph equipped with a PDA detector.
  • the measurement conditions are as follows: the injection volume is 10 ⁇ L, reversed-phase ODS-2Hypersil C 18 column (4.6nm ⁇ 250nm, Thermo Fisher Scientific) was used for HPLC analysis. The column temperature was 30°C, the flow rate was 1mL/min, and the mobile phase was 0.5% phosphoric acid aqueous solution (A) and methanol (B).
  • FIG. 1 shows the corresponding total ion chromatogram.
  • four types of Eucommia ulmoides cylinoids were identified, including trifoliolin, aucubin, genipin, and genipinic acid. Their secondary mass spectra are shown in Figures 2-5.
  • the characteristic secondary mass spectrometry fragments of aucubin were only identified in the anion mode ( Figure 3), and the characteristic secondary mass spectrum fragments of geniposide were only identified in the cation mode ( Figure 4).
  • the four iridoid components were quantitatively analyzed by HPLC.
  • the model organism Caenorhabditis elegans was fed with 100 ⁇ g/mL Eucommia male flower extract (abbreviated as EUFE) in Example 1, and compared with the vehicle (water) to compare the nematode movement ability and body composition under natural aging conditions (1, 5, and 9 days). Morphological changes in wall muscle fibers.
  • EUFE Eucommia male flower extract
  • Movement ability Before the nematodes are transferred, the movement status of the nematodes is artificially recorded as follows: moving independently is marked as A, moving only after being touched is marked as C, and the state between A and C is marked as B. Then pick the nematodes belonging to the A state to the blank board and record the video. Use WormLab to analyze average movement speed. For each experiment, each group contains at least 30 worms. A more accurate description of C. elegans motility was achieved by combining these two metrics.
  • Body wall muscle fiber morphology The body wall muscle fiber morphology of C. elegans RW1596 strain was observed under bright field (BF) and GFP channels (excitation and emission wavelengths are 488nm and 510-540nm respectively).
  • M9 buffer containing 5mM levamisole On days 1 and 5, different groups of nematodes were anesthetized using M9 buffer containing 5mM levamisole and placed on 1% agarose gel on thin glass slides. Use a Zeiss LSM880 confocal microscope equipped with a 40x water immersion objective to photograph the head, middle and tail of the nematodes respectively.
  • Mitochondrial morphology Wild-type nematodes were treated with vehicle or EUFE for 9 days. After collection and cleaning, prepare TEM samples. Fix nematodes overnight using 2.5% glutaraldehyde. After fixation, rinse the sample with 0.1M, pH 7.4 phosphate buffer, post-fix it with 1% osmium tetroxide for 1 to 2 hours, and then rinse with buffer. They were then dehydrated using a gradient concentration series of ethanol and acetone, immersed in a mixture of acetone and Spurr embedding medium, and then embedded in 100% Spurr embedding medium overnight. Cure at 70°C for 36 hours.
  • Ultrathin sections (70-90nm) were made with an ultramicrotome and transferred to a 200-mesh copper grid. Grids were stained with lead citrate and uranyl acetate (saturated solution in 50% (v/v) ethanol). Sections were observed with a Hitachi H-7650 transmission electron microscope.
  • Mitophagy activity C. elegans IR1511 strain was used for mitophagy detection. This strain expresses GFP-labeled DCT-1 and DsRed-labeled LGG-1 in body wall muscles. The colocalization of GFP and DsRed indicates the fusion of mitochondria and lysosomes, that is, the occurrence of mitophagy. DsRed is excited with a 561nm laser. The treatment time of both vehicle and EUFE was 1 day, and CCCP (carbonyl cyanide-3-chlorophenylhydrazone), a mitophagy inducer, was used as a positive control. Photographed using a Zeiss LSM 880 confocal microscope equipped with an 83x oil immersion objective.
  • CCCP carbonyl cyanide-3-chlorophenylhydrazone
  • EUFE treatment for 1 day increased the expression of LGG-1 in the C. elegans body wall muscles. More importantly, EUFE adds LGG-1 Co-localization with DCT-1, indicating enhanced mitophagy activity.
  • DCT-1 NIX/BNIP3L homolog
  • the culture medium was replaced until the cell density reached 80-90%, and then the cells were digested and passaged into T25 bottles. After growth, the transfection efficiency was observed using a fluorescence microscope. Use complete culture medium containing 0.5 ⁇ g/mL puromycin hydrochloride solution to select cells that were successfully transfected, and maintain them with 0.25 ⁇ g/mL to obtain mt-Keima cells.
  • Each confocal dish was inoculated with 1 mL, and the cell density was 2 ⁇ 10 5 cells/mL. After standing for 5 to 10 minutes, it was gently placed in the incubator. Dosage is given after the cells have grown to 30-40%. After treating mt-Keima cells with 10 ⁇ M of different Eucommia ulmoides ether monomer components or 10 ⁇ M CCCP for 6 hours, they were replaced with fresh complete culture medium and directly subjected to live cell confocal observation. Confocal parameters: excitation wavelength is 440nm and 596nm respectively, emission wavelength is 620nm, 40X water mirror observation.
  • mice 18-month-old ICR male elderly mice purchased from the Animal Research Center of East China Normal University were housed in single cages and 5-6 month-old BALB/c male young mice were housed in shared cages in a specific pathogen-free Zhejiang Traditional Chinese Medicine Animal House. in this issue During this time, mice could eat and drink freely. The animal room had a 12-h dark/light cycle, and the temperature and humidity were maintained at 23 ⁇ 2 °C and 50% ⁇ 5%, respectively. All procedures and protocols in this experiment were approved by the Animal Ethics Committee of Zhejiang University of Traditional Chinese Medicine.
