Composition containing sanguisorba officinalis root extract and application thereof

Abstract

The invention relates to a composition containing a sanguisorba officinalis root extract and application thereof. The present invention relates to a root extract of Sanguisorba officinalis and its use as an active ingredient for skin care and / or scalp care.

Description

Field of the Invention

[0001] The present invention relates to a root extract of the plant Sanguisorba officinalis for promoting light-related human mood. The present invention also relates to a Sanguisorba officinalis root extract rich in feruloylated tormentic acid derivatives and a method for preparing such an extract. Furthermore, the present invention relates to a composition comprising such a root extract, and the use of such a root extract as an active ingredient for skin care and / or scalp care for promoting light-related human mood. Background of the Invention

[0003] Sunlight is essential for many organisms, including humans, and exerts several powerful biological effects in addition to visual responses. It includes circadian rhythm regulation and brain activity, thus regulating sleep and learning. In addition, sunlight directly affects human mood without causing circadian rhythm disorders or sleep disruptions (LeGates et al., Nature Reviews Neuroscience, Vol. 15, pp. 443-454, 2014). There is ample evidence that lack of light is associated with depressive symptoms and cognitive dysfunction. The retina is the key organ involved in light detection, and opsins are the key light-conducting molecules found therein. As the organ most exposed to light, the skin also expresses photoreceptors called opsins. In addition to the retina, opsins are also expressed in the epidermis, dermis, and hair follicles. Different opsins have been described, each of which responds to different wavelengths of light. In the eye, one of the most abundant opsins is peropsins (RRH), which is also expressed in human skin. The biological role of opsins in skin physiology is not yet fully understood, but certain opsins have been associated with skin differentiation, circadian rhythm, dermal matrix remodeling through matrix metalloproteinase (MMP) regulation, or wound healing stimulation (Suh et al., Photodermatol Photoimmunol Photomed, Vol. 36(5), pp. 329-338, 2020). The ability of the skin to independently sense light and conduct it to the eye through the retina by irradiating the skin has been demonstrated in humans. It has been shown that illumination behind the knee affects the circadian rhythm, or that UV-A exposure with opaque goggles leads to an increase in serotonin and a more balanced and less tense feeling.

[0004] Serotonin (the body's natural mood booster) is an important mediator of the bidirectional interaction between the neuroendocrine system and the skin. The human skin has a serotonin system capable of producing serotonin. This places the skin at the center of the link between light and good mood, where serotonin plays an important role. Serotonin is also a precursor of melatonin, i.e., the "sleep hormone".

[0005] Melatonin is a neurohormone affected by the light / dark cycle. Light may inhibit or synchronize melatonin production according to the light schedule, and melatonin may stabilize and possibly strengthen the coupling of the circadian rhythm. It has been confirmed that daylight exposure stimulates melatonin levels in the morning without acting on melatonin levels in the evening, while improving a person's sleep. It is also known that melatonin is synthesized and metabolized in the skin, where it plays multiple roles. Some of its functions are related to antioxidant properties and include protection from ultraviolet and X-ray radiation. Specifically, melatonin is a powerful protector against ultraviolet radiation, fundamentally against UV-B radiation. In addition, melatonin also activates the antioxidant cascade, thereby reducing free radicals and the DNA repair system in the skin. Finally, melatonin enhances the skin barrier by stimulating the expression of epidermal proteins keratin-10 and keratin-14, and also promotes skin wound healing.

[0006] Another beneficial effect related to daylight may be the production of the essential nutrient vitamin D, often referred to as the "sunshine" vitamin. The skin is the main source of vitamin D in the human body after exposure to daylight. Unfortunately, due to spending long hours indoors, approximately 75% of the world's population is vitamin D deficient. After absorbing UV-B, the precursor 7-dehydrocholesterol (7-DHC) is converted to vitamin D3 in the skin, and this process is accelerated by heat energy. These reactions are non-enzymatic and depend on the UVB dose and temperature. 7-DHC in keratinocytes and dermal fibroblasts is converted to previtamin D. Vitamin D is enzymatically hydroxylated to 25-hydroxyvitamin D (25(OH)D), and then converted to its biologically active metabolite 1,25(OH)2D. Vitamin D synthesized in the skin can be released from the plasma membrane and enter the systemic circulation, binding to the vitamin D-binding protein (DBP). The vitamin D produced in the skin supplies more than 90% of the body's vitamin D requirements. Vitamin D status affects cognitive, behavioral, and mood disorders. In addition, people deficient in vitamin D are more likely to have any type of sleep disorder, poorer sleep quality, shorter sleep duration, and / or excessive daytime sleepiness.

[0007] The skin itself is capable of responding to the active metabolites of vitamin D3. The major genomic effects and biological responses of vitamin D3 metabolites in the skin are mediated through their binding to the nuclear vitamin D receptor (VDR). In fact, the skin expresses VDR and serves as a site of action for vitamin D. The VDR activated by classical 1,25(OH)2D3 induces rapid response signal transduction either through a non-genomic membrane-associated mechanism based on an alternative ligand-binding site or by acting on the 1,25D3-MARRS receptor. Vitamin D can have a variety of different effects on major skin cells (keratinocytes and fibroblasts) and immune cells by activating nuclear VDR. Vitamin D also plays a key role in skin homeostasis, which promotes skin barrier function and is beneficial for skin differentiation. Its deficiency has been associated with many proliferative and inflammatory skin disorders. In addition, as an important part of the functional immune system, the active form of vitamin D can regulate skin immunity and exert antioxidant, antifibrotic, and anti-inflammatory properties.

[0008] Sunlight is also beneficial for maintaining a balanced circadian rhythm, which is crucial for regulating various cellular, metabolic, physiological, and behavioral activities in mammals. The central circadian clock is not only greatly affected by light but also by the environment. Focusing on light, its effect on the phase of the circadian clock depends on the timing of light exposure. In this regard, several key circadian genes have been identified in the skin.

[0009] Due to these proven beneficial effects of light, phototherapy or photobiomodulation is used to treat mood disorders and various dermatological conditions such as psoriasis, atopic dermatitis, hair regrowth, wound healing, and tissue regeneration.

[0010] On the other hand, sunlight can also have harmful effects on the skin, called photoaging, such as hyperpigmentation, melanoma due to DNA damage, premature aging, weakened mechanical properties (i.e., loss of elasticity), and hardening due to degradation of extracellular matrix (ECM) components, dehydration, and changes in skin color. It has been demonstrated that broad protection against the entire solar spectrum of UV-B, UV-A, visible light, and short infrared rays is required to prevent sunlight-related skin damage.

[0011] Carbonylated proteins (CP) are synthesized through the reaction between the amino groups in proteins and the reactive aldehyde compounds generated by lipid peroxidation induced by reactive oxygen species. In the skin, CP is detected at a higher frequency at the sites of sun-exposed skin in elderly subjects [1]. Although the radiant energy of winter sunlight is weak, CP is also detected in the stratum corneum (SC) collected from the skin in winter [2]. It has been reported that CP is associated with a decrease in skin water content and transepidermal water loss (TEWL), changes in the dermal matrix, an increase in the darkness around facial pores, and a change in skin color from yellow to dark, all of which alter skin tone (Masaki et al., J. Dermatol Science, Vol. 84, 1: pp. 05-16 [01-04], 2016).

[0012] Sanguisorba officinalis, also known as great burnet or sanguisorbe officinale, is a perennial herbaceous plant of the Rosaceae family and is distributed in the Northern Hemisphere, Europe, Asia, and North America.

[0013] The root extract of Sanguisorba officinalis is known to contain metabolites such as ellagitanin, flavonoids, and polyphenols. These extracts have been reported to have antiviral, antibacterial, hemostatic, anti-inflammatory, and anticancer activities, and the activities are only partially explained by tormentic acid I and II and sanguisorbine H-6 (Jang et al., A Review, The American Journal of Chinese Medicine, Vol. 46, No. 2, 1-20, 2018).

[0014] The root extract of Sanguisorba officinalis also contains euscaphic acid and / or tormentic acid (Seongdae et al., Molecules, 23, 3001, 2018), ferulic acid or its esters. Ferulic acid exhibits antioxidant and anti-tyrosinase activities.

[0015] Tormentic acid belongs to triterpenoid compounds with six isoprene units. It is known to have anticancer, anti-atherosclerotic, anti-inflammatory, antidiabetic, antimicrobial, cardioprotective, and neuroprotective properties. Tormentic acid is widely present in various plants, but it also exists in various derivative forms that are more specific to certain plant families and certain tissues. If euscaphic acid or glycosylated forms of tormentic acid are the most common, these forms often have original activities different from the basic form (tormentic acid).

[0016] Feruloylated derivatives of tormentic acid are rare natural molecules that may be of interest in the cosmetic field. Specifically, 3-O-trans-feruloyl tormentic acid is only present in extracts of the plants Vitex rotundifolia and Vitex trifolia, and 3-O-trans-feruloyl euscaphic acid is present in the leaf extract of Eriobotrya japonica. Another compound, oryzanol (which has a structure similar to ferulic acid coupled to a triterpene and is different from tormentic acid), has not shown any toxicity in mice, suggesting that these molecules may be of interest in cosmetics.

[0017] It has been found that the roots of Sanguisorba officinalis can contain ferulic acid coupled to tormentic acid via an ester bond, which is different from the aerial parts. Therefore, obtaining the roots of Sanguisorba officinalis is key to recovering these compounds of interest. However, such an operation involves the destruction of all or part of the plant, which does not comply with the Nagoya Protocol and the guidelines of the International Standard (ISO 26000) for controlling access to biodiversity and ensuring sustainable development and social responsibility.

[0018] The use of Sanguisorba officinalis extract in cosmetics is already known. For example, CN-B105193680 and KR-A20190003011 disclose the use of Sanguisorba officinalis root extract for promoting skin whitening properties. According to CN-B 105193680, the root extract is obtained by using a resin fractionation step in which many active components (including feruloylated tormentic acid derivatives) are determinately adsorbed onto the resin. EP-B1 0993826 discloses a Sanguisorba officinalis root extract for stimulating melanogenesis in the skin. JP-B 3449967 discloses a Sanguisorba officinalis root extract for stimulating collagen synthesis after UV-B exposure. WO-A1 2018000060 discloses a Sanguisorba officinalis root extract as a sebum regulator for oily skin. EP-A2 1051965 discloses a Sanguisorba officinalis root extract for improving ceramide production and thus enhancing skin moisturizing effects.

[0019] It has been reported that sanguisorboside I (a triterpene compound saponin) isolated from the ethanol extract of Sanguisorba officinalis roots exhibits activities such as anti-inflammatory and anti-wrinkle (Young Heui KIM et al., Bioscience, Biotechnology and Biochemistry, 72:2, 303 - 311, 2014, DOI: 10.1271 / bbb.70268).

