WO2025219065A1

WO2025219065A1 - Herbal extracts based on scarlet beebalm, methods of extraction and uses thereof

Info

Publication number: WO2025219065A1
Application number: PCT/EP2025/058825
Authority: WO
Inventors: Julia Baumann; Franziska WANDREY; Florian HOFER; Torsten Grothe
Original assignee: Mibelle AG
Current assignee: Mibelle AG
Priority date: 2024-04-17
Filing date: 2025-04-01
Publication date: 2025-10-23
Anticipated expiration: 2026-10-17

Abstract

Method for the manufacturing of a powdered extract based on scarlet beebalm, and correspondingly obtained powders, 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, 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, wherein 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.

Description

TITLE

HERBAL EXTRACTS BASED ON SCARLET BEEBALM, METHODS OF EXTRACTION AND USES THEREOF

TECHNICAL FIELD

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.

PRIOR ART

Various molecular processes are understood to contribute to the aging of tissues, commonly known as the so-called hallmarks of aging. One key hallmark describes epigenetic alterations, which includes age-related DNA methylation (DNAm) changes occurring at various regions across the genome. These DNAm biomarkers of aging can be used to estimate the biological age of a tissue, as opposed to the chronological age determined by the lifespan (measured in time) of the tissue. These DNAm-dependent age predictors are also known as epigenetic age clocks. One of the first DNA methylation aging clock was described by Steve Horvath in 2013. Since then, numerous clocks have been developed, specific to tissue, disease, or population. DNA methylation-based clocks are proposed as biomarkers of early disease risk as well as predictors of life expectancy and mortality. DNA methylation and demethylation processes are not only important for transcription regulation but also play a crucial role during development and cell differentiation.

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.

US- A-2007129282 provides 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.

Yao et al (Molecules 2018, 23, 2547; doi:10.3390/molecules23102547) report, that didymin (isosakuranetin 7-O-rutinoside) is an orally bioactive dietary flavonoid glycoside first found in citrus fruits. T radi tional ly , this flavonoid has long been used in Asian countries as a dietary antioxidant. Studies have provided newer insights into this pleiotropic compound, which could regulate multiple biological activities of many important signaling molecules in health and disease. Emerging data also presented the potential therapeutic application of dietary flavonoid glycoside didymin against cancer, neurological diseases, liver diseases, cardiovascular diseases, and other diseases. 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, geraniol and extracts of Beta vulgaris cv. altissima, and mixtures of two or more of the same. WO-A-2009056208 discloses food compositions, comprising aqueous extracts of Melissa officinalis (Lemon balm) or its active principle rosmarinic acid.

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.

Howes et al in "Role of phytochemicals as nutraceuticals for cognitive functions affected in ageing" (doi: 10.1111/bph.14898) report that cognitive decline can occur with normal ageing and in age-related brain disorders, such as mild cognitive impairment and dementia, including Alzheimer's disease, with limited pharmacological therapies available. Other approaches to reduce cognitive decline are urgently needed, and so, the role of dietary interventions or nutraceuticals has received much attention in this respect. In this review, we examine the evidence for dietary plants and their chemical constituents as nutraceuticals, relevant to both cognitive decline in normal ageing and in dementia. Pharmacological (in vitro and in vivo), clinical and epidemiological evidence is assessed for both frequently consumed plants and their dietary forms, including tea, coffee, cocoa (chocolate), red wine, grapes, citrus and other fruits; in addition to plants used less frequently in certain diets and those that cross the blurred boundaries between foods, nutraceuticals and medicinal plants. For the latter, turmeric, saffron, sage, rosemary and lemon balm are examples of those discussed.