  • mice After the mice adapted for two weeks, the mice were divided into the following five groups, including the young mouse group, the control elderly group, and the low, medium, and high treatment groups of Eucommia male flower extract (respectively administered by gavage, 25, 50, 100 mg/ kg/d), 8 mice in each group.
  • the young mouse group and the control group were gavaged with equal volumes of physiological saline. Except for the young mouse group that was fed normal standard feed, the remaining four groups of mice were fed 42% high-fat feed.
  • mice in each group were sacrificed, and four muscles of the left hind limb of the mice in each group were carefully removed, including gastrocnemius (GA), soleus (SOL), and tibialis anterior (TA). ) and extensor digitorum longus (EDL), were weighed separately.
  • GA gastrocnemius
  • SOL soleus
  • TA tibialis anterior
  • EDL extensor digitorum longus
  • mice were trained monthly to grasp the horizontal bar of the grip meter and gently pull back until they could no longer resist the pulling force and released the horizontal bar. The force is recorded by a dynamometer.
  • mice to perform adaptive running on a treadmill at a speed of 10m/min and 0° for two days. On the third day, an exhaustive running test was conducted. Starting at 5m/min, the slope angle is 0°, then the speed and slope angle increase by 5m and 5° every 5min until they reach 20m/min and 14° respectively. When the mouse does not return to the track for more than 20 seconds and shows a significantly diminished response to external stimuli, record the running time in detail.
  • * represents the comparison between the elderly control group and the young group, * represents P ⁇ 0.05, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001;
  • # represents the comparison between the low, medium and high dose groups of Example 1 and the elderly control group, # represents P ⁇ 0.05, ## represents P ⁇ 0.01, ### represents P ⁇ 0.001;
  • the ATP content was detected using the ATP detection kit produced by Biyuntian Company. According to every 20mg shin Add approximately 100 to 200 ⁇ L of lysis solution to the preosseous muscle (TA) tissue, and then homogenize it. Homogenize thoroughly to ensure complete tissue lysis. After lysis, centrifuge at 12,000g for 5 minutes at 4°C, and take the supernatant for subsequent determination. Preparation for standard curve measurement: Melt the reagents to be used in an ice bath, and dilute the ATP standard solution with ATP detection lysate into an appropriate concentration gradient. The specific concentration depends on the concentration of ATP in the tissue sample.
  • Preparation of ATP detection working solution Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 ⁇ L ATP detection working solution for each sample or standard. Thaw reagents to be used in an ice bath. Take an appropriate amount of ATP detection reagent and dilute the above detection reagent with diluent at a ratio of 1:9. Add 100 ⁇ L ATP detection working solution into the detection tube. Leave it at room temperature for 3 to 5 minutes so that all the background ATP is consumed, thereby reducing the background. Add 20 ⁇ L of sample or standard into the detection tube, mix quickly with a gun (micropipette), and after at least 2 seconds, measure the RLU value with a chemiluminescence meter. Convert the corresponding ATP content according to the standard curve. The results are shown in Table 4.
  • # represents the comparison between the low, medium and high dose groups of Example 1 and the elderly control group, ### represents P ⁇ 0.001.
  • Mitochondria are the main sites for oxidative phosphorylation and synthesis of adenosine triphosphate (ATP) in cells, providing chemical energy for cellular activities.
  • the ATP content in skeletal muscle reflects the normal level of mitochondrial function in this tissue to a certain extent.
  • Table 4 the ATP content in the tibialis anterior muscle of aged mice decreased under the influence of aging and high-fat diet, and the intervention of Eucommia male flower extract effectively prevented the reduction of ATP, indicating that Eucommia male flower extract can effectively prevent bone loss during aging. Decline in muscle mitochondrial function.
  • mice Young ICR male mice aged 5 to 6 months purchased from the Animal Research Center of East China Normal University were maintained in a specific pathogen-free Zhejiang Traditional Chinese Medicine Animal House. During this period, mice had free access to food and water. The animal room had a 12-h dark/light cycle, and the temperature and humidity were maintained at 23 ⁇ 2 °C and 50% ⁇ 5%, respectively. All procedures and protocols in this experiment were approved by the Animal Ethics Committee of Zhejiang University of Traditional Chinese Medicine.
  • mice After the mice adapted for two weeks, the mice were divided into the following five groups, including the control group, the model group, and the low, medium, and high intervention groups of Eucommia male flower extract after modeling (respectively administered by gavage, 25, 50, 100 mg /kg/d), 8 mice in each group. Among them, the control group was intragastrically administered with an equal volume of normal saline. All mice were fed standard chow. The modeling method was to perform d-galactose-induced aging modeling, and d-galactose (60 mg/kg body weight/day, intraperitoneally 0.5 mL) was administered for 1.5 months. After Eucommia male flower extract intervened in the above-mentioned aging mouse model for 3 months, the indicators of activity detection examples 4 and 5 were measured. The results are shown in Table 5-8.
  • * represents the comparison between the elderly control group and the young group, * represents P ⁇ 0.05, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001;
  • # represents the comparison between the low, medium and high dose groups of Example 1 and the elderly control group, # represents P ⁇ 0.05, ## represents P ⁇ 0.01, ### represents P ⁇ 0.001;
  • Experimental data tables 5 to 8 show that the reduction in skeletal muscle mass induced by d-galactose is mainly reflected in the gastrocnemius (GA), tibialis anterior (TA), and soleus (SOL) muscles.
  • the reduction in soleus muscle mass is related to natural aging. There is a difference.
  • the Eucommia male flower extract prepared in Example 1 can effectively reduce the loss of gastrocnemius (GA), tibialis anterior (TA), and soleus (SOL) muscles.