[0020] Since a majority of the world's population spends most of its time indoors, there is an unmet need for products of natural origin that can be used to at least partially mimic the beneficial effects that sunlight may have on the body (preferably the skin), even in the absence of sunlight exposure and / or under low sunlight exposure conditions. In addition, it has in fact been observed that an increasing number of cosmetic consumers are seeking products of natural origin that meet these two criteria. The first criterion relates to the visual effects observed after applying these products. The second criterion is more related to the emotional aspect, in particular the well-being that the user can feel after applying these products.

[0021] Therefore, there is an unmet need in providing a product of natural origin, namely an extract of Sanguisorba officinalis root, which can bring visual beneficial effects to the body (especially the skin and / or scalp), and also enable the subject to exhibit a feeling of well-being.

[0022] Sunlight can also have well-known harmful effects on the skin. Therefore, there is also an unmet need for skin care products of natural origin, preferably ones that can prevent light-related skin damage and thus delay the signs of photoaging.

[0023] It has surprisingly been found that an extract of Sanguisorba officinalis root can mimic sunlight-related effects on the human body (especially on the skin), even in the absence of light exposure and / or under low light exposure conditions. In this regard, the extract of Sanguisorba officinalis root can be used, for example, in light-like therapies, for improving light-related human mood, for obtaining light-related beneficial effects on the body, and thus for obtaining a feeling of relaxation, even in the absence of light exposure and / or under low light exposure conditions.

[0024] It has also surprisingly been found that an extract of Sanguisorba officinalis root can be rich in certain target components that may naturally occur in small amounts. In fact, the Sanguisorba officinalis root can be rich in feruloylated derivatives of tormentic acid, for example, by cultivating the plant under certain conditions without compromising the development of the whole plant. The enriched root extract can be advantageously used for skin care and / or scalp care. Summary of the Invention

[0026] One aspect of the present invention relates to an extract of the root of Sanguisorba officinalis, which comprises:

[0027] - tormentic acid, which accounts for at least 1% by weight of the total weight of the dry extract,

[0028] - feruloylated derivatives of tormentic acid having the general formula (I)

[0029]

[0030] - feruloylated derivatives of deoxytormentic acid having the general formula (II)

[0031]

[0032] The present invention further relates to a method for preparing a root extract of Sanguisorba officinalis, said method comprising the following steps:

[0033] a) cultivating Sanguisorba officinalis under soil-free conditions, in particular by aeroponics,

[0034] b) stimulating the roots of said plant,

[0035] c) performing solid / liquid extraction by macerating the roots obtained in step b),

[0036] d) recovering the extract obtained in step c), and

[0037] e) optionally, diluting and / or clarifying the extract recovered in step d) by successive filtration.

[0038] The present invention further relates to a cosmetic or dermatological or nutritional composition comprising a root extract of Sanguisorba officinalis as defined above and optionally one or more excipients, which are preferably cosmetically or dermatologically or nutritionally acceptable.

[0039] The present invention further relates to the (cosmetic) use of a root extract of Sanguisorba officinalis as defined above as an active ingredient for skin care and / or scalp care.

[0040] The present invention also relates to a non-therapeutic method for preventing or delaying the appearance of skin aging effects and for promoting light-related human mood, said method comprising applying a root extract of Sanguisorba officinalis as defined above to at least a part of the body.

[0041] Another aspect of the present invention relates to the (cosmetic) use of a root extract of Sanguisorba officinalis for promoting light-related human mood.

[0042] All documents cited or mentioned herein (“documents cited herein”) and any manufacturer's instructions, descriptions, product specifications and product inserts of any product mentioned in this text or in any document incorporated herein by reference are hereby incorporated herein by reference and may be used to practice the present invention. More specifically, all reference documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0043] Description of the Drawings

[0044] Figure 1A and 1BShows the stimulation of the production of well-being hormone in skin explants treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention. Figure 1A Shows the concentration of serotonin released by skin cells in culture medium after 3 days of treatment. Figure 1B Shows the concentration of melatonin released by skin cells in culture medium after 5 days of treatment.

[0045] Figure 2A and 2B Shows the clinical evaluation related to the improvement of the well-being feeling of volunteers topically treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention. Figure 2A Confirms the improvement of relaxation between the 1st day and the 5th day after treatment. Figure 2B Confirms the improvement of relaxation 7 days after treatment.

[0046] Figure 3 Shows the self-evaluation questionnaire related to the well-being effect of a group of 36 volunteers topically treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention.

[0047] Figure 4A and 4B Shows the regulation of the downstream biological pathway of vitamin D in the skin using the root extract of Sanguisorba officinalis according to one aspect of the present invention. Figure 4A Shows the regulation of vitamin D receptor. Figure 4B Shows the regulation of vitamin D binding protein.

[0048] Figure 5 Shows the induction and enhancement of phototransduction signals on skin explants treated with tormentic acid and feruloylated tormentic acid derivatives isolated from the root extract of Sanguisorba officinalis according to one aspect of the present invention.

[0049] Figure 6 Shows the improvement of skin elasticity observed during the clinical study of volunteers topically treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention.

[0050] Figure 7 Shows the improvement of skin color observed during the clinical study of volunteers topically treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention.

[0051] Figure 8 Shows the content of carbonylated proteins in keratinocytes of volunteers treated with the root extract of Sanguisorba officinalis according to one aspect of the present invention after 28 days of application.

[0052] The elements of the present invention will be described in more detail. These elements are listed with specific embodiments; however, they can be combined in any way and in any number to create additional embodiments. Each described embodiment and implementation should not be construed as limiting the present invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed elements. In addition, any arrangement and combination of all the elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0053] Throughout this specification and the claims, unless the context requires otherwise, the word "comprising" and its variants such as "including" and "containing" should be understood to imply the inclusion of the stated members, integers or steps or groups of members, integers or steps, but not the exclusion of any other members, integers or steps or groups of members, integers or steps. The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) should be construed to cover the singular and the plural, unless otherwise indicated herein or the context clearly contradicts. The recitation of a range of values herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as", "for example") provided herein is merely intended to better illustrate the present invention and does not pose a limitation on the scope of the present invention that is otherwise claimed. The language of the specification should not be construed to indicate that any unclaimed element is essential for the practice of the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although the methods and materials described herein are preferred, other methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention. Detailed Description of the Invention

[0056] One aspect of the present invention relates to a root extract of Sanguisorba officinalis L., which comprises:

[0057] - tormentic acid, which accounts for at least 1% by weight of the total weight of the dry extract,

[0058] - an feruloylated derivative of tormentic acid having the general formula (I),

[0059]

[0060] - Feruloylated derivatives of deoxypotentillic acid having the general formula (II)

[0061]

[0062] According to one embodiment, potentillic acid accounts for 1 to 10% by weight, preferably 1.2 to 8% by weight, more preferably 1.5 to 7% by weight, especially 1.8 to 5% by weight of the total weight of the dry extract.

[0063] According to one embodiment, the feruloylated derivative of potentillic acid accounts for at least 0.1% by weight of the total weight of the dry extract. Advantageously, the feruloylated derivative of potentillic acid accounts for 0.1 to 5% by weight, preferably 0.2 to 4.5% by weight, more preferably 0.3 to 4% by weight, even more preferably 0.4 to 3.5% by weight, especially 0.5 to 3% by weight of the total weight of the dry extract.

[0064] According to one embodiment, the feruloylated derivative of deoxypotentillic acid accounts for at least 0.05% by weight of the total weight of the dry extract. Advantageously, the feruloylated derivative of deoxypotentillic acid accounts for 0.05 to 3% by weight, preferably 0.06 to 2.8% by weight, more preferably 0.07 to 2.6% by weight, even more preferably 0.08 to 2.4% by weight, most preferably 0.09 to 2.2%, especially 0.1 to 2% by weight of the total weight of the dry extract.

[0065] In a preferred embodiment, the feruloylated derivative of potentillic acid accounts for at least 0.1% by weight and the feruloylated derivative of deoxypotentillic acid accounts for at least 0.05% by weight, all weights being relative to the total weight of the dry extract.

[0066] According to one embodiment, potentillic acid, the feruloylated derivative of potentillic acid and the feruloylated derivative of deoxypotentillic acid together account for at least 1.15% by weight of the total weight of the dry extract. Advantageously, potentillic acid, the feruloylated derivative of potentillic acid and the feruloylated derivative of deoxypotentillic acid together account for 1.15 to 18% by weight, preferably 1.30 to 15% by weight, even more preferably 1.40 to 12% by weight, especially 1.60 to 10% by weight of the total weight of the dry extract.

[0067] According to one embodiment, the root extract may further contain one or more ellagitannins.

[0068] According to one embodiment, the root extract may be in liquid form and may contain a solvent selected from water, lower alcohols, diols or mixtures thereof, and dioctyl ether. The lower alcohol may preferably be selected from methanol and ethanol. The diol may preferably be selected from dipropylene glycol, 1,2 - propylene glycol, 1,3 - propylene glycol, 1,3 - butanediol, pentylene glycol, and glycerol. Preferably, the solvent may be selected from 1,3 - propylene glycol, 1,3 - butanediol, and glycerol. More preferably, the solvent is 1,3 - propylene glycol. Particularly preferably, the solvent is bio - based 1,3 - propylene glycol.

[0069] Thus, the liquid root extract may correspond to the original liquid extract obtained after solid / liquid extraction by maceration of the roots (optionally after stimulation) in step c) and after step d). Thus, the "maceration solvent" is the solvent used to obtain the root extract. Such a solvent needs to be selected from specific solvents to achieve the desired contents of the feruloylated derivatives of tormentic acid and the feruloylated derivatives of deoxytormentic acid.

[0070] According to one embodiment, after drying the root extract in liquid form in another step e), the root extract may be in solid or viscous form, wherein the drying is carried out according to any method known in the art, for example, by placing the root extract in liquid form in a hot and dry atmosphere to evaporate the maceration solvent.

[0071] The solid or viscous form of the root extract can be further diluted in a dilution solvent to obtain another type of liquid - form root extract. Thus, the "dilution solvent" is the solvent used to dilute the already obtained solid or viscous form of the root extract. Advantageously, such a root extract contains tormentic acid, the feruloylated derivatives of tormentic acid, and the feruloylated derivatives of deoxytormentic acid. The dilution solvent may particularly be selected from alcohols, diols, ethyl lactate, isopropyl myristate, triglycerides, triethyl citrate, dioctyl ether, glyceryl isostearate, glyceryl stearate, ethyl acetate, vegetable oils, or mixtures thereof. The alcohol is preferably selected from methanol and ethanol. In fact, although a specific solvent needs to be used as the maceration solvent to achieve the desired contents of the feruloylated derivatives of tormentic acid and the feruloylated derivatives of deoxytormentic acid, other solvents can be used to dilute the already obtained solid or viscous form of the root extract before using the extract. Specifically, the dilution solvent does not need to be the same as the maceration solvent used to obtain the extract of the present invention. Thus, the dilution solvent may preferably be selected from methanol, ethanol, 1,3 - propylene glycol, pentylene glycol, glycerol, dioctyl ether, especially bio - based dioctyl ether.

