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WO2025219065A1 - Extraits d'herbes à base de monarde, leurs procédés d'extraction et leurs utilisations - Google Patents

Extraits d'herbes à base de monarde, leurs procédés d'extraction et leurs utilisations

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Publication number
WO2025219065A1
WO2025219065A1 PCT/EP2025/058825 EP2025058825W WO2025219065A1 WO 2025219065 A1 WO2025219065 A1 WO 2025219065A1 EP 2025058825 W EP2025058825 W EP 2025058825W WO 2025219065 A1 WO2025219065 A1 WO 2025219065A1
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WO
WIPO (PCT)
Prior art keywords
range
scarlet
beebalm
extract
cyclodextrin
Prior art date
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Pending
Application number
PCT/EP2025/058825
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English (en)
Inventor
Julia Baumann
Franziska WANDREY
Florian HOFER
Torsten Grothe
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Mibelle AG
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Mibelle AG
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Publication date
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Publication of WO2025219065A1 publication Critical patent/WO2025219065A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0216—Solid or semisolid forms
    • A61K8/022—Powders; Compacted Powders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/738—Cyclodextrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • 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
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

  • the present invention relates a method of manufacturing herbal extracts based on scarlet beebalm, as well as to corresponding extracts, compositions containing corresponding extracts and uses of corresponding extracts.
  • DNAm age-related DNA methylation
  • Scarlet beebalm (Monarda didyma) is an aromatic herb in the family Lamiaceae, native to eastern North America. Its odor is considered similar to that of the bergamot orange, or to lemon balm (Melissa officinalis), which is a perennial herbaceous plant also in the Lamiaceae family.
  • US-A-2002132021 discloses how intact living plants or plant parts can be contacted with water to extract from the plant or plant part exuded chemical compounds, with the extracted chemical compounds subsequently being recovered from the water.
  • the plant is treated with an elicitor or inducer to initiate or increase production of a chemical compound.
  • the roots may be harvested for recovery of the chemical compounds.
  • Valuable substances exuded from or onto a plant surface, such as a plant cuticle or the root of a plant can be identified as biologically active. Libraries of substances exuded or secreted from various plant species can be elicited or induced to produce one or more of such substances.
  • compositions comprising formula 1 steroids, e.g., 16alpha- bromo-3beta-hydroxy-5alpha-androstan-17-one hemihydrate and one or more excipients, including compositions that comprise a liquid formulation comprising less than about 3% v/v water.
  • the compositions are useful to make improved pharmaceutical formulations.
  • the invention also provides methods of intermittent dosing of steroid compounds such as analogs of 16alpha-bromo-3beta-hydroxy-5alpha-androstan-17-one and compositions useful in such dosing regimens.
  • the invention further provides compositions and methods to inhibit pathogen replication, ameliorate symptoms associated with immune dysregulation and to modulate immune responses in a subject using the compounds.
  • the invention also provides methods to make and use these immunomodulatory compositions and formulations.
  • didymin in this review, they briefly introduce the source and extraction methods of didymin, and summarize its potential therapeutic application in the treatment of various diseases, with an emphasis on molecular targets and mechanism that contributes to the observed therapeutic effects.
  • the dietary flavonoid didymin can be used to affect health and disease with multiple therapeutic targets, and it is anticipated that this review will stimulate the future development of this potential dietary medicine.
  • Certain cough suppressant throat lozenges such as available from Ricola, Switzerland, and hair removal creams such as available from Seoul Cosmetics comprise monarda didyma as well as melissa officinalis ingredients.
  • CA-A-3 087 839 concerns a solid feed composition for use as nourishment for bees and for the prevention and treatment of acariosis, and, in particular, of infestation by Varroa destructor, as well as the relative treatment method, comprising: a) nutritional and tonic ingredients, consisting of algae containing vegetal proteins and yeasts; sugars and lower organic acids; b) natural antioxidants and antiseptics contained in the extracts of Origanum vulgare and of Pelargonium graveolens or essential oil of geranium and in the essential extracts of one or more aromatic or medicinal plants selected from: Crocus sativus, Monarda citriodora, Melissa officinalis, Myristica fragrans, and Origanum majorana; and c) curative substances for bees, comprising at least one of thymol and essential extracts of Thymus vulgaris, and at least one of oxalic acid, extracts of Aloe vera or Aloe arborescens, geranio
  • KR-A-2014 0119879 relates to a functional food composition for relieving stress containing an extract of Melissa officinalis.