Kennedy et al in "The psychopharmacology of European herbs with cognition-enhancing properties" (doi: 10.2174/138161206779010387) report, that extensive research suggests that a number of plant-derived chemicals and traditional Oriental herbal remedies possess cognition-enhancing properties. Widely used current treatments for dementia include extracts of Ginkgo biloba and several alkaloidal, and therefore toxic, plant-derived cholinergic agents. Several non-toxic, European herbal species have pan-cultural traditions as treatments for cognitive deficits, including those associated with ageing. To date they have not received research interest commensurate with their potential utility. Particularly promising candidate species include sage (Salvia lavandulaefolia/officinalis), Lemon balm (Melissa officinalis) and rosemary (Rosmarinus officinalis). In the case of sage, extracts possess anti-oxidant, estrogenic, and anti-inflammatory properties, and specifically inhibit butyryl- and acetyl-cholinesterase. 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. In the case of Melissa officinalis, extracts have, most notably, been shown to bind directly to both nicotinic and muscarinic receptors in human brain tissue. This property has been shown to vary with extraction method and strain. Robust anxiolytic effects have also been demonstrated following acute administration to healthy humans, with mnemonic enhancement restricted to an extract with high cholinergic binding properties. Chronic regimes of aromatherapy and essential oil respectively have also been shown to reduce agitation and attenuate cognitive declines in sufferers from dementia. Given the side effect profile of prescribed cholinesterase inhibitors, and a current lack of a well tolerated nicotinic receptor agonist, these herbal treatments may well provide effective and well-tolerated treatments for dementia, either alone, in combination, or as an adjunct to conventional treatments.

SUMMARY OF THE INVENTION

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 (Melissa officinalis), rich in didymin. While both herbs belong to the mint family (/am/aceae), lemon balm is utilized as a technical process stabilizer. The powdered extract is preferably standardized on a didymin content >4%.

According to a first aspect of the present invention, it relates to 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. Preferably according to the invention, 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.

The expression "lemon balm" in the present context is designating the species Melissa officinalis.

The expression "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. However, the problem with didymin is that it has the tendency to precipitate in the process, reducing the final content in the powdered extract.

So, if scarlet beebalm is extracted only, didymin is tending to precipitate during the manufacturing process, especially after extraction, during I after solvent evaporation and during I after heat treatment.

If the extraction however is performed with a combination of scarlet beebalm and lemon balm, precipitation level is surprisingly and significantly much lower.

Furthermore, the “dilution” (on a mass balance) by adding the lemon balm does surprisingly not result in lower didymin yields. Corresponding evidence of these surprising findings is given in the experimental section below.

Anti-aging effects of the extract have been investigated in several cellular assays and in a randomized, placebo-controlled, double blind, human clinical trial. Telomere attrition and length is considered as an effecting marker of cellular aging; once telomeres become critically short, cells cease dividing and enter a state of senescence. The treatment of human fibroblasts with the proposed extract significantly reduced the telomere shortening rate, suggesting a protective effect on telomeres. This effect could be confirmed in a human clinical trial (improvement of Leukocyte Telomere Length (LTL) after 12 weeks supplementation in comparison to placebo group). Furthermore, its ability to reduce epigenetic age - a measure of an individual's aging status determined by DNA methylation patterns - is impressive. Treatment of human fibroblasts with the extract led to a 40% reduction in epigenetic age, which is equivalent to a rejuvenation of 12 years.

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. Surprisingly, 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.

According to a first preferred embodiment, the cyclodextrin is selected as gammacyclodextrin (i.e. a cyclodextrin consisting of eight glucose units). Preferably 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.

Further stabilization can be achieved, if 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.

Preferably the modified starch is chemically modified starch, preferably modified by derivatization with octenyl succinic anhydride.

Typically, the modified starch has an average molecular weight in the range of 300,000- 400,000.

According to a particularly preferred embodiment, 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%.

Preferably, extraction takes place at a temperature in the range of 25-60°C, preferably in the range of 25-40°C.

Preferably, 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.

Preferably, 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. During the standing time, e.g. 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.

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.

After 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.

As for 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.

In 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.

Preferably, 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.

In the powdered extract 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.

In the powdered extract 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.

Preferably the carrier consists of said cyclodextrin and said modified starch.

According to another aspect of the present invention, it relates to a cosmetic, food or pharmaceutical composition comprising a powdered extract as detailed above.

According to yet another aspect of the present invention, it relates to a therapeutic or non- therapeutic, e.g. cosmetic use of a powdered extract or a composition as detailed above, in particular for antiaging.

Further embodiments of the invention are laid down in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

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.

DESCRIPTION OF PREFERRED EMBODIMENTS

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").

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).

Then follows a step of filtration, if need be assisted by centrifugation, to remove precipitations. Typically, a filter retaining particles larger than 4 pm is used ("Filtration/centrifugation").

Subsequently, 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.

In the following the solvent is evaporated ("Evaporate") to lead to a 40-50% w/w solids content.