[0072] Depending on whether the root extract is in liquid form, whether the original liquid root extract is in an impregnation solvent, or whether the dried root extract is diluted in a dilution solvent, the liquid form of the root extract may contain the impregnation solvent disclosed above or the dilution solvent disclosed above.

[0073] "Tormentic acid" refers to tormentic acid itself and its stereoisomers.

[0074] "Feruloylated derivatives of tormentic acid" refer to compounds corresponding to the molecular formula C40H56O8 and having the following general formula (I).

[0075]

[0076] Formula (I): Feruloylated derivatives of tormentic acid

[0077] "Feruloylated derivatives of deoxytormentic acid" refer to compounds corresponding to the molecular formula C40H56O7 and having the following general formula (II).

[0078]

[0079] Formula (II): Feruloylated derivatives of deoxytormentic acid

[0080] "Roots rich in feruloylated derivatives of tormentic acid" and "enriched roots" refer to root extracts of the plant containing a higher amount of at least one feruloylated derivative of tormentic acid (formula (I) or formula (II)) compared to the corresponding root extracts of the same plant that can be found in nature.

[0081] "Feruloylated derivatives of tormentic acid" refer to feruloylated derivatives of tormentic acid and feruloylated derivatives of deoxytormentic acid.

[0082] Preferably, in order to obtain a root extract of Sanguisorba officinalis according to one aspect of the present invention, when the roots are considered to be fully developed, the roots are brought into contact with an extraction solvent by maceration or preferably by maceration, and then the extraction solvent is recovered and processed to extract the secondary metabolites released by the roots therefrom, including feruloylated derivatives of tormentic acid. This preferred method is adapted from the "PAT" method developed by a company named Plant Advanced Technologies (PAT) and described in International Application WO 01 / 33942. The teachings of this document are incorporated by reference into the specification of the present invention.

[0083] Accordingly, the present invention further relates to a method for preparing a root extract of the plant Sanguisorba officinalis according to one aspect of the present invention, the method comprising the following steps:

[0084] a) Cultivating Sanguisorba officinalis under soilless conditions, especially by aeroponics,

[0085] b) Stimulating the roots of the said plant,

[0086] c) Performing solid / liquid extraction by impregnating the roots obtained in step b),

[0087] d) Recovering the extract obtained in step c), and

[0088] e) Optionally, diluting and / or clarifying the extract recovered in step d) by successive filtration.

[0089] In one embodiment, the root extract of Sanguisorba officinalis according to one aspect of the present invention can be obtained by the above method.

[0090] "Cultivating a plant under soilless conditions" means any cultivation method in which the development of the roots of the plant does not take place in soil. More precisely, soilless cultivation is a type of cultivation in which the roots of the plant grow detached from the soil in a reconstituted culture medium. This medium is regularly irrigated with a well-known nutrient solution suitable for the plant being cultivated.

[0091] There are known several soilless cultivation techniques, such as substrate-free systems and substrate-based systems that require an oxygen-rich nutrient solution. Substrate-free systems include: aquaculture, in which the nutrient solution does not circulate and is contained in a culture tank; nutrient film technique (NFT), in which the nutrient solution is enriched with dissolved oxygen by exchanging with air during its movement; and aeroponics, in which the roots of the plant are neither in contact with a solid medium nor with a liquid medium. In fact, the roots are nourished by a nutrient mist obtained by atomizing the nutrient solution in a closed medium. Substrate-based systems include sub-irrigation, in which the nutrient solution penetrates the substrate through the lower part of the substrate; and percolation, in which the nutrient solution is distributed at the upper surface of the system by discontinuous irrigation and then penetrates to the bottom of the substrate. Mineral or organic substrates are neutral and inert, such as sand, clay or rockwool. The substrate may also be of synthetic origin.

[0092] The terms "aeroponic plant cultivation" and "aeroponics" denote a soilless cultivation method in which the roots of the plant are not in permanent contact with a solid medium or a liquid nutrient medium.

[0093] The term "nutrient solution" denotes a solution containing essential mineral salts (nitrogen - N, phosphorus - P, potassium - K), which are mixed with each other in optimal amounts and optimal ratios to obtain maximum root growth and maximum production of secondary metabolites (including feruloylated derivatives of tormentic acid having formula I and / or formula II).

[0094] The term "stimulating nutrient solution" refers to a nitrogen-deficient solution containing essential mineral salts (nitrogen - N, phosphorus - P, potassium - K), said mineral salts being in optimal amounts to obtain maximum root growth and maximum production of secondary metabolites, including feruloylated derivatives of tormentic acid of formula I and formula II.

[0095] According to a preferred embodiment, the plants can be fed with a nutrient solution mist obtained by spraying a nutrient solution in a closed medium using a nebulizer.

[0096] According to one embodiment, the plants can be placed on a tray with the aerial part of the plants above the tray and the root part below the tray, the tray is placed on a table that forms a retention area to collect the excess liquid diffusing towards the plants, and the tray is transferred to the tables at various workstations. The teachings regarding this technique suitable for aeroponic plant cultivation are described in more detail in international application WO2018054704A1, which is also incorporated by reference into the specification of the present invention.

[0097] In a preferred embodiment, in step a), the sanguisorba is fed by spraying a nutrient solution of essential mineral salts (nitrogen - N, phosphorus - P, potassium - K) on the roots of the sanguisorba cultivated by aeroponics to obtain maximum root growth and maximum concentration of the above-mentioned secondary metabolites without compromising the survival of the plants. Using their general knowledge, a person skilled in the art knows how to adjust the proportions and concentrations of the various mineral salts to optimize plant growth, especially root growth. In this case, the mineral salt concentration of the nutrient solution is usually in the low conductivity range, advantageously between 0.4 and 1.6 mS / cm, preferably between 0.8 and 1.2 mS / cm, to promote a greater diversity of secondary metabolites (including tormentic acid and feruloylated derivatives of tormentic acid) in the root extracts.

[0098] Advantageously, the above-mentioned aeroponic cultivation conditions allow the obtaining of certain secondary metabolites, including feruloylated derivatives of tormentic acid, in amounts greater than those of these same compounds obtained in soil cultivation.

[0099] In a preferred embodiment, the method can include a step of stimulating the roots. In this case, step b) of stimulating the roots of the plants comprises the step of bringing the roots into contact with a nitrogen-deficient stimulating nutrient solution, which can be a solution in which the proportion of nitrogen it contains is less than the proportion of nitrogen generally considered optimal for plant growth, especially root growth. Advantageously, the nutrient solution can contain less than 15% nitrogen, and more advantageously, does not contain any nitrogen, and steps a) and b) are consecutive or simultaneous.

[0100] In a preferred embodiment, the root stimulation of the plant in step b) comprises the step of bringing the roots into contact with a nitrogen-deficient nutrient solution. Contacting the plant with a nitrogen-deficient nutrient solution creates a "nitrogen stress", which stimulates the production of secondary metabolites, in particular feruloylated derivatives of tormentic acid.

[0101] In a particular embodiment, the nitrogen-deficient stimulating nutrient solution is a solution that generally contains less than 15% nitrogen, preferably less than 10% nitrogen, advantageously less than 8%, more advantageously less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% nitrogen and even more advantageously 0% nitrogen.

[0102] In one embodiment, the root stimulation step b) allows a significant increase in the content of secondary metabolites in the roots, in particular feruloylated derivatives of tormentic acid, and thus promotes the flow of said metabolites from the roots into the solvent selected for extraction, and does so without completely losing the viability of the plant, such that the plant can be returned to cultivation and then reused. In other words, the plant stimulation step promotes the biosynthesis of the above secondary metabolites.

[0103] According to one embodiment, the root stimulation in step b) can be carried out by supplying to the roots a nitrogen-deficient N / P / K stimulating nutrient solution that is evaporated or sprayed onto the roots.

[0104] According to one embodiment, step b) can be carried out by spraying or dipping the roots with an N / P / K nutrient solution containing less than 6% nitrogen, more preferably less than 3% nitrogen, and the solution is preferably evaporated or sprayed onto the roots.

[0105] According to one embodiment, the step b) of stimulating the roots by feeding the roots with a nitrogen-deficient N / P / K nutrient solution evaporated onto the roots is advantageously carried out for a time between 1 week and 8 weeks, more specifically between 1 week and 3 weeks, preferably 2 weeks.

[0106] According to one embodiment, in step b), the mineral salt concentration of the nutrient solution can be in the low conductivity range, advantageously between 0.4 and 1.2 mS / cm, preferably between 0.6 and 1.0 mS / cm, to promote a greater diversity of secondary metabolites (including feruloylated derivatives of tormentic acid).

[0107] According to one embodiment, step b) and step a) can be carried out simultaneously, and then the nutrient solution can be replaced with the stimulating solution.

[0108] According to a preferred embodiment, the method for preparing the root extract of the plant Sanguisorba officinalis of the present invention comprises:

[0109] a) cultivating Sanguisorba officinalis under soilless conditions, in particular by aeroponics,

[0110] b) Stimulate the roots of the plant,

[0111] c) Perform solid / liquid extraction by macerating the roots obtained in step b),

[0112] d) Recover the extract obtained in step c), and

[0113] e) Optionally, dilute and / or clarify the extract recovered in step d) by successive filtration.

[0114] Then, generally, step c) is carried out on the cultivated and stimulated plants, that is, solid / liquid extraction by root maceration is carried out under the conditions of a given solvent, temperature and extraction time, so as to obtain a root extract rich in feruloylated derivatives of tormentic acid and of course containing other secondary metabolites. Solid / liquid extraction is a known extraction technique based on solvents. The secondary metabolites released by the roots are recovered in the extraction solvent.

[0115] According to a preferred embodiment, an additional washing step can be carried out before step c) of the solid / liquid extraction of the roots stimulated in step b), wherein the solvent diffused into the plant is clear water. Thus, in the step of soaking in the solvent, the supply of the elements contained in the nutrient solution or the stimulating nutrient solution in the extraction solvent is limited.

[0116] According to an embodiment, root maceration can be carried out on the cut and then dried and optionally ground roots. The drying method can be any suitable typical known drying method, in particular by placing the root biomass at a temperature between 30 °C and 60 °C for 24 hours to 72 hours, preferably in a drying environment. The root biomass can in particular be dried in a ventilated oven. The grinding of the root biomass can be carried out by implementing any typical known grinding method, in particular by placing the root biomass in a ball mill or a knife mill or a hammer mill. After the step of cutting the roots, the plants are returned to the aeroponic culture according to step a) and optionally b) to restart their root growth and promote the production of secondary metabolites by the roots.

[0117] According to a preferred embodiment, the method can comprise the steps of cutting the roots and drying the cut roots before the maceration step, wherein the maceration is carried out by bringing the cut and dried roots into contact with a solvent.