  • the composition of the present invention effectively inhibits density of corticosterone, beta-endorphin, and MHPG-SO4, which are hormones induced by physical and mental stress, from increasing. Therefore, the composition can be effectively used for functional food compositions for alleviating diseases associated with stress.
  • Particularly promising candidate species include sage (Salvia lavandulaefolia/officinalis), Lemon balm (Melissa officinalis) and rosemary (Rosmarinus officinalis).
  • sage Salvia lavandulaefolia/officinalis
  • Lemon balm Menelissa officinalis
  • rosemary Rosmarinus officinalis
  • Acute administration has also been found to reliably improve mnemonic performance in healthy young and elderly cohorts, whilst a chronic regime has been shown to attenuate cognitive declines in sufferers from Alzheimer's disease.
  • these herbal treatments may well provide effective and well-tolerated treatments for dementia, either alone, in combination, or as an adjunct to conventional treatments.
  • Scarlet beebalm is native to Eastern North America and was introduced to Europe in the 18 th century. This aromatic herb is used in various food applications such as tea, syrups, and herbal candies. Scarlet beebalm contains phenolic monoterpenoids, including thymol and carvacrol, but also bioflavonoids, including didymin ((S)-5,7-Dihydroxy-4'-methoxy- flavanon-7-p-rutinosid, lsosakuranetin-7-O-rutinosid, Neoponcirin): and iso-sakuranetin (2,3-Dihydro-5,7-dihydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4- on, 5,7-Dihydroxy-4'-methoxyflavanon):
  • the present invention relates to a unique, natural, and flavonoid-rich powder extract prepared from scarlet beebalm (Monarda didyma) and lemon balm (Mel
  • a method for the manufacturing of a powdered extract based on scarlet beebalm which is characterized in that a) a mixture of scarlet beebalm and lemon balm natural starting material is provided, b) that mixture is subjected to extraction in an ethanol/water mixture followed by separation of the extracted natural starting material from the liquid, c) at least one carrier is added to the liquid resulting from step b), said carrier comprising at least one cyclodextrin, d) solvent is evaporated to lead to a dry solids content in the range of 35-60% w/w, e) the resulting solution is dried using spray drying leading to the powdered extract.
  • the mixture of scarlet beebalm and lemon balm natural starting material contains at least 40% w/w of scarlet beebalm natural starting material, relative to the total weight of scarlet beebalm and lemon balm natural starting material.
  • scarlet beebalm in the present context is including the species Monarda didyma as well as Monarda fistulosa, preferably Monarda didyma is used.
  • the compound of interest in the present context is didymin. This compound is only contained in the scarlet beebalm starting material.
  • the aim in these extraction processes is to achieve an as high as possible didymin content in the final powdered product.
  • didymin is that it has the tendency to precipitate in the process, reducing the final content in the powdered extract.
  • Maltodextrin and gum arabic are the normally used carrier materials for plant powder extracts. For this case here both surprisingly do not work and cannot encapsulate the didymin during the manufacturing process.
  • cyclodextrin, and in particular a combination of a specific cyclodextrin and modified starch, preferably in a specific ratio can reduce the precipitation significantly during the manufacturing process.
  • the cyclodextrin is selected as gammacyclodextrin (i.e. a cyclodextrin consisting of eight glucose units).
  • gammacyclodextrin i.e. a cyclodextrin consisting of eight glucose units.
  • 20-40% w/w of cyclodextrin, preferably gamma-cyclodextrin, are added, wherein the % w/w are calculated with respect to the powdered extract total weight.
  • the carrier comprises, in addition to cyclodextrin, , preferably gamma-cyclodextrin, modified starch.
  • This modified starch preferably makes up 10-30% w/w, wherein the % w/w are calculated with respect to the powdered extract total weight.