  • low, medium and high dose intervention of Eucommia male flower extract can improve d-galactose induction Reductions in forelimb grip strength and running time to failure.
  • Eucommia male flower extract has the effect of alleviating aging-related muscle dysfunction.
  • Eucommia male flower extract 34.9% (prepared in Example 1), xylitol 40%, fructooligosaccharide 5%, inulin 5%, maltodextrin 15%, citric acid 0.1%.
  • the uniformly stirred materials are quantitatively packaged and then sterilized by microwave.
  • the microwave sterilization frequency is 2000MHz.
  • the temperature of the beverage is controlled at 65°C.
  • the sterilization time is determined according to the beverage flow and microwave power. After sterilization, the total number of colonies and yeast content of the beverage do not exceed the national standard.

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Abstract

公开了杜仲雄花提取物在制备抗肌肉衰老的食品或药物中的应用，属于植物提取物技术领域。杜仲雄花提取物主要活性成分为环烯醚萜类，其组成包括桃叶珊瑚苷、京尼平苷、京尼平苷酸和车叶草苷。首次公开杜仲雄花提取物及其含有的环烯醚萜类化合物具有线粒体自噬激活活性，通过有效刺激线粒体自噬而维持线粒体功能正常。该提取物具有较强的预防或缓解衰老相关肌肉功能减退能力。

Description

杜仲雄花提取物在制备抗肌肉衰老的食品或药物中的应用 技术领域
本发明涉及植物提取物技术领域，具体涉及含环烯醚萜类成分的杜仲雄花提取物在制备抗肌肉衰老的食品(包括宠物食品)或药物中的应用。
背景技术
骨骼肌是人体运动系统的动力，其衰老是机体衰老的重要标志。作为脊椎动物中最丰富的塑性器官，它在新陈代谢、运动、呼吸、保护、日常身体活动以及保持姿势、平衡方面发挥着重要作用。2016年肌肉减少症(特征是渐进性和全身性的骨骼肌质量和功能丧失)被正式确认为一种疾病，代码ICD-10-CM(M62.84)，引起了人们对这种年龄相关疾病的额外关注。其中衰老相关肌肉功能下降是肌肉减少症发生、发展的重要特征。
肌肉减少症，尤其是衰老相关的肌肉功能下降与骨骼肌的线粒体功能障碍密切相关。清除功能障碍线粒体以维持衰老过程中线粒体稳态主要通过线粒体特异性自噬方式，即线粒体自噬进行。但线粒体自噬活性在肌肉衰老过程中会降低。这些都表明了线粒体自噬对衰老过程中肌肉功能维持的重要性。近年来，研究表明，通过施用单一天然化合物(如尿石素A、亚精胺、番茄碱)来增强线粒体自噬，对老化的骨骼肌、心脏和神经元产生有益影响(Urolithin A induces mitophagy and prolongs lifespan in C.elegans and increases muscle function in rodents,Nat Med 2016；22:879-88.Mitophagy inhibits amyloid-βand tau pathology and reverses cognitive deficits in models of Alzheimer’s disease,Nat Neurosci 2019；22:401-12.Cardioprotection and lifespan extension by the natural polyamine spermidine,Nat Med 2016；22:1428-38.Tomatidine enhances lifespan and healthspan in C.elegans through mitophagy induction via the SKN-1/Nrf2 pathway,Sci.Rep 2017；7:46208.)。这些研究表明线粒体自噬增强剂可能为预防和缓解衰老肌肉功能减退提供新的选择。