[0118] According to a preferred embodiment, step c) of the solid / liquid extraction may comprise contacting the roots with a solvent selected from water, alcohols, diols or mixtures thereof, and dioctyl ether, preferably a bio-based dioctyl ether. Thus, the alcohol may preferably be selected from ethanol and methanol and used in the form of a pure substance or an aqueous solution, which contains 10% to 99.9%, more preferably 40% to 90%, especially 50% to 85% alcohol. The diol may be selected from dipropylene glycol, propane-1,3-diol, propane-1,2-diol, pentanediol, 1,3-butanediol and glycerol, and may be used in the form of a pure substance or an aqueous solution of the diol, which contains 10% to 99.9%, preferably 70% to 99.9% diol.

[0119] Preferably, the solvent may be a diol, which may be, for example, 1,3-propanediol, 1,3-butanediol or glycerol. In a preferred embodiment, the solvent may be selected from 1,3-propanediol. Particularly preferably, the solvent is a bio-based 1,3-propanediol.

[0120] According to one embodiment, step c) may comprise contacting the roots with the solvent (e.g., by maceration) for a time of 30 minutes to 48 hours, preferably 1 hour to 24 hours. In such a case, the operating temperature may be between 20 °C (room temperature) and 80 °C, preferably between 40 °C and 60 °C.

[0121] According to a specific embodiment, for the maceration of dry roots, the ratio of the amount of dry roots to the amount of solvent may be between 1 kg of dry roots / 10 kg of solvent and 1 kg of dry roots / 100 kg of solvent, more preferably between 1 kg of dry roots / 20 kg of solvent and 1 kg of dry roots / 40 kg of solvent.

[0122] For one aspect of the present invention, "macerating the roots in a solvent" means such root maceration where the solvent in contact with the roots is a solvent which may be, for example, pure ethanol or ethanol used in the form of an aqueous solution (which contains 50% to 85% ethanol), pure dioctyl ether, pure 1,3-butanediol or 1,3-butanediol used in the form of an aqueous solution (which contains 50% to 85% 1,3-butanediol), pure 1,3-propanediol or 1,3-propanediol used in the form of an aqueous solution (which contains 50% to 85% 1,3-propanediol). More preferably, the solvent is pure bio-based 1,3-propanediol or bio-based 1,3-propanediol used in the form of an aqueous solution (which contains 50% to 85% bio-based 1,3-propanediol).

[0123] According to one embodiment, the method may comprise one or more additional steps of treating the plant root extract, which may be selected from the following known methods:

[0124] - Dilution, concentration,

[0125] - One or more filtrations, especially clarification filtration and / or sterilizing filtration,

[0126] - Solid / liquid extraction,

[0127] - Purification,

[0128] - Bleaching of the liquid root extract.

[0129] The present invention further relates to a root extract of Sanguisorba officinalis obtained by the method as detailed above.

[0130] The present invention further relates to a cosmetic or dermatological composition, which comprises:

[0131] - A root extract of Sanguisorba officinalis according to the above aspect of the present invention, or a root extract of Sanguisorba officinalis obtained from the method defined above; and

[0132] - At least one cosmetic or dermatologically acceptable ingredient other than the extract of Sanguisorba officinalis,

[0133] wherein the composition is a composition for topical use selected from the following: solution, suspension, emulsion, cream, paste, gel, lotion, powder, soap, surfactant-containing water, oil, shampoo and spray, or wherein the composition is a nutritional composition for oral administration.

[0134] According to a preferred embodiment of the composition, the root extract of Sanguisorba officinalis may account for 0.0001 to 15% by weight, preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the cosmetic or dermatological or nutritional composition.

[0135] The mode of administration, dosage and optimal dosage form of the cosmetic composition according to the present invention can be determined according to the criteria usually considered in formulating a cosmetic treatment suitable for the subject (such as, for example, skin type). According to the desired type of administration, the cosmetic composition according to the present invention may further comprise at least one cosmetic acceptable excipient. The cosmetic composition according to the present invention may further comprise at least one cosmetic adjuvant known in the art, which is selected from thickeners, preservatives, fragrances, dyes, chemical or mineral filters, moisturizers, thermal spring water, etc.

[0136] Therefore, the cosmetic composition of the present invention may further comprise other cosmetic active agents other than the root extract of Sanguisorba officinalis as defined above, such as other anti-aging agents or moisturizers, agents having sedative, soothing or relaxing activities, agents stimulating skin microcirculation, sebum regulators for oily skin care, cleansing or purifying agents, anti-free radical agents, anti-inflammatory agents, chemical or mineral sunscreens, etc.

[0137] Suitable cosmetic excipients are those known in the art. For example, suitable cosmetic excipients may be selected from polymers, silicone compounds, surfactants, rheology modifiers, humectants, penetrants, oily components, waxes, emulsifiers, film formers, and fragrances, electrolytes, pH regulators, antioxidants, preservatives, dyes, pearlescent agents, pigments, and mixtures thereof.

[0138] Thus, the cosmetic composition of the present invention is advantageously intended for topical application. It may specifically be in the form of a cream, lotion, wash, gel, serum, spray, mousse, solution, ointment, emulsion, patch, or mask.

[0139] Suitable dermatological excipients are also those known in the art. For example, suitable dermatological excipients may be the same as those intended for use in cosmetics.

[0140] Suitable nutritional excipients are known in the art. Examples may include water-soluble polymers such as cellulosic polymers, acrylate polymers and copolymers, polyvinylpyrrolidone, water-soluble polyethylene glycols, vinyl copolymers. The nutritional composition may be, for example, a food additive in the form of a solid, coated or uncoated tablet, liquid, powder, soft or hard gelatin capsule, etc.

[0141] It should be understood that the definitions and preferred embodiments made in the context of the root extract of Sanguisorba officinalis according to one aspect of the present invention apply, mutatis mutandis, to the compositions containing said root extract.

[0142] The present invention also relates to the (cosmetic) use of the root extract of Sanguisorba officinalis according to one aspect of the present invention as an active ingredient as defined above for skin care and / or scalp care, in particular for promoting light-related human mood by stimulating the production of at least one well-being hormone in skin cells.

[0143] Advantageously, the root extract of Sanguisorba officinalis according to one aspect of the present invention as defined above is applicable in the context of well-ageing or healthy ageing, which is a new lifestyle concept, since said extract is capable of stimulating well-being hormones to prevent the harmful effects on the skin due to exogenous (e.g., UV-rays) and endogenous factors and thus affect overall skin homeostasis and impair health and beauty.

[0144] Thus, even in the absence of light exposure conditions and / or under low light exposure conditions, the root extract of Sanguisorba officinalis of the present invention is capable of reproducing or mimicking the beneficial effects of light (preferably sunlight) on the body (preferably the skin). These light-related beneficial effects are obtained even in the absence of light exposure conditions and / or under low light exposure conditions. In fact, under light exposure conditions, the beneficial effects are much better.

[0145] According to one embodiment, the use can be for promoting the production of at least one happiness hormone in skin cells, for promoting or stimulating the expression of phototransduction molecules (preferably peropsins (RRH)) in skin cells, for maintaining or restoring the circadian rhythm of clock proteins in skin cells, for stimulating the downstream biological pathway of vitamin D, or a combination thereof.

[0146] Thus, the use for skin care can also be for preventing or delaying the signs of skin photoaging, for preventing or treating UV-related skin damage, or a combination thereof.

[0147] According to one embodiment, the use for skin care can be for reducing and / or preventing protein carbonylation in skin cells.

[0148] According to one embodiment, preventing or delaying the signs of skin photoaging can be for improving the mechanical properties of the skin, particularly for improving skin elasticity, for improving skin complexion, for increasing dermal density, for maintaining or restoring the integrity of skin cells, for maintaining or restoring skin barrier function, for promoting skin hydration, for improving skin tone, for preventing the formation of fine lines and wrinkles, or a combination of two or more thereof.

[0149] The terms "happiness hormone" or "good mood hormone" or "pleasure hormone" used herein can be understood interchangeably in the broadest sense and, as commonly understood in the art, are molecules (e.g., hormones composed of proteins or peptides, or can be chemical molecules) capable of providing a happiness or soothing effect in a subject.

[0150] Throughout the context of the present invention, "low light exposure conditions" means that the light intensity received by the body, skin or scalp may exceed 5 lux, preferably exceed 200 lux, and even more preferably exceed 5000 lux. Conversely, during, for example, phototherapy, the light intensity may exceed approximately 5000 lux, preferably exceed approximately 10000 lux.

[0151] Advantageously, the light can be any light, which can be of natural origin such as sunlight, daylight or artificial light emitted by any light source, such as artificial light emitted by a lamp or the like. Preferably, the light is sunlight or daylight.

[0152] Throughout the context of the present invention, "light-related beneficial effects" means, for example, happiness promotion, mood control and a sense of relaxation. For example, these effects can be obtained by measuring the levels of serotonin and melatonin in plasma and the expression level of peropsins (RRH) in skin cells. It may also include circadian rhythm regulation, brain activity promotion and sleep regulation. In addition, it means increasing the activity of vitamin D in skin cells to supply the needs of the body.

[0153] Throughout the context of the present invention, "downstream biological pathway" refers to the transport of the active form of vitamin D through its nuclear receptor (VDR: vitamin D - receptor) and its carrier (DBP: vitamin D - binding protein), thereby exerting its biological effects on the body. Thus, vitamin D can be distributed from the skin and / or scalp to all organs of the body through DBP.

[0154] It should be understood that the definitions and preferred embodiments made in the context of the use of the root extract of Sanguisorba officinalis in the above - mentioned aspects of the present invention apply to the use of the root extract of Sanguisorba officinalis with necessary modifications in details. Specifically, throughout the context of the present invention, the term "active ingredient" can be understood in the broadest sense as a component that can individually exhibit the desired and expected above - mentioned activities, or a component that can exhibit activities in combination with one or more carriers that are inactive per se.

[0155] The present invention also relates to a non - therapeutic method for preventing or delaying the appearance of skin aging effects and / or for promoting a sense of well - being and / or relaxation, the method comprising applying the root extract of Sanguisorba officinalis according to the above - mentioned aspects of the present invention to at least a part of the skin and / or scalp. Examples

[0156] In the following examples, the root extract of Sanguisorba officinalis according to one aspect of the present invention, which is cultivated under soilless conditions (specifically aeroponics), will be referred to as SORE.

[0157] SORE was prepared from plants whose seeds were purchased from a UK supplier (Seedaholic) from 2015 to 2018. SORE was prepared from plants whose seeds were purchased from a French supplier ("Les Semences du Puy") from 2019 to 2022. Since 2019, the Sanguisorba officinalis plants have been preserved and propagated by the applicant.

[0158] Example 1: Preparation and Characterization of SORE

[0159] SORE was obtained according to the following method:

[0160] a) Aeroponically cultivate Sanguisorba officinalis using a nutrient medium with an N / P / K composition corresponding to 15 / 10 / 30 respectively and an electrical conductivity between 0.4 and 1.6 mS / cm for a period of 2 to 6 weeks,

[0161] b) Stimulate the plants with nitrogen stress using a nutrient solution with an N / P / K composition containing less than 6% nitrogen, 15% phosphorus and 40% potassium and an electrical conductivity between 0.4 and 1.2 mS / cm for 1 to 3 weeks,

[0162] b’) Rinse with clear water, then drain the roots stimulated in step b),

[0163] b”) Cut the roots and dry them in a ventilated oven at a temperature of 30 °C to 60 °C for 24 to 72 hours,

[0164] c) Perform solid / liquid extraction for 2 to 24 hours by immersing the dried roots in a pure 1,3-propanediol solution at a temperature of 50 °C,

[0165] d) Recover the root extract obtained in step c),

[0166] e) Clarify by filtration.