  • the modified starch is chemically modified starch, preferably modified by derivatization with octenyl succinic anhydride.
  • the modified starch has an average molecular weight in the range of 300,000- 400,000.
  • the carrier consists of said cyclodextrin, preferably gamma-cyclodextrin, and said modified starch.
  • the scarlet beebalm and lemon balm natural starting material are preferably harvested leaves of the corresponding origin, which are preferably, before extraction, cut to a size in the range of 0.2-1 cm.
  • the scarlet beebalm natural starting material preferably has a didymin content in the range of 5-10 % w/w, preferably in the range of 6-8 % w/w.
  • Extraction typically takes place in an ethanol/water mixture having an ethanol content in the range of 30-70 %, preferably in the range of 30-50%.
  • extraction takes place at a temperature in the range of 25-60°C, preferably in the range of 25-40°C.
  • extraction takes place for a time span of at least one hour, preferably in the range of 2-5 hours.
  • Separation of the extracted natural starting material from the liquid in step b) preferably comprises at least one step of sedimentation/decantation and/or sieving and/or pressing, preferably pressing using over a fabric mesh with 0.5-5 mm, preferably 1-3 mm mesh width, and/or filtration and/or centrifugation.
  • separation of the extracted natural starting material from the liquid in step b) may be followed by a standing time, normally at room temperature, preferably for a duration of 3-24 hours, or in the range of 8-16 hours.
  • the standing time is decoupled from the extraction and takes place after separation and before filtration/centrifugation. This is a safety mechanism, like some 'maturation', so that no problems occur later in the process, especially if raw material with a high flavonoid content is used.
  • any excess didymin in the liquid extract precipitates and can be separated from the extract during filtration/centrifugation (together with residual plant raw material). In this way, crystallisation nuclei that could initiate further precipitation at a later stage can be removed and, in combination with the further steps, this ensures a stable process.
  • step c) Before step c), preferably immediately before step c) there is preferably a step of filtration and/or centrifugation, preferably to remove particles having an average diameter larger than 20 pm, preferably larger than 10 pm or larger than 4 or larger than 5 pm.
  • the aim of this additional separation step is to remove residual plant or other particulate material, which for example was not held back in the pressing process of the separation within step b), and/or to remove precipitated extracted materials which have too large a particle size for the following process.
  • step d) there can be a step of heat treatment, followed by re-dilution with water, or a mixture of water and ethanol, to a solids content in the range of 40-60% w/w.
  • the heat treatment can be a pasteurization, typically at a temperature of at least 80°C within at time span in the range of 10-60 minutes, or an ultrahigh temperature treatment at a temperature in the range of 110-130°C for only 0.1-3 minutes.
  • the heat treatment aims at controlling the microbiology in the product.
  • the re-dilution step this is preferably carried out using a solvent which does not cause problems during spray drying (the solvent or solvent mixture is preferably non-explosive), therefore preferably water or a water/ethanol mixture with sufficiently low ethanol content is used.
  • Re-dilution can be used for controlling the viscosity of the starting material for the spray drying.
  • step c) before step e) or after step e) at least one anti-caking agent can be added.
  • This can be selected as silica, preferably in a proportion of 0.01-0.2 % w/w, wherein the % w/w are calculated with respect to the powdered extract total weight.
  • the powdered extract has a scarlet beebalm herb extract content in the range of 20-30% w/w, a lemon balm herb extract content in the range of 20-30% w/w, a carrier content in the range of 40-60% w/w, and optionally a content of 0.01-0.5 of further additives, wherein the % w/w are calculated with respect to total of the powdered extract total weight making up 100% w/w.
  • the present invention also relates to a powdered extract, preferably obtained using a method as detailed above, having a scarlet beebalm herb extract content in the range of 20-30% w/w, a lemon balm herb extract content in the range of 20-30% w/w, a carrier content in the range of 40-60% w/w, and optionally a content of 0.01-0.5 further additives, wherein the % w/w are calculated with respect to total of the powdered extract total weight making up 100% w/w, and wherein the carrier at least contains at least one cyclodextrin.
  • the cyclodextrin is preferably selected as gamma-cyclodextrin, and preferably 20-40% w/w thereof are present, wherein the % w/w are calculated with respect to the powdered extract total weight.