天然产物尤其植物化学素是线粒体自噬调节和抗肌肉衰老活性化合物的潜在来源。近年来，食用花卉成为食物资源消费和植源性天然产物开发的新趋势之一。突出的是，食用花卉资源具有潜在的延缓衰老及其相关疾病价值。作为我国卫生部批准的 一种新食品原料，杜仲雄花(male flowers of Eucommia ulmoides Oliver)具有降血脂、抗肥胖、抗氧化、抗疲劳等生理活性。作为杜仲雄花主要的功能活性成分，环烯醚萜类化合物在传统中药中分布广泛，包括杜仲科、茜草科等植物，具有抗炎、抗肥胖、抗菌、抗肿瘤、神经保护等药理作用，也是中药中重要的一类质控成分。但是，杜仲雄花及其功能活性成分对肌肉衰老及其相关功能减退的预防和缓解作用未见报道。
发明内容
本发明的目的在于挖掘杜仲雄花在预防和缓解骨骼肌衰老功效方面的新功能，并对杜仲雄花中的有效成分进行提取，以填补目前杜仲雄花提取物在上述功效和功效成分的研究空缺。
为实现上述目的，本发明采用如下技术方案：
本发明利用秀丽隐杆线虫对30种国内常见食用花卉进行筛选发现，杜仲雄花提取物有效延长了线虫健康寿命，尤其是改善了年龄相关的肌肉功能下降。利用高分辨质谱定性、定量分析表明，上述杜仲雄花提取物中主要抗衰老活性成分为环烯醚萜类，包括车叶草苷、桃叶珊瑚苷、京尼平苷和京尼平苷酸。利用体外人源mt-Keima HEK 293T细胞，发现这些环烯醚萜类具有线粒体自噬激活活性。进一步地，在哺乳动物模型上明确了杜仲雄花提取物对衰老相关肌肉功能减退的预防和缓解作用。
因此，本发明提供了杜仲雄花提取物在制备预防或缓解衰老相关肌肉功能减退的食品或药物中的应用，所述杜仲雄花提取物中活性成分包括环烯醚萜类化合物。
所述食品包括人类食品和宠物食品。
所述杜仲雄花提取物为利用有机溶剂水溶液或纯水溶液对杜仲雄花进行浸提后获得的主要活性成分为环烯醚萜类的提取物。具体的，浸提温度为20～65℃。
进一步的，所述环烯醚萜类化合物包括桃叶珊瑚苷、京尼平苷、京尼平苷酸、车叶草苷中的一种或多种。
所述预防或缓解衰老相关肌肉功能减退包括改善骨骼肌质量、骨骼肌力量、线粒体功能和机体运动能力。本发明通过线虫和小鼠体内实验对杜仲雄花提取物进行功效研究，该提取物能够改善骨骼肌质量、力量、线粒体功能和机体运动能力等，表明其具有预防或缓解衰老相关肌肉功能减退等抗肌肉衰老功效。
所述衰老相关肌肉功能减退与骨骼肌的线粒体功能障碍密切相关，相关疾病包括肌肉减少症。
进一步的，所述杜仲雄花提取物通过激活肌肉细胞线粒体自噬活性、促进ATP 的合成和/或维持线粒体形态的正常来改善线粒体功能以延缓肌肉功能减退。杜仲雄花提取物通过改善骨骼肌的线粒体功能以延缓肌肉衰老。
本发明对杜仲雄花中线粒体自噬活性成分进行鉴定，明确杜仲雄花提取物中的主要线粒体自噬增强活性成分为环烯醚萜类，包括车叶草苷、桃叶珊瑚苷、京尼平苷和京尼平苷酸。为了保证提取物中含有上述活性成分，本发明以杜仲雄花环烯醚萜成分易溶于水的性质为指导对提取方法进行具体规定。
所述杜仲雄花提取物的制备方法包括以下步骤：
(1)将杜仲雄花干制品按照1g：20mL～60mL的料液比加入到体积比为0％～90％的有机溶剂水溶液中，于20～65℃条件下浸提，分离得到萃取液；
(2)去除萃取液中有机溶剂，再加水充分复溶后分离得到提取液，干燥后制得杜仲雄花提取物。
所述杜仲雄花指杜仲(Eucommia ulmoides Oliver)雄株树花蕊。
本发明采用的原料为杜仲雄花干制品，指新鲜杜仲雄花或普通晒干杜仲雄花经干燥至水分含量不超过10％的一类原料。原料可进行粉碎预处理，也可不经过粉碎预处理。优选的，对杜仲雄花干制品进行粉碎，有利于活性物质的溶出。
步骤(1)中，通过控制料液比、提取温度以及原料含水量，综合实现较高的提取率。
优选的，所述杜仲雄花干制品的含水量≤10％，料液比为1g：40mL。
本发明采用的提取剂可以为含有剂溶剂的水溶液，也可以为纯水。优选的，所述有机溶剂为乙醇、甲醇或正丁醇，有机溶剂水溶液的体积比浓度为65％～80％。
优选的，浸提的温度为45～60℃，浸提的时间为20～30h。
优选的，浸提过程中结合超声处理，超声条件为：超声频率200～500kHz，超声强度5～15W/cm2，时间为1.5～3.0h，超声结束后静置。
步骤(2)中，利用减压蒸发去除有机溶剂，得到浓缩液，加入浓缩萃取液体积的5～15倍水充分复溶后，过滤分离得到提取液，再经冷冻干燥后得到杜仲雄花提取物。
由上述方法制备得到的杜仲雄花提取物的活性成分以车叶草苷、桃叶珊瑚苷、京尼平苷和京尼平苷酸为主。其中车叶草苷的重量含量为0.05％～5.0％；桃叶珊瑚苷的重量含量为0.05％～10.0％；京尼平苷的重量含量为0.1％～5.0％；京尼平苷酸的重量含量为0.1％～10.0％。考虑到杜仲雄花提取物及其主要活性成分环烯醚萜类化合物具有预防和缓解衰老相关肌肉功能障碍等抗肌肉衰老功效，因此可用于制备相关的食品和药品。
所述食品为具有预防或缓解衰老相关肌肉功能减退功效的功能性食品或饮品。将所述杜仲雄花提取物或其主要活性成分配以食品上可接受的辅料或辅助性成分制备成 功能性食品或饮品。
所述药品为具有预防或缓解衰老相关肌肉功能减退功效的药物制剂。将所述杜仲雄花提取物或其主要活性成分配以药品上可接受的辅料或辅助性成分制备成药物制剂。所述制剂可以为片剂、粉剂、颗粒剂、胶囊、口服液或缓释剂等。
本发明具备的有益效果：