[0167] The feruloylated derivatives of tormentic acid having the general formula I and formula II are hereinafter named FDTA-I and FDTA-II respectively. Tormentic acid is named TA.

[0168] Quantify the amounts of TA, FDTA-I, and FDTA-II in the SORE according to the protocol described in Example 2.

[0169] - Dry extract content: 9.9 g / kg to 13.4 g / Kg

[0170] - TA content: 119.9 mg / Kg to 217.2 mg / Kg (i.e., 1.21% to 1.67% of the dry extract)

[0171] - FDTA-I content: 30.21 mg / Kg to 45.42 mg / Kg (i.e., 0.3% to 0.35% of the dry extract)

[0172] - FDTA-II content: 7.32 mg / KgL to 11.90 mg / Kg (i.e., 0.07% to 0.09% of the dry extract)

[0173] The obtained SORE has 9 g to 13.4 g of dry extract / kg of extract and contains approximately 2% by weight of total markers (FTA-I and FTA-II) relative to the total weight of the dry extract.

[0174] Example 2: Quantitative Methods for FDTA-I and FDTA-II

[0175] A Kinetex EVO C18 reverse-phase column (150 mm x 2.1 mm, 2.6 μm, Phenomenex, USA) maintained at 40 °C throughout all analytical procedures was used, and all samples were analyzed using a UHPLC Shimadzu Nexera X2 system (Shimadzu, Japan) equipped with a PDA detector coupled to a mass spectrometer LCMS2020 (negative ion mode electrospray ionization, m / z 100 - 1000). The mobile phase consisted of ultrapure water (Mili-Q, Merck Millipore) + 0.1% formic acid (Carlo Erba, France) (phase A) and pure acetonitrile (Sigma-Aldrich Chemie, Germany) (phase B), delivered at 0.5 ml / min, and the gradient of phase B was as follows: 5 - 95% (0 - 10 min); 95% (10 - 13.5 min); 95 - 5% (13.5 - 13.55 min), 5% (13.55 - 15.1 min).

[0176] Ferulic acid standards were used to quantify FDTA-I and FDTA-II in different extracts of Sanguisorba officinalis, and the standards were prepared at a concentration of 100 mg / L in an ethanol / water mixture (70 / 30, v / v). The contents of FDTA-I and FDTA-II were expressed as ferulic acid equivalents in each extract sample.

[0177] In the context of the present disclosure, the concentrations of FDTA-I and FDTA-II in the extracts of Sanguisorba officinalis expressed in mmol / L were determined by measuring the peak areas corresponding to FDTA-I and FDTA-II on the HPLC chromatogram (at 330 nm) of the extracts. Then, the peak areas of FDTA-I and FDTA-II were divided by the peak area of the standard solution containing 0.515 mmol / L of ferulic acid, and then multiplied by the molar concentration of the ferulic acid standard (0.515 mmol / L). The molar concentration values of FDTA-I and FDTA-II (expressed in mol / L) were converted to mass concentrations (expressed in g / L) by multiplying by the molecular weights of the two compounds.

[0178] Quantification of FDTA-I and FDTA-II was carried out according to the following equation:

[0179]

[0180] Where:

[0181] - The concentrations of FDTA-I and FDTA-II (FDTA) in sample X;

[0182] - Concentration of ferulic acid standard solution (0.515 mmol / L);

[0183] - Peak area of compound FDTA in sample X at 330 nm;

[0184] - Peak area of ferulic acid standard at 330 nm, corresponding to the standard solution concentration of 0.515 mmol / L;

[0185] MW FDTA - Molecular weight of FDTA

[0186] Potentillaic acid in different extracts of Sanguisorba officinalis was quantified using a TA standard prepared at a concentration of 100 mg / L in absolute ethanol. In the context of the present disclosure, the concentration of TA in the extract of Sanguisorba officinalis was determined by measuring the peak area corresponding to TA on the HPLC chromatogram (190 nm) of the extract. According to the following equation, the peak area of the TA peak in the extract sample was divided by the peak area of the 100 mg / L TA standard solution, and then multiplied by the mass concentration of the standard (100 mg / L):

[0187]

[0188] Where:

[0189] - Concentration of potentillaic acid (TA) in sample X

[0190] - Concentration of TA standard solution (100 mg / L)

[0191] - Peak area of TA in sample X at 190 nm

[0192] - Peak area of TA standard at 190 nm, corresponding to a concentration of 100 mg / L

[0193] The above-described quantification methods for TA and FDTA-I and FDTA-II were used for all the following examples.

[0194] Example 3: Phytochemical Analysis of FDTA-I and FDTA-II Contents in Extracts of Different Sanguisorba officinalis in Different Cultivation Methods and in Different Tissues

[0195] The Sanguisorba officinalis plants were cultivated by aeroponics for 4 weeks using a 15 / 10 / 30 (N / P / K) medium and a conductivity between 1.0 and 1.2 mS / cm, and then a root stimulation step was carried out for 2 weeks using a defined solution nutrient in which the N / P / K composition corresponded to 0 / 15 / 40 and the conductivity was between 1 and 1.2 mS / cm. The roots harvested before changing the medium corresponded to the non-stimulated roots. In addition, roots of commercially available dried Sanguisorba officinalis (Xuewen Tang Bozhou Swanf Commerce&Trade), which consisted almost entirely of rhizomes, were purchased for evaluation of their phytochemical composition.

[0196] The roots and aerial parts were cut, harvested, dried in a ventilated oven at 50 °C for 48 h, and ground. To prepare each extract, 25 mg of the ground root powder or 25 mg of the ground aerial part powder was impregnated in 0.5 mL of ethanol / water (70 / 30 v / v) and stirred at ambient temperature for 1 h. The samples were centrifuged and analyzed by HPLC-UV-MS.

[0197] The results are summarized in Table 1 below:

[0198] Table 1: Concentrations of TA and FDTA-I and FDTA-II in different extracts of the roots or aerial parts of Sanguisorba officinalis cultivated under different conditions (n = 3).

[0199]

[0200]

[0201] It was observed that the root extracts of Sanguisorba officinalis cultivated by aeroponics contained FDTA-I and FDTA-II.

[0202] In addition, the concentrations of FDTA-I and FDTA-II were significantly increased by the stimulation step:

[0203] - In the plants cultivated under aeroponic conditions, the difference in FDTA-I concentration between the non-stimulated and stimulated roots reached 2.17-fold.

[0204] - In the plants cultivated under aeroponic conditions, the difference in FDTA-II concentration between the non-stimulated and stimulated roots reached 10.66-fold.

[0205] Interestingly, it was observed that the amount of TA in the stimulated roots was reduced compared to the non-stimulated roots. This suggests that TA may be conjugated with ferulic acid after stimulation, thereby increasing FDTA-I and FDTA-II.

[0206] This study also confirmed that:

[0207] - Whether cultivated in soil or under aeroponic conditions, the aerial parts of Sanguisorba officinalis do not contain FDTA-I and FDTA-II.

[0208] - According to the protocol studies conducted, FDTA-I and FDTA-II were not detected in commercially available dried roots (only rhizomes).

[0209] These results highlight one of the advantages of cultivating Sanguisorba officinalis roots under aeroponic conditions.

[0210] Example 4: Method for Extracting TA, FDTA-I and FDTA-II from SORE

[0211] According to Example 3, an extract was prepared from the roots of Sanguisorba officinalis cultivated by an aeroponic method with a stimulation step.

[0212] A) Comparison of different extraction solvents

[0213] According to step c) of the method for preparing the root extract, the different solvents selected for solid / liquid extraction by maceration were a 70 / 30 (v / v) ethanol / water mixture, pure water, a 30 / 70 (v / v) or 50 / 50 (v / v) and 70 / 30 (v / v) propan-1,3-diol / water mixture, pure propan-1,3-diol, an 80 / 20 (v / v) 1,3-butanediol / water mixture, and pure dioctyl ether.

[0214] The extraction rates of TA, FDTA-I, and FDTA-II were compared with the extraction rate of ethanol extraction carried out under the same conditions as explained in Example 3, except for the maceration time (4 hours in this example). The contents of TA, FDTA-I, and FDTA-II in each sample were measured according to the protocol described in Example 2. Table 2 below describes the results:

[0215] Table 2: Results of the extraction rates of TA, FDTA-I, and FDTA-II using different solvents, with the extraction rate calculated relative to ethanol (70%). (PD: propylene glycol; BD: butanediol; DE: dioctyl ether)

[0216]

[0217] For those skilled in the art, the 70 / 30 ratio ethanol / water mixture can be regarded as the reference solvent. The results shown in Table 2 indicate that among all the solvents tested, pure propan-1,3-diol has the best extraction rates for TA, FDTA-I, and FDTA-II. Pure propan-1,3-diol was selected for the following examples.

[0218] B) Comparison of different extraction methods independent of solvents

[0219] To improve the extraction of TA, FDTA-I, and FDTA-II in SORE, pure propane-1,3-diol was used as the impregnation solvent, and different extraction methods were compared. Except for the extraction temperatures (25 °C and 50 °C) and extraction durations of 2 hours, 4 hours, and 24 hours, the extraction conditions were the same as those described in Example 3.

[0220] The contents of TA, FDTA-I, and FDTA-II in each sample were measured according to the protocol described in Example 2. The extraction yields were calculated relative to 50 °C or relative to 24-hour extraction.

[0221] For the extraction temperature experiment, the reference extract (100) had the following characteristics:

[0222] - Dry extract content: 7.06 g / Kg

[0223] - TA content: 105.85 mg / Kg (i.e., 1.50% of the dry extract)

[0224] - FDTA-I content: 40.10 mg / Kg (i.e., 0.57% of the dry extract)

[0225] - FDTA-II content: 29.27 mg / Kg (i.e., 0.41% of the dry extract)

[0226] For the extraction duration experiment, the reference extract (100) had the following characteristics:

[0227] - Dry extract content: 13 g / Kg

[0228] - TA content: 217.20 mg / Kg (i.e., 1.67% of the dry extract)

[0229] - FDTA-I content: 45.42 mg / Kg (i.e., 0.35% of the dry extract)

[0230] - FDTA-II content: 10.64 mg / Kg (i.e., 0.08% of the dry extract)

[0231] The following Tables 3 and 4 describe the results:

[0232] Table 3 : Results of the extraction yields of TA, FDTA-I, and FDTA-II using two different extraction temperatures. The extraction lasted for 2 hours.

[0233]

[0234] These results confirm that the optimal temperature for extracting TA, FDTA-I, and FDTA-II is 50 °C. Compared with 25 °C, it achieves the following gains:

[0235] - Yield of increased FDTA-I: Multiply by 3.