  • the carrier preferably comprises, in addition to cyclodextrin, modified starch, preferably 10-30% w/w of starch, wherein the % w/w are calculated with respect to the powdered extract total weight.
  • the modified starch is preferably chemically modified starch, preferably modified by derivatization with octenyl succinic anhydride, and/or the modified starch has an average molecular weight in the range of 300,000-400,000.
  • the carrier consists of said cyclodextrin and said modified starch.
  • a cosmetic, food or pharmaceutical composition comprising a powdered extract as detailed above.
  • a therapeutic or non- therapeutic e.g. cosmetic use of a powdered extract or a composition as detailed above, in particular for antiaging.
  • Fig. 1 schematically shows the steps of the extract generation, wherein it is to be noted that some steps may also be omitted without departing from the invention
  • Fig. 2 shows an overview of the experimental setup
  • Fig. 3 shows the inhibition of SA-p-galactosidase activity
  • Fig. 4 shows the visual reduction of SA-p-galactosidase
  • Fig. 5 shows the reduction of DNAmAge with the extract
  • Fig. 6 shows the decrease in telomere shortening rate after 6 weeks of extract treatment
  • Fig. 7 shows the effects of the extract on LTL after 12 weeks
  • Fig. 8 shows the prevention of cellular aging with the extract
  • Fig. 9 shows the increase in QOL after extract supplementation
  • Fig. 10 shows the improved movement and sleep index after extract supplementation
  • Fig. 11 shows the increase of the extraction yield of soluble didymin in dependence on scarlet bee balm I lemon balm ratio
  • Fig. 12 shows the stabilization of didymin in a thermally stressed intermediate product with a combination of g-cyclodextrin and modified starch.
  • Extract powder manufacturing according to the invention see also Fig. 1:
  • a mixture of 50 weight percent of scarlet beebalm leaves and 50 weight percent lemon balm leaves (dried starting material, typically water content less than 12 %) is cut to a size in the range of 0.2-1 cm ("Herbal raw material preparation").
  • the starting material is added to ethanol (40 weight percent in water) at a drug to solvent ratio (DSR) of 1 :10-1 :30 at slightly elevated temperature, typically up to 35°C for 2-5 hours ("Extraction").
  • DSR drug to solvent ratio
  • Extraction is followed by a "Separation” step to remove the extracted herbal raw material, by sedimentation/decantation, sieving or pressing. Pressing is carried out over a fabric mesh with 0.5-5 mm mesh width, preferably 1-3 mm, and is then in a standing time allowed to rest for another 6-24 hours at room temperature (normally 24°C).
  • carrier material is added ("Adding carrier”). Specifically, 25-40% by weight gamma-cyclodextrin and 15-25% by weight modified starch (chemically modified starch obtained by derivatization with octenyl succinic anhydride, having an average molecular weight in the range of 300,000-400,000). The weight percent values are calculated based on the powder final product.
  • Heat treatment typically at a temperature in the range of at least 80°C for a time span in the range of 10-60 minutes, or at higher temperatures in a window of 110-130°C for 0.1-3 minutes).
  • the resulting material is re-diluted with water, if needed, to a solids content of 50% w/w, and is subjected to "Spray drying" to lead to the "Final powder". If needed, before spray drying or after, silica (e.g. 0.1 % w/w) can be added.
  • silica e.g. 0.1 % w/w
  • Didymin tends to precipitate if an intermediate product is stressed thermally e.g. while evaporation, or drying steps. The result is a strong increase in viscosity and also reduction of water soluble and therefore bioavailable didymin.
  • the addition of cyclodextrin, in particular of y-cyclodextrin increases the stability of didymin within the intermediate product. While modified starch on its own does not have a stabilizing effect, the combination with y-cyclodextrin is more potent than equivalent levels of y- cyclodextrin in combination with e.g. acacia gum.
  • the samples were thermally stressed by applying the following conditions:
  • the stress test takes place in a glass round flask with applied water cooler and heating via an oil bath.
  • Concentrated liquid extract (intermediate product between step ‘evaporation’ and step ‘heat treatment’) is heated up to 100°C under constant stirring and kept boiling at that temperature for 60 minutes.