(1)本发明首次报道杜仲雄花提取物及其含有的车叶草苷、桃叶珊瑚苷、京尼平苷和京尼平苷酸具有线粒体自噬激活活性，这些成分能有效刺激线粒体自噬而维持线粒体功能正常。该提取物具有较强的预防或缓解衰老相关肌肉功能减退能力，在人类食品、宠物食品、药品等领域中具有极大的应用前景。
(2)通过本发明技术实现了杜仲雄花中延缓肌肉衰老功效成分的有效提取，严格控制原料含水量以及浸提条件以提高提取物得率，进一步的，在浓缩萃取液后，增加复溶工艺有助于水溶性的杜仲雄花环烯醚萜成分溶解，增加其含量，得到的杜仲雄花提取物质量好、品质稳定，并且工艺操作简便，对生产设备要求较低，利于产业化发展。
附图说明
图1为实施例1中杜仲雄花提取物总离子流图，其中A为阴离子模式，B为阳离子模式。
图2为提取物中车叶草苷的二级质谱图，其中A为阴离子模式，B为为阳离子模式。
图3为提取物中桃叶珊瑚苷的阴离子模式下二级质谱图。
图4为提取物中京尼平苷的阳离子模式下二级质谱图。
图5为提取物中京尼平苷酸的二级质谱图，其中A为阴离子模式，B为阳离子模式。
图6为提取物中环烯醚萜类成分定量谱图，1-桃叶珊瑚苷，2-京尼平苷，3-京尼平苷酸，4-车叶草苷。其中A为标准品溶液，B为提取物样品溶液。
图7为提取物中环烯醚萜类成分的结构式。
图8为杜仲雄花提取物组和载体组饲喂线虫1,5,9天后运动状态变化图，其中Vehicle表示载体组，EUFE表示杜仲雄花提取物组，下同。
图9为杜仲雄花提取物组和载体组饲喂线虫1,5,9天后运动平均速率变化图。
图10为杜仲雄花提取物组和载体组饲喂线虫1和5天后体壁肌纤维形态变化图。
图11为杜仲雄花提取物组和载体组饲喂线虫9天后肌肉线粒体形态变化图。
图12为杜仲雄花提取物组、载体组、阳性对照组饲喂线虫1天肌肉线粒体自噬活性变化图。
图13为车叶草苷体外激活线粒体自噬图。
图14为杜仲雄花环烯醚萜类成分线粒体自噬激活活性比较图，其中，Asp-车叶草苷，Auc-桃叶珊瑚苷，Gen-京尼平苷，Gen a-京尼平苷酸。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例仅用于说明本发明，不用来限制本发明的适用范围。在不背离本发明精神和本质的情况下，对本发明方法、步骤或条件所做的修改或替换，均属于本发明的范围。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例中采用的原料杜仲雄花购自湖南张家界，统一经40℃烘制24h以上。
定量分析和活性例中采用的杜仲雄花环烯醚萜单体成分车叶草苷(CAS：14259-45-1)、桃叶珊瑚苷(CAS：479-98-1)、京尼平苷(CAS：24512-63-8)和京尼平苷酸(CAS：27741-01-1)购自成都植标化纯生物技术有限公司，纯度均≥98％。
实施例1
1、杜仲雄花提取物的制备
制备方法：将1kg干燥的水分含量≤10％(按照GB 5009.3-2016直接干燥法进行测定，若不符合水分含量要求则继续40℃烘制干燥)的杜仲雄花花蕊按1g：40mL料液比，加入80％(v/v)的乙醇水溶液，控制温度为45℃，于超声频率400kHz，超声强度10W/cm2，进行浸提2h以充分萃取并静置24h。
通过离心将萃取液与沉淀物分离。通过减压蒸发回收乙醇，得到浓缩液。再加入浓缩液体积的10倍水后，搅拌充分进行复溶，通过离心将提取液与不溶物分离，提取液经冷冻干燥后即得到杜仲雄花提取物。
2、对所得提取物进行定性、定量分析
1)提取物的定性分析—UPLC-QE高分辨质谱
通过UPLC-QE高分辨质谱仪对提取物进行成分定性分析。测定条件为ACQUITY BEH C18柱(2.1mm×100mm，Waters)用于UPLC分析，柱温为40℃，流速 为0.4mL/min，流动相为0.1％甲酸水溶液(A)和0.1％甲酸-乙腈溶液(B)。流动相的梯度程序如下：0min(A:B＝95:5)、3min(A:B＝75:25)、4min(A:B＝35:65)和10min(A:B＝35:65)。进样量为10μL。QE在正负离子模式下工作。操作参数设定为：锥电压30v，毛细管电压2kv，源温度100℃。在95～1400的质荷比(m/z)范围内记录数据，扫描时间为0.25s，扫描间隔为0.02s，持续10min。在此基础上，结合质谱数据库Compound DiscovererTM检索匹配和文献查询，确定可能的成分。
2)提取物中环烯醚萜成分的定量分析—UPLC-PDA色谱
通过配备PDA检测器二等HPLC色谱仪对提取物进行环烯醚萜成分定量分析。测定条件为进样量为10μL，反相ODS-2Hypersil C18柱(4.6nm×250nm，Thermo Fisher Scientific)用于HPLC分析，柱温为30℃，流速为1mL/min，流动相为0.5％磷酸水溶液(A)和甲醇(B)。流动相梯度程序如下：0min(A:B＝95:5)、30min(A:B＝85:15)和55min(A:B＝70:30)。