[0236] - Yield of increased FDTA-II: Multiply by 4.5.

[0237] - Yield of increased TA: Multiply by 1.25.

[0238] - Yield of increased FDTA-I and FDTA-II: Multiply by 1.7

[0239] Table 4: Results of the extraction rates of TA, FDTA-I, and FDTA-II using different extraction durations. The extraction temperature was 50 °C.

[0240]

[0241] These results confirm that the optimal duration for extracting TA, FDTA-I, and FDTA-II is 24 h. Compared with 2 h and 4 h, it will achieve the following gains:

[0242] - Yield of increased FDTA-I: Multiply by 1.5 and 1.25 respectively.

[0243] - Yield of increased FDTA-II: Multiply by 1.45 and 1.3 respectively.

[0244] - Yield of increased TA: Multiply by 1.8 and 1.3 respectively.

[0245] - Yield of increased TA, FDTA-I, and FDTA-II: Multiply by 1.8 and 1.3 respectively.

[0246] Example 5: SORE provides feelings of happiness and relaxation by upregulating the expression of serotonin and melatonin

[0247] Serotonin (also known as the "good mood hormone" or "happiness hormone") is a natural mood booster in the body and has been shown to be upregulated by sunlight. The skin may play a central role in the link between light and mood, and serotonin is very important in this regard. Therefore, experiments have shown that SORE can mimic the light transduction signal in the skin. Wondering whether topically applied SORE on the skin in the absence of sunlight exposure can upregulate serotonin expression. For this purpose, according to the manufacturer's instructions, a commercially available ELISA kit (Biovison, Abcam) was used to evaluate the serotonin in the culture medium of skin explants treated with 1% SORE for 3 days in the absence of light. Interestingly, it was confirmed that SORE significantly stimulated the production of serotonin in skin explants, with an increase of +55% ( Figure 1A ).

[0248] For transcriptome analysis, after 24 hours of tissue culture, total RNA was extracted using the RNeasy Mini kit from Qiagen. Their concentration and integrity were analyzed by spectrophotometry and capillary electrophoresis. Transcriptome analysis was performed on the Affymetrix Human Clariom S array according to the Affymetrix user manual. To analyze the data, all statistically significantly regulated genes were analyzed using the DAVID bioinformatics resource. This tool identifies functional regulatory pathways from large gene or protein datasets.

[0249] Transcriptome data (Table 5) indicated that SORE may upregulate the mRNA expression of serotonin receptor 3A, one of several receptors for serotonin.

[0250] Table 5: Upregulation of mRNA expression of HTR3A

[0251] Gene Fold Change p-Value HTR3A (5-Hydroxytryptamine Receptor 3A) 1.57 0.021

[0252] Serotonin is a precursor of melatonin, and melatonin is related to sleep quality, which itself is related to well-being. Melatonin secretion depends on the light / dark cycle. Since melatonin also stimulates antioxidant and DNA repair systems, and as its precursor, serotonin is upregulated by SORE, melatonin in the medium of skin explants treated with SORE for 5 days under light-free conditions was evaluated using a commercially available ELISA kit (Abbexa) according to the manufacturer's instructions. Intriguingly, it was confirmed that SORE significantly stimulated melatonin production in skin explants on day 5, with an increase of +120% ( Figure 1B ).

[0253] Then the well-being effect of SORE was demonstrated at the clinical level in two clinical studies.

[0254] A gel cream containing 1% SORE (see Table 9) was used in double-blind and vehicle-controlled clinical studies. In this regard, the recommendations of the Declaration of Helsinki and the guidelines of the International Conference on Harmonization Good Clinical Practice were considered applicable to non-drug studies.

[0255] - In the first study (proof-of-concept study): 10 Caucasian volunteers, aged between 32 and 58 years old, consisting of 4 females and 6 males, applied a gel cream containing SORE on the entire face twice a day for 5 days. In this study, the same volunteers tested the gel cream containing SORE and a placebo. There was a 5-day washout period between the two evaluations of the gel cream containing SORE. The order of use of the gel cream containing SORE and the placebo was randomly assigned to the volunteers.

[0256] - In the second study: 36 Caucasian females, aged between 30 and 50 years old. All the volunteers participating in this study claimed to suffer from depression. The facial skin color of the volunteers was poor and the skin lacked brightness. The group members were randomly divided into two groups: a placebo preparation (21 volunteers) and a formulated SORE (15 volunteers), and were required to use the gel cream containing SORE or the placebo on the entire face twice a day for 28 days.

[0257] To study the improvement of the mood (happiness) of the volunteers in these two studies, a headset device produced by MyBrainTechnologies was used to measure the electrical signals emitted by the brain during activity. These electroencephalogram signals were measured on the head surface by the capturer of the headset. A proprietary algorithm calculates the relaxation index (the higher the value, the more relaxed the volunteer). The measurement was carried out at least 1 hour after the application of the product to avoid the influence of the sensitive characteristics (fragrance, texture,...) of the preparation. Therefore, the observed effect is due to SORE itself.

[0258] For Study 1, the volunteers were measured on Day 0 (before applying the gel cream containing SORE), and then measured twice a day on Day 1 and Day 5 after applying the gel cream containing SORE.

[0259] For Study 2, the measurement was carried out on Day 0 and 7 days after applying the gel cream containing SORE twice a day.

[0260] To normalize the initial state values of each volunteer, the percentage change of each volunteer with respect to their initial state was calculated according to the following formula:

[0261] (Final value - Value on Day 0) / Value on Day 0 * 100

[0262] Compared with the placebo, the formulated SORE topically applied to the face improved the relaxation of the volunteers after 1 day of use. In addition, this effect was maintained after 5 days of use. Figure 2A It was shown that SORE improved relaxation: +112% on Day 1, +97% on Day 5, while the placebo did not improve relaxation: +17% on Day 1, and -50% on Day 5.

[0263] In the second study, the well-being effect of the formulated SORE observed in the first clinical study was confirmed. In fact, compared to the placebo control, relaxation parameters improved after 7 days of using the formulated SORE. Figure 2B It was shown that the formulated SORE was able to significantly improve relaxation on day 7 (+279% #p<0.07), while the placebo did not improve relaxation (-123.6% ns). The effect of SORE was significantly different compared to the effect of the placebo (*p<0.05). It was shown that the effects on serotonin secretion and well-being observed in vitro were confirmed in in vivo relaxation. In addition, the self-evaluation of the volunteers confirmed the well-being effect of the formulated SORE ( Figure 3 ). The volunteers found themselves refreshed and showed an improvement in the feeling of well-being. In addition, the volunteers found that their skin was more relaxed and more revitalized, two signs that change in the case of depression.

[0264] Example 6: SORE Upregulates the Downstream Biological Pathway of Vitamin D

[0265] In fact, since the skin cannot produce vitamin D in the absence of sunlight (especially UVB), it was wondered whether SORE could enhance the action of sunlight by upregulating the downstream biological pathways of vitamin D. In fact, the active form of vitamin D requires VDR and the carrier DBP (vitamin D-binding protein DBP) to exert its biological action. Vitamin D can be distributed from the skin to systemic organs via vitamin D-binding protein (DBP). Vitamin D may also have a direct beneficial effect on the skin through its nuclear receptor (VDR). Therefore, the effect of SORE on the downstream biological pathways of vitamin D (VDR and DBP) was evaluated at the transcriptome level in skin explants 24 hours after SORE treatment and at the proteomic level in skin explants 5 days after SORE treatment (with or without phototherapy exposure).

[0266] As described in Example 5, the related effect of 1% formulated SORE (see Table 9) on vitamin D-binding protein (DBP) was studied in the transcriptome analysis, and the phototherapy coupling effect on the vitamin D receptor (VDR) was studied.

[0267] Table 6: Skin explants treated with SORE for 24 hours showed a significant stimulation of DBP mRNA expression

[0268] Gene Fold Change p-Value GC Vitamin D-Binding Protein (DBP) 1.72 0.004

[0269] Figure 4 shows that in skin explants treated with light (without UV-B rays) and 1% formulated SORE (see Table 9) simultaneously for 5 days, the protein VDR (+15%: Figure 4A) and DBP (+35%): Figure 4B ) expression was upregulated. All these results confirmed that SORE can improve the distribution of skin-produced vitamin D to other tissues by upregulating its carrier (DBP). In addition, experiments showed that SORE can also improve the biological effects of vitamin D in the skin, as it was confirmed to upregulate VDR.

[0270] Example 7: Mixture of FDTA-I and TA from SORE Simulates Photoeffects on the Skin

[0271] SORE was formulated at a concentration of 0.75% by weight in a gel cream (Table 7). The biological effects of SORE compared to placebo were evaluated ex vivo on human skin explants from a 39-year-old female Caucasian donor by transcriptomic analysis. Briefly, the formulation was applied topically to the skin explants at 2 mg / cm 2 . The explants were cultured at 37 °C and 5% CO2 for 24 hours.

[0272] Table 7: Gel cream formulation containing 0.75% by weight of SORE

[0273]

[0274] SORE induces phototransduction signals similarly to visible light (but in the absence of light). Transcriptomic studies as described in Example 5 showed that SORE induces the expression of photoreceptors known to be induced by visible light in the skin, thus confirming that SORE can trigger light signal transduction even in the absence of light exposure. In fact, visible light has been described as stimulating opsin photoreceptors such as Perospin (RRH) or rhodopsin to transduce light signals. Transcriptomic skin analysis showed an enrichment of transcripts involved in phototransduction signals and photoreceptor homeostasis typically found in the retina (Table 8):

[0275] Table 8: Stimulation of light perception-related genes in the skin induced by formulated SORE

[0276] Gene Fold Change p-Value RRH (Visual Pigment-Like Receptor Peropsins) 1.63 0.022 OR56A5 (Olfactory Receptor Family 56 Subfamily A Member 5) 1.61 0.063 SAG (S-Antigen Visual Rhodopsin-Inhibiting Protein) 1.66 0.004

[0277] -RRH plays a role in retinal pigment epithelium physiology by directly detecting light or by monitoring the concentration of photoreceptor-derived compounds. It is also expressed in the skin and may be involved in the phototransduction of short-wavelength violet light, suggesting that the skin may carry photoreceptors that can actually perceive and distinguish various light qualities.

[0278] -OR56A5 was described as silenced in retinitis pigmentosa, a disease associated with vision loss. ORA5 is involved in the signal transduction of ultraviolet light.

[0279] -SAG binds to photo-activated phosphorylated Rho and terminates Rho signaling through G proteins by competing with G proteins for the same binding site on Rho. It plays a role in preventing light-dependent degeneration of retinal photoreceptor cells.

[0280] Then, it was demonstrated that SORE was able to induce phototransduction signals on skin explants without light exposure similar to visible light. In other words, SORE could induce the expected biological effects of light on the skin without light.