  • the soluble Didymin content is analyzed by HPLC, comparing the total Didymin content in the extract and the share of soluble Didymin in the sample by centrifugating the sample preparation (1 % of extract solids in water).
  • This enzyme is commonly increased in senescent fibroblasts, therefore increased SA-p-galactosidase activity correlates with the senescent status of the fibroblasts.
  • the biological age was determined according to Horvath’s epigenetic age clock (Horvath S, Oshima J, Martin GM, Lu AT, Quach A, Cohen H, et al. Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY). 2018;10(7):1758-75).
  • a stock solution of Extract made as described above was prepared by dissolving the test compound in water and subsequent sterile filtration leading to a concentration of the extract in water in the range of 0.01 - 0.1 mg/mL.
  • Human dermal fibroblasts were cultured at 37°C and 5 % CO2 in culture medium (DMEM with 10 % serum). Young fibroblasts (early passage, P7) were seeded and grown for 24 hours. The medium was then replaced by culture medium containing or not (control) the test compound and the cells incubated for 6 days, with treatment renewal after 72 hours. After, the cells were passaged and seeded to a new plate, to obtain a subsequent “older” passage of fibroblasts (P8) and held in culture for 24 hours. The cells were again treated with or without (control) the test compounds for 6 days, with treatment renewal after 72 hours. This process was repeated multiple times over several weeks in order to obtain different passages of aged fibroblasts, up to P17 (Fig. 2).
  • TGF-P Transforming growth factor beta
  • gDNA was sequenced using Illumina EPIC methylation arrays (Illumina, USA).
  • Illumina EPIC methylation arrays Illumina, USA.
  • the array used in this study is a genome-wide methylation screening tool that targets over 935,000 CpG* sites in the most biologically significant regions of the human methylome. Alterations in such CpG islands can be correlated with up- or downregulation of genes (for example related to aging).
  • the data was then analysed and aligned according to the Horvath “Skin and Blood Clock”, an epigenetic clock developed to determine the biological age specifically of skin and blood samples.
  • Epigenetic clocks are based on specific sets of CpG sites (CpG islands are short stretches of palindromic DNA with the sequence “CpG” that code for the same sequence in the complementary strand - i.e., repeated cytosine and guanine nucleotides with the “p” representing the linking phosphate) whose altered DNA methylation levels yield the organism’s age. These clocks are acknowledged as a highly accurate molecular tool that correlate with the chronological age in humans and other vertebrates.
  • the proposed extract based on scarlet beebalm is a potent active in preventing the senescent phenotype during cell aging, and capable of reducing the biological age of aging cells.
  • telomere attrition and length is considered as a hallmark of aging and also effecting of cellular aging: once telomeres become critically short, cells cease dividing and enter senescence. Such telomere shortening is further associated with age-related diseases and can be induced by oxidative stress and inflammation.
  • cells were treated with the extract or vehicle control before being passaged multiple times for 6 weeks (representing replicative senescence). The measured parameter was telomere length, which was assessed with a high-throughput (HT) quantitative fluorescence in situ hybridization (Q-FISH) technology.
  • HT high-throughput
  • Q-FISH quantitative fluorescence in situ hybridization
  • a stock solution of Extract made as described above was prepared by dissolving the test compound in water and subsequent sterile filtration leading to a concentration of the extract in water in the range of 0.005 - 0.5 mg/mL.
  • Fibroblasts Primary human fibroblasts were grown in fibroblast medium (HEPES and bicarbonate based, pH 7.4) supplemented with 2 % fetal bovine serum (FBS), growth factors and essential nutrients at 5 % CO2 I 95 % ambient air. Fibroblasts were seeded at 5x103 cells/cm2 prior to experimental start. At the onset of the experiment, cells were treated with extract or left untreated (control). The media and treatment was renewed every 2-3 days and cells passaged at sub-confluence (70-80 %) every 7 days for a total of 6 weeks.