使用UHPLC-QE-MS在阴、阳离子模式下对杜仲雄花上述提取物进行了定性分析，图1为相应的总离子流图。其中杜仲雄花环烯醚萜共鉴定出四种，包括车叶草苷、桃叶珊瑚苷、京尼平苷、京尼平苷酸，它们的二级质谱图分别为图2-5。其中桃叶珊瑚苷特征性二级质谱碎片只在阴离子模式下鉴定出(图3)，京尼平苷特征性二级质谱碎片只在阳离子模式下鉴定出(图4)。进一步的，通过HPLC对这四种环烯醚萜成分进行了定量分析。标准溶液和提取物样品溶液的HPLC色谱图分别如图6A和6B所示。需要强调的是，桃叶珊瑚苷在236nm处几乎没有吸收，而206nm是对其定量的合适的紫外吸收设置条件。结果表明，京尼平苷酸含量最高，为6.39±0.33％，桃叶珊瑚苷含量为6.05±0.22％，京尼平苷含量为1.85±0.16％，车叶草苷含量最低，为0.56±0.01％。上述四种环烯醚萜类成分的结构式如图7所示。
活性检测例1
通过模式生物秀丽隐杆线虫饲喂100μg/mL实施例1中杜仲雄花提取物(缩写为EUFE)，和载体(水)对照比较自然衰老情况下(1，5，9天)线虫运动能力和体壁肌纤维形态变化。具体方法如下：
1、运动能力：线虫在转移前，人为记录线虫的运动状态如下：自主移动，记为A，仅在被触摸后移动，记为C，A和C之间的状态，记为B。然后将属于A状态的线虫挑到空白板上并进行视频拍摄。使用WormLab分析平均移动速度。对于每个实验，每组至少包含30条蠕虫。通过结合这两个指标更准确地描述线虫运动能力。
结果如图8、9所示，与载体相比，EUFE处理9天后，更多的线虫群体(A状态)能够自主移动，并且它们的平均移动速度明显更快。
2、体壁肌纤维形态：线虫RW1596株系在明场(BF)和GFP通道(激发和发射波长分别为488nm和510～540nm)下进行体壁肌纤维形态观察用载体或100μg/mL EUFE预处理蠕虫1天和5天，使用含有5mM左旋咪唑的M9缓冲液对不同组别线虫进行麻醉，并将其放置在薄载玻片上1％琼脂糖胶上。使用配备40x水浸物镜的Zeiss LSM880共聚焦显微镜分别拍摄线虫头部、中间和尾部。
结果如图10所示，对于体壁肌纤维形态，第1天两组线虫的头、中、尾部体壁肌纤维组织完整，排列有序。然而，到第5天，如红色箭头所示，载体组的线虫在头部、中部和尾部均出现明显的断裂、缺失或排列紊乱。相比之下，EUFE处理组线虫的肌肉形态保持相对良好的状态。
活性检测例2
杜仲雄花提取物饲喂对线虫体壁肌细胞中线粒体健康和线粒体自噬活性影响研究。具体方法如下：
1、线粒体形态：野生型线虫用载体或EUFE处理9天。收集和清洁后，制备透射电镜样品。使用2.5％戊二醛过夜固定线虫。固定后，用0.1M，pH 7.4磷酸盐缓冲液冲洗样品，用1％四氧化锇后固定1～2h，再用缓冲液冲洗。然后用乙醇系列梯度浓度和丙酮脱水，浸入丙酮和Spurr包埋剂的混合物中，然后嵌入100％Spurr包埋剂中过夜。在70℃下固化36小时。用超薄切片机进行超薄切片(70～90nm)并转移到200目铜网格上。网格用柠檬酸铅和乙酸双氧铀(50％(v/v)乙醇中的饱和溶液)染色。切片由日立H-7650透射电镜观察。
结果如图11所示，载体组线虫生长9天后，它们的肌肉线粒体外膜出现缺陷，线粒体脊出现减少和模糊。相反，EUFE处理很好地保持了线粒体形态。
2、线粒体自噬活性：线虫IR1511株系用于线粒体自噬检测，这株系在体壁肌肉中表达GFP标记的DCT-1和DsRed标记的LGG-1。GFP和DsRed的共定位表明线粒体与溶酶体的融合，即线粒体自噬的发生。DsRed用561nm激光激发。载体和EUFE的处理时间均为1天，CCCP(羰基氰酯-3-氯苯基腙)是一种线粒体自噬诱导剂作为阳性对照。使用配备83x油浸物镜的Zeiss LSM 880共聚焦显微镜进行拍摄。
通过使用IR1511株系，如图12所示，与线粒体自噬诱导剂CCCP一致地，EUFE处理1天增加了线虫体壁肌肉中LGG-1的表达。更重要的是，EUFE增加了LGG-1 与DCT-1的共定位，表明增强线粒体自噬活性。DCT-1(NIX/BNIP3L同源物)位于线粒体外膜，是线粒体自噬的关键调节因子。
活性检测例3
杜仲雄花环烯醚萜类成分包括车叶草苷、桃叶珊瑚苷、京尼平苷和京尼平苷酸体外激活线粒体自噬活性研究。具体方法如下：
按正常细胞培养、传代方式培养293T细胞，于24孔板接种，每孔接种0.5mL，细胞密度为5×104cell/mL，共计接入2.5×104cell，接板前把离心管里的细胞吹打均匀，以保证开始和最后的细胞密度一致，静置5～10分钟后，轻轻放置培养箱中。隔夜后，移走原完全培养基，每孔加入含有1.5×105TU慢病毒完全培养基溶液0.3mL，37℃培育8h后每孔补200μL完全培养基。转染24h后更换培养基至细胞密度达到80-90％后消化传代到T25瓶中。长满后用荧光显微镜观察转染效率。使用含0.5μg/mL嘌呤霉素盐酸盐溶液的完全培养基筛选转染成功的细胞，并用0.25μg/mL进行维持，即获得mt-Keima细胞。
共聚焦皿每皿接种1mL，细胞密度为2×105cell/mL，静置5～10min后，轻轻放置培养箱中。待细胞长至30～40％给药。用10μM不同杜仲雄花环烯醚萜单体成分或10μM CCCP处理mt-Keima细胞6h后，换成新鲜完全培养基后直接进行活细胞共聚焦观察。共聚焦参数：激发波长分别为440nm和596nm，发射波长为620nm，40X水镜观察。