[0281] An additional ex vivo experiment (skin from a 41-year-old female donor) was conducted using a commercially available phototherapy device (Dayvia White072) under light or no-light conditions. This device is specifically used to treat people with seasonal affective disorder. Briefly, a gel cream containing 1% SORE or placebo (see Table 9) was topically applied to the skin explants at 2 mg / cm 2 The explants were exposed to the phototherapy device. The device emits 10,000 lux of light at a distance of 21 cm and does not contain ultraviolet and infrared rays. 10,000 lux is the light intensity recommended for phototherapy. The skin explants received placebo or SORE and were then treated with the light of the device for 20 minutes at a distance of 21 cm. Non-exposed topically treated explants were also cultured for reference. The exposure was carried out daily, as was the daily topical application of placebo or SORE. The topical application was performed immediately after light exposure. The explants were cultured at 37 °C, 5% CO2 for 5 days.

[0282] At the end of the experiment, the explants were fixed with formalin and then embedded in paraffin. Thin sections were processed for RRH immunofluorescence examination. The expression levels were estimated by fluorescence quantification using Image J. Figure 5 It was shown that, as expected, light upregulated the expression of RRH. In addition, SORE could stimulate the expression of RRH (+17%) without light, indicating its ability to mimic the effect of light on this photoreceptor and potentially trigger light-like effects. This result confirmed the transcriptome data. Furthermore, SORE may also be able to enhance light perception through RRH (+54%) as its expression in light-exposed skin explants treated with SORE was higher than that in light-exposed skin explants treated with placebo.

[0283] Table 9: Gel cream containing 1 wt% of SORE and placebo

[0284]

[0285] Example 8: SORE Regulates the Circadian Rhythm of Skin Cells

[0286] Results were obtained from the human skin explant experiment described in Example 5.

[0287] Table 10: SORE upregulated key circadian genes formulated at 0.75% (see Table 7).

[0288] Gene Fold Change p-Value TOP2A (DNA Topoisomerase IIα) 1.84 0.013 SUV39H2 (Heterochromatin Protein 1 Homolog 2) 1.58 0.009 SFPQ (Splicing Factor Rich in Proline and Glutamine) 1.38 0.030 PSPC1 (Paraspeckle Component 1) 1.28 0.038 TIMELESS (Perpetual Circadian Rhythm Regulator) 1.77 0.017 HNRNPU (Heterogeneous Nuclear Ribonucleoprotein U) 1.27 0.024 PASD1 (Repressor Protein Containing PAS Domain 1) 1.40 0.035

[0289] - TOP2A plays a role in regulating the period length of the ARNTL / BMAL1 transcriptional oscillation and affects the circadian rhythm period.

[0290] - SUV39H2 may be recruited by the large PER complex to the E - box element of circadian target genes such as PER2 itself or PER1.

[0291] - Both SFPQ and PSPC1 can regulate the circadian clock by inhibiting the transcriptional activation activity of the CLOCK - ARNTL / BMAL1 heterodimer. They are required for the transcriptional repression of circadian target genes such as PER1, mediated by the large PER complex through histone deacetylation.

[0292] - TIMELESS is involved in determining the period length and in the phase advance of the DNA damage - dependent circadian clock. Timeless negatively regulates the trans - activation of PER1 induced by CLOCK|NPAS2 - ARTNL / BMAL1|ARTNL2 / BMAL2, possibly through the translocation of PER1 into the nucleus.

[0293] - HNRNPU has multiple functions, including involvement in the circadian regulation of the transcription of the core clock component ARNTL / BMAL1.

[0294] - PASD1 acts as an inhibitor of the biological clock, which drives the daily circadian rhythm of body cells. It may act as a nuclear repressor of the transcriptional activation of core clock components mediated by the CLOCK - ARNTL / BMAL1 heterodimer.

[0295] All these results suggest that SORE may regulate the circadian rhythm of skin cells, which is crucial for regulating various cellular, metabolic, physiological, and behavioral activities in mammals.

[0296] Example 9: SORE Maintains Cell Integrity by Inducing DNA Repair Mechanisms

[0297] Results were obtained from the human skin explant experiment described in Example 5. SORE showed a cytoprotective effect by upregulating DNA repair factors (Table 11).

[0298] Table 11: Upregulation of DNA repair-related genes on SORE-treated skin explants formulated at 0.75% (see Table 7).

[0299] Gene Fold Change p-Value MBD4 (Methyl-CpG Binding Domain 4, DNA Glycosylase) 1.47 0.014 TREX1 (Three Prime Repair Exonuclease 1) 1.37 0.029 BOD1L1 (Chromosome Biorientation Protein 1-Like 1 in Cell Division) 1.30 0.041 RAD51B (RAD51 Paralog B) 1.61 0.020 RAD51AP1 (RAD51-Associated Protein 1) 1.68 0.045 KIN (Kin17 DNA and RNA Binding Protein) 1.55 0.040 MSH2 (mutS Homolog 2) 1.63 0.048 FANCD2 (FA Complementation Group D2) 1.97 0.002 RPA3 (Replication Protein A3) 1.69 0.033 POLI (DNA Polymerase ι) 1.43 0.041 SLF1 (SMC5-SMC6 Complex Localization Factor 1) 2.04 0.005 XPA (XPA, DNA Damage Recognition and Repair Factor 1.71 0.009 NSMCE4A (NSE4 Homolog A, SMC5-SMC6 Complex Component) 1.69 0.045 ATR (ATR Serine / Threonine Kinase) 1.62 0.033 PRIMPOL (DNA-Directed Primase / Polymerase Protein) 1.69 0.033

[0300] - MBD4 is a mismatch-specific DNA N-glycosylase involved in DNA repair. MDB4 has thymine glycosylase activity and specifically targets G:T mismatches within methylated and unmethylated CpG sites. It also removes uracil or 5-fluorouracil in G:U mismatches.

[0301] - TREX1 is a major cellular 3'-to-5' DNA exonuclease that can digest single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with a mismatched 3' end.

[0302] - BOD1L1 is a component of the fork protection mechanism that is responsible for protecting stalled / damaged replication forks from runaway DNA2-dependent resection. It acts by stabilizing RAD51 at stalled replication forks and protecting RAD51 nucleofilaments from anti-recombinase activity.

[0303] - RAD51B is involved in the homologous recombination repair (HRR) pathway of double-stranded DNA breaks that occur during DNA replication or are induced by DNA-damaging agents. It promotes the assembly of presynaptic RAD51 nucleoprotein filaments.

[0304] - KIN is involved in DNA replication and the cellular response to DNA damage. It is involved in DNA replication factories and bridges the gap between DNA replication and repair mediated by high molecular weight complexes.

[0305] - MSH2 is a component of the post-replicative DNA mismatch repair system (MMR) that binds to DNA mismatches to initiate DNA repair.

[0306] - FANCD2 is essential for maintaining chromosomal stability and is involved in DNA double-strand break repair by homologous recombination and single-strand annealing.

[0307] - RAP3 is part of the heterotrimeric replication protein A complex (RPA / RP-A) that binds and stabilizes single-stranded DNA intermediates formed during DNA replication or after DNA stress. Thus, it plays an important role in DNA replication and the cellular response to DNA damage.

[0308] - POLI is an error-prone DNA polymerase specifically involved in DNA repair.

[0309] Some upregulated DNA repair systems are highly specific for environmental damage and especially UV damage.

[0310] -XPA is involved in DNA excision repair. It initiates repair by binding to damaged sites with various affinities, which depend on the photoproduct and the transcriptional status of the region. Additionally, it is also required for UV-induced CHEK1 phosphorylation and the recruitment of CEP164 to cyclobutane pyrimidine dimers (CPDs), the DNA damage sites after UV irradiation.

[0311] -SLF1 plays a role in the DNA damage response (DDR) pathway by regulating post-replication repair of UV-damaged DNA and maintaining genomic stability. Complementarily, NSMCE4A (non-structural maintenance of chromosomes element 4 homolog A) is a component of the SMC5-SMC6 complex, which is involved in DNA double-strand breaks by homologous recombination.

[0312] -ATR activates checkpoint signaling after genotoxic stress such as ionizing radiation (IR), ultraviolet (UV), or DNA replication stalling, thus acting as a DNA damage sensor.

[0313] -PRIMPOL is a DNA primase and DNA polymerase that is essential for tolerating replication stalling damage by bypassing it. When DNA replication stalls, it provides different translesion synthesis alternatives. It is able to synthesize DNA primers downstream of the damage, such as ultraviolet (UV) damage, R-loops, and G-quadruplexes, to allow DNA replication to continue.

[0314] Therefore, SORE acts on several DNA repair components involved in conventional or environmentally induced DNA damage. It may be a powerful protector that safeguards DNA integrity, which may be compromised by the aging process and / or environmental insults such as sunlight.

[0315] Example 10: Anti-Collagenase Inhibitory Activity of FDTA-I and FDTA-II Purified from SORE

[0316] Purify FDTA-I and FDTA-II from stimulated Sanguisorba officinalis roots, where the plants have been stimulated for 1 to 3 weeks using a nitrogen-free nutrient medium (N / P / K 0 / 15 / 40). Immerse 10 g of dried and ground (1 mm) roots in 100 ml of ethyl acetate at 50 °C for 24 h. Obtain purified FDTA-I and FDTA-II fractions by preparative HPLC and characterize them by mass spectrometry. Commercially purchase ferulic acid and TA. Additionally, include Sanguisorbic acid I in the experiments, which is an active compound isolated from Sanguisorba officinalis roots and is known to have various activities, including increased expression of type I collagen. For comparison, obtain SORE as shown in Example 1. Measure the anti-collagenase inhibitory activity by tracking the degradation kinetics of a collagenase-driven collagenase substrate: FALGPA (N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala) degrades to FAL (N-[3-(2-furyl)acryloyl]-Leu) and Gly-Pro-Ala.

[0317] Briefly, a solution of 100 μL volume of 1.5 mM FALGPA in 50 mM tris (hydroxymethyl) methylglycine buffer (containing 10 mM calcium chloride and 400 mM sodium chloride, pH 7.5) (Bachem, 4006713.0025) was mixed with 10 μL volume of the test sample or the pure solvent of the test sample (positive control), and then 10 μL volume of 0.05 mg / mL Clostridium histolyticum collagenase (type IA, Sigma-Aldrich, C9891) was added. To determine the enzymatic conversion rate, the content of FAL was quantified by stopping the enzymatic conversion of FALGPA with EDTA at the start of the reaction and 30 minutes after the start of the reaction. To quench the enzymatic conversion, 50 μL volume of the test sample was mixed with 50 μL volume of EDTA (0.2 M aqueous solution). Then, the FAL content in the sample was quantified by UHPLC method for separating FALGPA and FAL with optimized components. Analysis was performed using a Kinetex Biphenyl reverse-phase column (150 mm x 2.1 mm, 2.6 μm, Phenomenex, Torrance, USA) maintained at 40 °C. The mobile phase consisted of water (A) containing 0.1 vol% formic acid and pure acetonitrile (B), delivered at 0.5 ml / min, and the gradient of phase B was as follows: 5 - 41% (0 - 9 min); 41 - 90% (9 - 9.05 min); held at 90% (9.05 - 11.50 min); 90 - 5% (11.50 - 11.55 min), held at 5% (11.55 - 14.50 min). The sample injection volume was 5 μL, and FALGPA / FAL detection was performed at 338 nm. Then, the residual activity and inhibition degree of collagenase in the presence of test sample X were calculated using the following equations:

[0318]

[0319] Inh% EchX = 100% - ActR% EchX

[0320] Where:

[0321] ActR% - residual activity; Inh% - inhibition degree; AUC - peak area of FAL; EchX - test sample; BL - blank.