  • FBS fetal bovine serum
  • PD Population doubling
  • Telomere length analysis was performed with High-throughput (HT) Q-FISH. This method is based on a quantitative fluorescence in-situ hybridization method. Brieftly, telomeres are hybridized with a fluorescent (Alexa-488) peptide nucleic acid (PNA) probe that recognizes three telomere repeats. The images of the nuclei and telomeres are captured by a high-content screen system and the intensity of the fluorescent signal detected from the probes can be translated to telomere length. Cells from each weekly passage were seeded to black-walled 384-well plates, with 5 replicates of each treated sample and 8 replicates of untreated each control.
  • the cells were treated with pepsin to digest the cytoplasm and the nuclei processed for in situ hypridizaiton with the PNA probe. After washing the cell nuclei were further stained with DAPI (4',6-diamidino-2-phenylindole) before continuing to image acquisition. For each well, 15 independent images were captured, and the telomere fluorescence intensity measured at the 488 nm wavelength. The results of fluorescence intensity were analysed and calculated with Life Length’s proprietary program. Statistical analysis was performed with Student’s T-test.
  • TAT® Telomere length analysis
  • telomere shortening rate Normalizing the median telomere length (initial - final) to the population doubling was used to determine the telomere shortening rate. Treatment with the extract significantly reduced the telomere shortening rate (see Fig. 6), which suggests a telomere protective effect and prevention against cell senescence.
  • Interventions to slow down biological aging and extend health span are major challenges for health, nutrition, and quality of life, given the social and healthcare costs of aging population.
  • Aging is an individual, natural, and biologically complex process, consequently people do not age at the same rate.
  • Excessive oxidative stress exposures and altered inflammation responses are related to biological aging and involved in the pathogenesis of age-related diseases.
  • telomeres A powerful marker of cellular aging in humans is the epigenetic age, also defined as DNA methylation age (DNAmAge). DNAmAge is assessed from methylation modifications of each of our individual DNA, and it is strongly correlated with chronological age.
  • Leukocyte telomere length (LTL) is a further estimator of cellular aging. Telomeres act as a mitotic clock, which is a measure of biological aging based on the number of times a cell has divided. Each cell division shortens the protective telomeres at the ends of chromosomes, which eventually leads to cellular senescence (replicative senescence) or cell death. This study aimed to explore the potential of the extract to reduce the biological aging of a susceptible stressed population.
  • Subject is of a susceptible working population which may include shift workers, nurses, landscape and construction workers;
  • Participant characteristics A total of 81 male and female participants were included in the trial, with 41 participants in the placebo group, and 40 in the intervention group.
  • the group demographics (measured at TO) are shown in Table 1.
  • Table 1 Demographics of extract and placebo groups. There were no significant differences between groups on any measure.
  • Treatments Daily dose: 100 mg extract or 100 mg Placebo (maltodextrin). Mode of administration: 1 capsule per day, with lunch and plenty of water
  • Adverse events There was no evidence of any adverse events related to taking the extract and all participants reported 100 % compliance in both groups, with no gastrointestinal issues.
  • Leukocyte Telomere Length (LTL ) Leukocyte telomere length (LTL) analysis provides an indication of biological aging, as shorter telomeres are associated with aging and various age-related diseases. This measurement can give insights into an individual's overall health status, potential longevity, and risk for chronic diseases.
  • LTL was measured by a real-time quantitative PCR method. This method measures the relative telomere length in genomic DNA by determining the ratio of telomere repeat copy number (T) to single-copy gene (S) in experimental samples relative to the T/S ratio of a reference sample. Hereby, the change in LTL could be measured and compared to initial conditions.
  • QOL Quality of Life
  • the Quality of Life (QOL) was assessed using the World Health Organization’s Quality of Life Assessment, BREF version (WHOQOL-BREF).
  • WHOQOL-BREF Quality of Life Assessment
  • This questionnaire is a self-report questionnaire developed by the World Health Organization to assess an individual's perceived quality of life across four domains: physical health, psychological health, social relationships, and environment. The questionnaire is a valid tool employed to understand an indivitual’s overall well-being and life satisfaction.