If needed, this is followed by "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.

Further extract powder manufacturing, also for comparison:

The same procedure as given above was followed for additional extract powder manufacturing, wherein in one case for comparison no lemon balm leaves were used for the herbal raw material preparation (so the ratio of scarlet beebalm leaves and lemon balm leaves was 1 :0), and wherein in another case the ratio of scarlet beebalm leaves to lemon balm leaves was chosen not to be 0.5/0.5 but 0.7/0.3.

In the table given below the water-soluble didymin extract yield is summarized for three different assays:

The results are also illustrated in Fig. 11 , showing that an increasing yield can be obtained with increasing lemon balm proportion. With the addition of lemon balm (which does not contain didymin) to scarlet bee balm, it is thus shown to be surprisingly possible to stabilize solubilized didymin after the extraction. The total amount of extracted water soluble didymin ranging from 32-53% when only scarlet bee balm is used can be increased to 69-79% when blended with lemon balm in a 0.5/0.5 ratio.

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. Unlike other carrier substances, 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.

This was experimentally verified by modifying the above process by using different carrier materials as given in the following table, also listing the resultant soluble didymin proportions in wt-%:

In these experiments 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. After cooling, 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).

The results are also illustrated in Fig. 12, showing that only when using cyclodextrin high soluble didymin proportions can be achieved.

Study of the effects of extract on cell senescence and epigenetic age:

In this part, the effects of a herbal extract based on scarlet beebalm on preventing aging is investigated in human dermal fibroblasts. The fibroblasts were passaged multiple times to induce replicative senescence according to the Hayflick model (Hayflick L. The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res. 1965;37:614-36), while being treated with said herbal extract. The efficacy of the active on preventing replicative senescence was evaluated by senescence-associate p-galactosidase staining (SA-p- galactosidase). This enzyme is commonly increased in senescent fibroblasts, therefore increased SA-p-galactosidase activity correlates with the senescent status of the fibroblasts. Further, 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).

Test samples:

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.

Cell cultivation and treatment:

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). At the start of the experiment (P7), at an intermediate timepoint (P13) and at the end (P17), a part of the cells, treated or not with the compound, was frozen and stored for genomic DNA (gDNA) extraction and SA- P-galactosidase activity. All experimental conditions were performed in triplicates.

Analysis of SA-p-galactosidase activity:

Young (P7) and aged fibroblasts (P13 and P17), previously harvested and frozen during the replicative senescence assay, were seeded to 96-well plates and cultured in culture medium for 24 hours. The medium was then replaced by assay medium containing or not (control) the test compounds or the reference (Transforming growth factor beta (TGF-P) at 10 ng/mL), and the cells incubated for 72 hours. After incubation, the SA-p-galactosidase activity was evaluated using the CellEventTM Senescence Green Detection kit (Invitrogen, USA) according to the manufacturers instructions. Briefly, the assay medium was discarded and the cells rinsed, fixed and permeabilized. Cells were then labeled with the fluorescent SA-p-galactosidase probe and Hoechst solution for the cell nuclei. Images of the cells were obtained using a fluorescent microscope (INCell AnalyzerTM, GE Healthcare, USA). Epigenetic Age Calculation According to Horvath Clock:

To determine the epigenetic age, and therefore the biological age of the samples, gDNA was sequenced using Illumina EPIC methylation arrays (Illumina, USA). The array used in this study (Infinium Methylation EPIC Bead Chip Kit) 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.

Results:

Treatment with 0.03 mg/mL of the extract significantly prevented the senescent phenotype occurring at P17, as measured by SA-p-galactosidase assay. Compared to the aged controls, the levels of SA-p-galactosidase were significantly reduced by 63 % (Fig. 3). This effect even outperformed the treatment with the control TGF-p. This was also evident from the images taken (Fig. 4).

These effects were also confirmed in the aging clock analysis. Quantifying the biological age according to the Skin and Blood Clock demonstrated, that with increasing passage number (and thereby increased senescent phenotype) the biological age increased (Fig. 5). Treatment with 0.03 mg/mL extract could however prevent this aging effect from occurring.

To summarize, 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. Study of the effect of the extract on Telomere Length in Human Fibroblasts:

In this part, the effect of the extract on telomere length in primary human fibroblasts and whether the extract demonstrates protective effects on telomere shortening rate is looked at. 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. For this experiment, 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.