[0322] Table 12: The anti - collagenase inhibitory activities of FDTA - I and FDTA - II, saponarin, TA and ferulic acid at a concentration of 25 μM and SORE at a concentration of 0.04%.

[0323]

[0324] SORE rich in FDTA-I and FDTA-II inhibited collagenase IA activity (50%) at a very low concentration (0.04%). FDTA-I and FDTA-II acted as collagenase IA inhibitors at 25 μM (76% and 36% inhibition (Inh%), respectively), while TA and ferulic acid alone had no effect on the enzyme at the same concentration. It emphasizes the relevance of the coupling of ferulic acid and TA via an ester bond in the context of their anti-collagenase bioactivity. Interestingly, saponarin had no enzyme inhibitory effect at 25 μM.

[0325] The results demonstrated that SORE containing FDTA-I and FDTA-II had the ability to inhibit collagenase activity. Therefore, SORE can be used to improve the mechanical properties of the skin, especially ECM components, by inhibiting the activity of collagenase. In addition, their activity against collagenase was apparently not related to the reported activity of saponarin.

[0326] Example 11: Anti-Hyaluronidase Inhibitory Activity of FDTA-I and FDTA-II Purified from SORE

[0327] As described in Example 10, FDTA-I and FDTA-II were purified from stimulated Sanguisorba roots. In addition, saponarin was also included in the test. For comparison, SORE was prepared as shown in Example 1.

[0328] Hyaluronidase catalyzes the degradation reaction of hyaluronic acid (HA). This test was based on the quantification of the intact HA content in the presence / absence of the extract and / or compound of interest. The degradation rate of the HA content is directly proportional to the enzyme activity of hyaluronidase and inversely proportional to the inhibitory ability of the extract.

[0329] Prepare the reaction mixture as follows: 37.5 μL of 20 mM PHB buffer pH 7, 187.5 μL of hyaluronidase solution (bovine hyaluronidase, type IS, Sigma) at 0.02 mg / mL, and 25 μL of sample (SORE or pure FDTA-I and FDTA-II to be tested). In the case of Table 13, pure FDTA-I and FDTA-II as well as SORE are in pure propane-1,3-diol. In the case of Table 14, pure FDTA-I and FDTA-II are in 100% ethanol. Incubate the mixture at room temperature for 10 minutes and then add 250 μL of HA substrate (0.3 mg / mL in phosphate buffer) to initiate the reaction. Incubate in a thermostatic mixer at 37 °C with stirring (450 rpm). Sample the reaction mixture (50 μL) every 15 minutes and monitor the reaction for 60 minutes. Place these samples into the wells of a microtiter plate containing 200 μL of 0.1% BSA solution (pH 3.75) and incubate in BSA for 10 minutes. HA forms aggregates with the BSA solution, which precipitate. Thus, the amount of precipitated BSA is proportional to the optical density at 600 nm, which allows monitoring of the enzyme reaction. Finally, insert the microtiter plate into a spectrophotometer and measure the optical density at this wavelength.

[0330] Table 13: Anti-hyaluronidase inhibitory activities of FDTA-I, TA, and ferulic acid at 15 μM concentration and SORE at 0.01% concentration.

[0331]

[0332] FDTA-I acts as a hyaluronidase inhibitor at 15 μM, while TA and ferulic acid alone have no effect on the enzyme at the same concentration. This result emphasizes the relevance of the ester bond coupling between ferulic acid and TA in the context of their anti-hyaluronidase biological activity.

[0333] Table 14: Anti-hyaluronidase inhibitory activities of saponin I, FDTA-I, and FDTA-II at 15 μM.

[0334]

[0335] FDTA-I and FDTA-II act as hyaluronidase inhibitors at 15 μM, while saponin I has no effect on the enzyme at the same concentration. This result demonstrates the ability of SORE containing FDTA-I and FDTA-II to inhibit hyaluronidase activity. Thus, SORE can be used to improve the mechanical properties of the skin, particularly ECM components, by inhibiting hyaluronidase activity.

[0336] Example 12: SORE Improves Skin Elasticity and Skin Tone

[0337] Another clinical study on Caucasian women showed that the use of SEM improved skin elasticity measured by a cutaneous elasticity meter (Courage&Khazaka), which allows the determination of skin elasticity properties by calculating the Ue parameter. There is sufficient evidence that skin elasticity properties decrease with aging.

[0338] Figure 6 showed that SORE improved skin elasticity, as demonstrated by an increase in the Ue parameter after 28 days of use (+15%*: p<0.05), while the placebo had no significant effect (+5% ns). This is consistent with the data obtained in vitro and described in Examples 10 and 11, indicating that FTDA-I and FTDA-II inhibit the activities of collagenase and hyaluronidase, which are known to alter skin mechanical properties such as elasticity and hardness.

[0339] Clinical study 2 also confirmed that SORE improved skin tone after 28 days of application. The uniformity of facial skin color was scored based on an unstructured scale. Photographs were taken with a high-resolution camera Nikon D300S with n°2. Finally, the self-evaluation of the volunteers on the cream was evaluated through a questionnaire. Figure 7 showed that SORE improved skin color, making it appear more uniform, with a +16% improvement in the uniformity score (*: p<0.05), while the placebo did not (+4% ns).

[0340] It has been confirmed that this beneficial effect has been recognized by the volunteers themselves. After 28 days of topical application of SORE, more than 73% of the volunteers found their skin color to be more uniform compared to the placebo (52%) (data not shown).

[0341] Example 13: SORE reduces protein carbonylation in keratinocytes

[0342] Since it is generally agreed that in the skin, an increase in protein carbonylation is associated with changes in skin quality such as skin tone or fragile dermis, it was decided to also analyze the effect of SORE on protein carbonylation in keratinocytes in the clinical study described in Example 5.

[0343] Keratinocytes were collected from the skin surface of volunteers before (day 0) and 28 days after using 1% SORE (see Table 9) or placebo. The D-squames adhesive was applied for 5 to 20 seconds using a dedicated applicator (stable pressure: 50 g). The first sampling was not retained, and only the second sampling was considered. Then, the keratinocytes were recovered from the strip by immersing the strip in the following dissociation solution and maintaining it at 100 °C for 10 minutes: 1% SDS, 20 mM DL-dithiothreitol, 5 mM EDTA, 0.1 M Tris-HCl buffer pH 8. The tubes were centrifuged. They were washed 3 times for 1 minute each in the dissociation solution at room temperature and then recentrifuged. The keratinocytes were labeled with 2 mM fluorescein-5-thiosemicarbazide in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES)-Na buffer (pH 5.5) for 1 hour at room temperature. After washing 3 times in phosphate-buffered saline (PBS), the suspension was placed on microscope glass slides for 12 hours. Five photos were taken of each slide. The fluorescence intensity was quantified using Image J software. The results showed that 28 days after application, the content of carbonylated proteins in the keratinocytes of volunteers using SORE was significantly lower than that of volunteers using placebo ( Figure 8 ). These results demonstrated that SORE was able to protect the skin from the harmful effects of carbonylated proteins and thus improve skin quality (including skin color).

Claims

1. The root extract of Sanguisorba officinalis, comprising: - Tormentic acid, which accounts for at least 1% by weight of the total weight of the dry extract, - Feruloylated derivatives of tormentic acid having the general formula (I), and - Feruloylated derivatives of deoxytormentic acid having the general formula (II) 2. The root extract according to claim 1, wherein the feruloylated derivative of deoxytormentic acid accounts for at least 0.05% by weight of the total weight of the dry extract.

3. The root extract according to any one of claims 1 or 2, wherein the feruloylated derivative of tormentic acid accounts for at least 0.1% by weight of the total weight of the dry extract.

4. The root extract according to any one of claims 1 to 3, wherein the tormentic acid, the feruloylated derivative of tormentic acid and the feruloylated derivative of deoxytormentic acid together account for at least 1.15% by weight of the total weight of the dry extract.

5. The root extract according to any one of claims 1 to 4, wherein the root extract further contains one or more ellagitannins.

6. A method for preparing the root extract of the plant Sanguisorba officinalis according to any one of claims 1 to 5, the method comprising the following steps: a) Cultivating Sanguisorba officinalis under soilless conditions, in particular by aeroponics, b) Stimulating the roots of the plant, c) Performing solid / liquid extraction by immersing the roots obtained in step b), d) Recovering the extract obtained in step c), and e) Optionally, diluting and / or clarifying the extract recovered in step d) by continuous filtration.

7. A cosmetic or dermatological composition, comprising: - The root extract of Sanguisorba officinalis according to any one of claims 1 to 5 or the root of Sanguisorba officinalis obtained from the method according to claim 6; and - At least one other cosmetic or dermatologically acceptable ingredient other than the root extract of Sanguisorba officinalis, wherein the composition is a composition for topical use selected from the following: solution, suspension, emulsion, cream, paste, gel, lotion, powder, soap, surfactant-containing water, oil, shampoo and spray, or wherein the composition is a nutritional composition for oral administration.

8. The cosmetic use of the root extract of Sanguisorba officinalis according to any one of claims 1 to 5 as an active ingredient for skin care and / or scalp care, for promoting light-related human mood.

9. The cosmetic use according to claim 8, for increasing and / or stimulating the production of at least one happiness hormone in skin cells.

10. The cosmetic use according to any one of claims 8 or 9, for promoting the production of at least one happiness hormone in skin cells.

11. The cosmetic use according to any one of claims 8 to 10, for promoting or stimulating the expression of phototransduction molecules, preferably peropsins (RRH), in skin cells.

12. The cosmetic use according to any one of claims 8 to 11, for maintaining or restoring the circadian rhythm of clock proteins in skin cells.

13. The cosmetic use according to any one of claims 8 to 12, for stimulating the downstream biological pathway of vitamin D.

14. The cosmetic use according to any one of claims 8 or 13, for preventing or delaying signs of skin photoaging, for preventing or treating UV-related skin damage, or a combination of two or more thereof.

15. The cosmetic use according to claim 14, for reducing and / or preventing carbonylation of proteins in skin cells.

16. The cosmetic use according to any one of claims 14 or 15, for improving the mechanical properties of the skin.

17. The cosmetic use according to any one of claims 14 to 16, for improving skin elasticity, for improving skin complexion, for increasing dermal density, for maintaining or restoring the integrity of skin cells, for maintaining or restoring skin barrier function, for promoting skin hydration, for improving skin tone, for preventing the formation of fine lines and wrinkles, or a combination of two or more thereof.

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