结果如图13和图14所示，CCCP阳性药物处理显著引起293T细胞线粒体自噬的发生。杜仲雄花中以车叶草苷为代表单体成分同样也具有线粒体自噬激活活性。且通过平均荧光密度比值比较，同浓度下车叶草苷效果优于其他三种单体成分。结合杜雄雄花提取物可能通过线粒体自噬活性激活改善线粒体功能以延缓肌肉衰老，因此上述具有线粒体自噬激活活性的环烯醚萜类成分是杜仲雄花提取物发挥预防或缓解肌肉衰老功效的主要活性成分。
活性检测例4
杜仲雄花提取物饲喂18月龄ICR老年小鼠延缓其肌肉功能减退活性研究。具体方法如下：
购买自华东师范大学动物研究中心的18月龄ICR雄性老年小鼠单笼饲养和5～6月龄BALB/c雄性年轻小鼠合笼饲养在无特定病原体的浙江中医药动物房中。在此期 间，小鼠可以自由进食和饮水。动物房的暗/光循环为12小时，温度和湿度分别保持在23±2℃和50％±5％。本实验中的所有程序和方案均经浙江中医药大学动物伦理委员会标准批准。
小鼠适应两周后，将小鼠分为以下五组，包括年轻小鼠组、对照老年组、杜仲雄花提取物低、中、高处理组(分别灌胃给药，25,50，100mg/kg/d)，每组小鼠8只。其中，年轻小鼠组和对照组用等体积生理盐水灌胃。除年轻小鼠组饲喂正常标准饲料外，其余四组小鼠饲喂42％高脂饲料。
1)骨骼肌质量：干预各组小鼠3个月后，经处死小鼠，小心切除各组小鼠左后肢四块肌肉，包括腓肠肌(GA)、比目鱼肌(SOL)、胫骨前肌(TA)和趾长伸肌(EDL)，分别进行称重。
2)前肢握力：干预过程中，每月训练小鼠抓住水平放置的抓力计单杠，并轻轻向后拉，直到它们无法抵抗拉力并释放单杠。力由测力计记录。
3)力竭跑步时间：训练小鼠在跑步机上以10m/min的速度和0°进行自适应跑步两天。第三天，进行了详尽的跑步测试。以5m/min开始，坡度角为0°，然后速度和坡度角每5min增加5m和5°，直至分别达到20m/min和14°。当小鼠超过20秒没有返回轨道并且表现出对外部刺激的反应明显减弱时，记录详尽的跑步时间。
结果见表1-3。
表1杜仲雄花提取物对自然衰老小鼠骨骼肌质量的变化影响(n＝8)
注：*表示为老年对照组与年轻组比较，*代表P<0.05，**代表P<0.01，***代表P<0.001；
#表示为实施例1低、中、高剂量组与老年对照组比较，#代表P<0.05，##代表P<0.01，###代表P<0.001；
下同。
表2杜仲雄花提取物对自然衰老小鼠前肢握力的变化影响(n＝8)
表3杜仲雄花提取物对自然衰老小鼠力竭跑步时间的变化影响(n＝8)
如表1所示，比目鱼肌(SOL)和趾长伸肌(EDL)受衰老和高脂饮食影响变化较小，而腓肠肌(GA)和胫骨前肌(TA)受衰老和高脂饮食影响变化较大。老年小鼠呈现了质量减少的趋势。实施例1的杜仲雄花提取物干预预防了上述肌肉类型质量的减少，并且呈现剂量依赖性。如表2和表3所示，除了骨骼肌质量外，骨骼肌功能随着衰老也出现了下降，具体表现为衰老小鼠高脂饮食干预三个月后，前肢握力和力竭跑步时间出现明显的下降。而低、中、高剂量的实施例1杜仲雄花提取物干预均能预防骨骼肌上述功能的下降。因此，杜仲雄花提取物具有预防衰老相关肌肉功能减退的功效。
活性检测例5
杜仲雄花提取物饲喂18月龄ICR老年小鼠对其肌肉ATP含量影响研究。具体方法如下：
采用碧云天公司生产的ATP检测试剂盒进行ATP含量的检测。按照每20mg胫 骨前肌(TA)组织加入约100～200μL裂解液，然后用进行匀浆。充分匀浆以确保组织被完全裂解。裂解后4℃，12000g离心5min，取上清，用于后续的测定。标准曲线测定的准备：冰浴上融解待用试剂，把ATP标准溶液用ATP检测裂解液稀释成适当的浓度梯度。具体的浓度需根据组织样品中ATP的浓度而定。ATP检测工作液的配制：按照每个样品或标准品需100μL ATP检测工作液的比例配制适当量ATP检测工作液。把待用试剂在冰浴上融解。取适量的ATP检测试剂，按照1:9的比例用稀释液稀释上述检测试剂。加100μL ATP检测工作液到检测管内。室温放置3～5min，以使本底性的ATP全部被消耗掉，从而降低本底。在检测管内加上20μL样品或标准品，迅速用枪(微量移液器)混匀，至少间隔2秒后，用化学发光仪测定RLU值。根据标准曲线换算相应ATP含量。结果见表4。
表4杜仲雄花提取物对自然衰老小鼠胫骨前肌中ATP含量变化影响(n＝8)
注：*表示为老年对照组与年轻组比较，***代表P<0.001；
#表示为实施例1低、中、高剂量组与老年对照组比较，###代表P<0.001。
线粒体是细胞内氧化磷酸化和合成三磷酸腺苷(ATP)的主要场所，为细胞的活动提供了化学能量。骨骼肌中ATP的含量一定程度上反映了该组织中线粒体功能正常水平。如表4所示，老年小鼠经衰老和高脂饮食影响，胫骨前肌中ATP含量减少，而杜仲雄花提取物干预有效预防了ATP的减少，表明杜仲雄花提取物能有效预防衰老过程中骨骼肌线粒体功能的下降。
活性检测例6
杜仲雄花提取物饲喂d-半乳糖诱导的5～6月龄ICR衰老小鼠缓解其肌肉功能减退活性研究。具体方法如下：
购买自华东师范大学动物研究中心的5～6月龄ICR雄性年轻小鼠饲养在无特定病原体的浙江中医药动物房中。在此期间，小鼠可以自由进食和饮水。动物房的暗/光循环为12小时，温度和湿度分别保持在23±2℃和50％±5％。本实验中的所有程序和方案均经浙江中医药大学动物伦理委员会标准批准。