Test samples:

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.

Materials:

• Cell growth: CountessTM cell counter (Invitrogen)

• Fluorescent peptide nucleic acid probe: TelC-Alexa488 (Panagene)

• High Content Screening Opera Phenix System (Perkin Elmer)

• Columbus software Version 2.9 (Perkin Elmer)

Cell cultivation and treatment:

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.

Measurement of Population Doubling:

At each passage, cell growth is monitored by counting the cell numbers in an automated cell counter. Population doubling (PD) was calculated with the formula PD = 3.322(Log (Cf) - Log (Ci)) + x. Cf represents the final cell number, Ci the initial cell number and X the PD of the previous passage. One PD is equivalent to one round of cell replication.

Telomere Length Detection and Analysis:

Telomere length analysis (TAT®) 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. After fixation with methanol/acetic acid 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.

Results:

Telomere length analysis (TAT®):

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.

Study of the effect of the extract on Epigenetic Age: A Randomized, Double-Blind, Placebo- Controlled Clinical Study:

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.

Our body has a biological aging clock in epigenetic signature and 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.

Study design: The effects of the extract on healthy aging were assessed in a randomized, double-blind, placebo-controlled study. All study participants were screened for eligibility and were informed of the purpose of the study during recruitment. Eligible subjects for study participation (n = 81 , aged 45-65 years), who provided written informed consent, were randomly allocated to two different groups at day of enrolment (TO): Intervention group with nutritional supplementation of extract or placebo control group, with placebo supplementation. The extract and placebo supplements were provided in capsules (hydroxypropylmethylcellulose) with the same shape and appearance to allow double blind performance. All study participants underwent clinical examination, an interview with structured questionnaires administered by trained interviewers, and collection of fasting blood for laboratory tests including biological age analyses, basic biochemistry, and inflammation markers. Clinical examination and sample collection, to assess all primary and secondary outcomes, were performed at enrollment (TO) and at the follow up (T1) after 12 weeks (84 days) of treatment with dietary supplementation.

Tracking of physical parameters (e.g., heart rate (HR), sleep, mobility) was carried out during the study period (from baseline (TO) to Follow up (T1)) by using MiBand 7 watch wearable devices. Participants also compiled a diary throughout the time course of the study to determine compliance and to further monitor participant well-being, health status, stress levels and treatment effects.

Participants:

Inclusion criteria

• Subject is able and willing to follow the study protocol procedures to sign the Informed Consent Form prior to screening evaluations;

• Subject is of a susceptible working population which may include shift workers, nurses, landscape and construction workers;

• Age: 45 - 65;

Exclusion criteria:

• Consumption of any dietary/nutritional supplements or functional foods;

• Smokers

• Known allergies against the mint family (labiate), especially lemon balm (cedronella; melissa, citronella)

• Pregnancy, breast feeding or intention to become pregnant during the study

• Participation in another clinical study within the last 4 weeks and concurrent participation in another clinical study • Blood donation within 4 weeks prior to study start (screening) or during study

• Anticipating any planned changes in lifestyle for the duration of the study

• Intake of any nutritional supplements (e.g. vitamins, antioxidant, herbal, or sports enhancing)

• Intake of medication that might affect the study outcome, including stress levels (psychologically and physiologically)

• Medical history or presence of any medical disorder potentially interfering with the study (gastrointestinal, renal, active cancer, diabetes, heavy depression, or cardiovascular disease)

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.

Measures:

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. In this study, 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.

Quality of Life (QOL): The Quality of Life (QOL) was assessed using the World Health Organization’s Quality of Life Assessment, BREF version (WHOQOL-BREF). 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: 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.

Results:

The extract prevents telomere shortening: After 12 weeks supplementation, Leukocyte telomere length (LTL), the biological biomarker of ageing, increased following daily supplementation with 100 mg extract (p=0.053), while it decreased even more significantly in the placebo group (p=0.0006). The comparison of LTL values between groups shows that LTL are significantly longer in the extract treated group compared to placebo (p=0.004144). These results highlight that extract supplementation has the potential to slow cellular aging, which leads to overall better health and possibly increased lifespan.

Fig. 7 shows the effects of the extract on LTL after 12 weeks.