  • DNAmAge values were determined by analysis the methylation levels from five selected markers (ELOVL2, C1orf122, KLF14, TRIM59 and FHL2) in genomic DNA isolated from the blood samples using bisulfite conversion and Pyrosequencing® methodology on PyroMark Q48 Autoprep (QIAGEN, Milano, Italy). The resulting Pyrograms® generated by the instrument are automatically analyzed using Pyromark Q48 Autoprep Software (QIAGEN, Milano, Italy). The levels of methylation are expressed as a percentage of methylated cytosines at the 5 CpG sites considered and are used for estimation of biological aging (years) as previously reported.
  • Salivary cortisol Morning saliva samples for cortisol levels were collected from the participants in Salivette device (SARSTEDT AG & Co, Numbrecht, Germany), and analyzed according to Laboratory Medicine Unit instructions (AOUP).
  • Wearables monitoring HR monitoring, sleep tracking, daily step count and energy expenditure data during the study period, were recorded through MiBand 7 wearable devices (Xiaomi). The participants were asked to avoid removing the device during the study period if not strictly necessary, and only for short periods.
  • the devices provided continuous heart rate monitoring, energy expenditure and sleep profile tracking throughout the study period in order to evaluate metabolic demands and exercise pattern. Calibration was performed by comparing the data obtained with laboratory tests and with questionnaires administered including data from, lifestyle, physical activity, life, and sleep quality. The final readouts analyzed include physical activity and sleep index.
  • LTL Leukocyte telomere length
  • Fig. 7 shows the effects of the extract on LTL after 12 weeks.
  • DNAmAge values which reflect cellular aging, remained stable after treatment with extract, while it exhibited a significant increase in the placebo group after 12 weeks (p ⁇ 0.001 , Figure 2).
  • Fig. 8 shows the prevention of cellular aging with extract.
  • cortisol levels Reduction of cortisol levels: Supplementation with extract led to decreased salivary cortisol levels. After 12 weeks, compared to initial conditions, cortisol levels were significantly decreased by 25.0 %. Treatment with placebo reduced cortisol levels by 18 %. This further highlights that extract supplementation can improve health parameters related to stress, such as cortisol levels.
  • Fig. 9 shows the increase in QOL after extract supplementation.
  • Fig. 10 shows improved movement and sleep index after extract supplementation.

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Abstract

L'invention concerne un procédé de fabrication d'un extrait en poudre à base de monarde, et des poudres correspondantes, caractérisés en ce que a) un mélange de matériaux de départ naturels à base de monarde et de mélisse est fourni, b) ce mélange est soumis à une extraction dans un mélange éthanol/eau suivi d'une séparation du matériau de départ naturel extrait du liquide, c) au moins un véhicule est ajouté, comprenant au moins une cyclodextrine, d) le solvant est évaporé pour atteindre une teneur en solides secs comprise entre 35 et 60 % m/m, e) la solution obtenue est séchée par séchage par pulvérisation pour obtenir l'extrait en poudre, le mélange de matériaux de départ naturels de monarde et de mélisse contenant au moins 40 % m/m de matériau de départ naturel de monarde par rapport au poids total de matériaux de départ naturels de monarde et de mélisse.
PCT/EP2025/058825 2024-04-17 2025-04-01 Extraits d'herbes à base de monarde, leurs procédés d'extraction et leurs utilisations Pending WO2025219065A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20020132021A1 (en) 1997-04-30 2002-09-19 Ilya Raskin Elicited plant products
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WO2009056208A1 (fr) 2007-10-17 2009-05-07 Cognis Ip Management Gmbh Compositions alimentaires comprenant des extraits de mélisse
KR20140119879A (ko) 2013-03-28 2014-10-13 (주)네추럴에프앤피 레몬밤 추출물을 함유하는 스트레스 완화용 기능성 식품 조성물
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US20070129282A1 (en) 1998-11-24 2007-06-07 Ahlem Clarence N Pharmaceutical treatments and compositions
WO2009056208A1 (fr) 2007-10-17 2009-05-07 Cognis Ip Management Gmbh Compositions alimentaires comprenant des extraits de mélisse
KR20140119879A (ko) 2013-03-28 2014-10-13 (주)네추럴에프앤피 레몬밤 추출물을 함유하는 스트레스 완화용 기능성 식품 조성물
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