小鼠适应两周后，将小鼠分为以下五组，包括对照组、模型组、经造模后杜仲雄花提取物低、中、高干预组(分别灌胃给药，25，50，100mg/kg/d)，每组小鼠8只。其中，对照组用等体积生理盐水灌胃。所有小鼠饲喂标准饲料。造模方法为进行d-半乳糖诱导衰老造模，给予d-半乳糖(60mg/kg体重/天，腹膜内0.5mL)1.5个月。杜仲雄花提取物干预上述衰老小鼠模型3个月后，进行活性检测例4和5各指标的测定。结果见表5-8。
表5杜仲雄花提取物对d-半乳糖诱导的衰老小鼠骨骼肌质量的变化影响(n＝8)
注：*表示为老年对照组与年轻组比较，*代表P<0.05，**代表P<0.01，***代表P<0.001；
#表示为实施例1低、中、高剂量组与老年对照组比较，#代表P<0.05，##代表P<0.01，###代表P<0.001；
下同。
表6杜仲雄花提取物对d-半乳糖诱导的衰老小鼠前肢握力的变化影响(n＝8)

表7杜仲雄花提取物对d-半乳糖诱导的衰老小鼠力竭跑步时间的变化影响(n＝8)
表8杜仲雄花提取物对d-半乳糖诱导的衰老小鼠胫骨前肌中ATP含量变化影响(n＝8)
实验数据表5～8显示，d-半乳糖诱导的骨骼肌质量减少主要体现在腓肠肌(GA)、胫骨前肌(TA)、比目鱼肌(SOL)上，其中比目鱼肌质量减少与自然衰老情况下有所不同。实施例1制备的杜仲雄花提取物能有效减少腓肠肌(GA)、胫骨前肌(TA)、比目鱼肌(SOL)的损失。对于骨骼肌功能，杜仲雄花提取物低、中、高剂量干预能改善d-半乳糖诱导 的前肢握力和力竭跑步时间的降低。同样地，杜仲雄花提取物低、中、高剂量干预改善了d-半乳糖诱导的小鼠胫骨前肌中ATP含量的下降。因此，杜仲雄花提取物具有缓解衰老相关肌肉功能减退的功效。
应用例1固体饮料制备
杜仲雄花提取物34.9％(实施例1所制)，木糖醇40％，低聚果糖5％，菊粉5％，麦芽糊精15％，柠檬酸0.1％。
其制备方法按照如上配方，依次进行如下步骤：
(1)按照上述配方比例称重、混料并均匀搅拌，干燥至水分含量≤5％，得到杜仲雄花提取物固体饮料制品。其余物料均为市售的质量合格产品。
(2)将搅拌均匀的物料定量包装后进行微波杀菌。其中微波杀菌频率为2000MHz，杀菌过程中饮料的温度控制在65℃，杀菌时间根据饮品流量及微波功率而定，灭菌后饮品菌落总数和酵母菌含量均不超过国标规定。
最后，还需要注意的是，以上列举的仅是本发明的具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联系到的所有变形，均应认为是本发明的保护范围。

Claims (10)

  1. 杜仲雄花提取物在制备预防或缓解衰老相关肌肉功能减退的食品或药物中的应用，所述食品包括人类食品和宠物食品，其特征在于，杜仲雄花提取物中活性成分包括环烯醚萜类化合物。
  2. 如权利要求1所述的应用，其特征在于，所述环烯醚萜类化合物包括桃叶珊瑚苷、京尼平苷、京尼平苷酸、车叶草苷中的一种或多种。
  3. 如权利要求1所述的应用，其特征在于，所述预防或缓解衰老相关肌肉功能减退包括改善骨骼肌质量、骨骼肌力量、线粒体功能和机体运动能力。
  4. 如权利要求1所述的应用，其特征在于，所述杜仲雄花提取物通过激活肌肉细胞线粒体自噬活性、促进ATP的合成和/或维持线粒体形态的正常来改善线粒体功能以延缓肌肉功能减退。
  5. 如权利要求1所述的应用，其特征在于，所述杜仲雄花提取物的制备方法包括以下步骤：
    (1)将杜仲雄花干制品按照1g：20mL～60mL的料液比加入到体积比为0％～90％的有机溶剂水溶液中，于20～65℃条件下浸提，分离得到萃取液；
    (2)去除萃取液中有机溶剂，再加水充分复溶后分离得到提取液，干燥后制得杜仲雄花提取物。
  6. 如权利要求5所述的应用，其特征在于，步骤(1)中，所述有机溶剂为乙醇、甲醇或正丁醇，有机溶剂水溶液的体积比浓度为65％～80％。
  7. 如权利要求5所述的应用，其特征在于，步骤(1)中，所述杜仲雄花干制品的含水量≤10％，料液比为1g：40mL。
  8. 如权利要求5所述的应用，其特征在于，步骤(1)中，浸提的温度为45～60℃，浸提的时间为20～30h。
  9. 如权利要求5所述的应用，其特征在于，步骤(1)中，浸提过程中结合超声处理，超声条件为：超声频率200～500kHz，超声强度5～15W/cm2，时间为1.5～3.0h，超声结束后静置。
  10. 环烯醚萜类化合物在制备预防或缓解衰老相关肌肉功能减退的食品或药物中的应用。
PCT/CN2023/098531 2022-07-26 2023-06-06 杜仲雄花提取物在制备抗肌肉衰老的食品或药物中的应用 Ceased WO2024021876A1 (zh)

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CN118512496A (zh) * 2024-05-09 2024-08-20 中国医学科学院药用植物研究所 杜仲雄花在制备调节生物昼夜节律产品中的用途

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