Effects of the extract on cellular aging: 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). Upon comparing post-treatment DNAmAge values between the two groups, it was evident that the DNAmAge of the placebo was significantly higher than that of the extract treated group (p=0.0162). This data highlights that extract can prevent epigenetic changes occurring during aging stress, resulting in a reduced biological age.

Fig. 8 shows the prevention of cellular aging with extract.

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.

Extract improves the Quality of Life (QOL): After 12 weeks, supplementation with extract demonstrated an an increase in the score of Quality of Life compared to initial conditions (p=0.0085), in particular in the physical domain. In contrast, supplemenation with the placebo yielded no such beneficial effects, with the QOL even decreasing compared to initial conditions (Figure 3).

Fig. 9 shows the increase in QOL after extract supplementation.

Improved sleep index and physical activity: The use of wearable devices that measure physical parameters including activity and sleep is a useful tool to further investigate the effectiveness of a treatment on the quality of life (6). When evaluating the physical activity, a significant increase was observed in the extract treated group. Similarly, the sleep index, which reflects the quality of sleep, was increased compared to baseline and placebo. These data further reflect the beneficial effects of extract on healthspan and quality of life, which can effectively contribute to healthy aging.

Fig. 10 shows improved movement and sleep index after extract supplementation.

Conclusion: This part demonstrates that supplementation of extract offers significant benefits across various health parameters, including, cellular aging, quality of life, physical activity, and sleep quality. This places extract as the first botanical active to effectively combat aging by preventing epigenetic changes and improving the quality of life.

Claims

1. Method for the manufacturing of a powdered extract based on scarlet beebalm, 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, 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, wherein 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.

2. Method according to claim 1 , wherein the cyclodextrin is selected as gammacyclodextrin, and wherein preferably 20-40% w/w of gamma-cyclodextrin are added, wherein the % w/w are calculated with respect to the powdered extract total weight.

3. Method according to any of the preceding claims, wherein the carrier 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, wherein preferably the modified starch is chemically modified starch, preferably modified by derivatization with octenyl succinic anhydride, and/or wherein the modified starch has an average molecular weight in the range of 300,000-400,000, wherein preferably the carrier consists of said cyclodextrin and said modified starch.

4. Method according to any of the preceding claims, wherein the scarlet beebalm and lemon balm natural starting material are harvested leaves of the corresponding plant, which are preferably, before extraction, cut to a size in the range of 0.2-1 cm.

5. Method according to any of the preceding claims, wherein the scarlet beebalm natural starting material has a didymin content in the range of 5-10 % w/w, preferably in the range of 6-8 % w/w.

6. Method according to any of the preceding claims, wherein extraction takes place in an ethanol/water mixture having an ethanol content in the range of 30-70 % w/w, preferably in the range of 30-50% w/w.

7. Method according to any of the preceding claims, wherein extraction takes place at a temperature in the range of 25-60°C, preferably in the range of 25-40°C, and/or for a time span of at least one hour, preferably in the range of 2-5 hours, and/or wherein separation of the extracted natural starting material from the liquid in step b) 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, optionally followed by a standing time at room temperature, preferably for a duration of 3-24 hours, or in the range of 8-16 hours.

8. Method according to any of the preceding claims, wherein before step c), preferably immediately before step c) there is 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 5 pm or larger than 4 pm.

9. Method according to any of the preceding claims, wherein after step d) there is 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.

10. Method according to any of the preceding claims, wherein in step c), before step e) or after step e) at least one anti-caking agent is added, preferably 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.

11. Method according to any of the preceding claims, wherein 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.

12. Powdered extract, preferably obtained using a method according to any of the preceding claims, 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.

13. Powdered extract according to claim 12, wherein the cyclodextrin is selected as gamma-cyclodextrin, and wherein preferably 20-40% w/w thereof are present, wherein the % w/w are calculated with respect to the powdered extract total weight, and/or wherein the carrier 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, wherein preferably the modified starch is chemically modified starch, preferably modified by derivatization with octenyl succinic anhydride, and/or wherein the modified starch has an average molecular weight in the range of 300,000-400,000, wherein preferably the carrier consists of said cyclodextrin and said modified starch.

14. Cosmetic, food or pharmaceutical composition comprising a powdered extract according to any of claims 12 and 13.

15. Therapeutic or non-therapeutic and cosmetic use of a powdered extract or a composition according to any of claims 12-14, in particular for antiaging.