"Combination of palmitoylethanolamide and Equisetum, food, nutraceutical and/or pharmaceutical compositions containing it, and use thereof in the prevention and treatment of osteoporosis"

It is an object of the present invention a synergistic combination of palmitoylethanolamide and Equisetum with anti-osteoporotic activity, food, nutraceutical and/or pharmaceutical compositions containing it, and use thereof as agents promoting bone formation and in the prevention and treatment of bone resorption.

Technical field

Osteoporosis is a widespread chronic disease characterized by decreased bone mineral density, impaired bone strength, and increased risk of fragility-related fractures that are associated with significant morbidity, mortality, and disability. Fractures are a major public health burden, as they are major causes of morbidity, impairment, decreased quality of life, and mortality. Osteoporosis therefore involves many medical, economic and social consequences. The total burden of osteoporosis is estimated to grow by 50 percent with more than 3 million incident fractures by 2025, a cost that translates to nearly $25.3 billion annually in the United States alone. Globally, 200 million people suffer from osteoporosis and 8.9 million fractures occur annually. By 2050, hip fractures could exceed 21 million cases. The prevalence of osteoporosis is 18.3 percent globally and is higher in women than men (23.1 and 11.7 respectively).

At the molecular level, bone homeostasis is compromised by the molecular disruption of communication between bone-forming osteoblasts and bone-resorbing osteoclasts. It follows that controlling the signaling network that drives osteoblast-osteoclast balance could lead to the identification of new therapeutic targets. In this context, the influence of nutritional factors on the development and progression of this disease could be significant but has yet to be elucidated.

It is well known that many female sufferers of osteoporosis would like to have a natural therapeutic approach in addition to or as an alternative to conventionally administered drugs; the same applies to perimenopausal and menopausal women who wish to prevent the disease or at least delay its onset.

It is therefore of great interest to seek new natural therapeutic solutions, alternatives to conventional drugs, for the prevention and treatment of osteoporosis.

Purposes of the invention

It is an purpose of the invention to make available a combination of active ingredients of natural origin having potent anti-osteoporotic activity through different mechanisms of action.

It is another purpose of the invention to make available food, nutraceutical, and/or pharmaceutical compositions comprising said combination and their use in therapy.

Brief description of the Drawings

Figure 1 shows the effect of PEA and Equisetum, alone and in combination on alkaline phosphatase (ALP) activity (A) and mineralization (Alizarin red) (B) on MC3T3 cells. Panel (C) shows the effect on tartrate-resistant acid phosphatase (TRAP) on RAW 264.7 cells. Results are expressed as mean ± SD (%) vs control (0% line) of 5 independent experiments, each performed in quadruplicate. * p<0.05 vs control; ** p<0.05 vs single agents. Figure 2 shows the effect of PEA and Equisetum, alone and in combination, on cannabinoid receptor 1 (CB1) (panel A) and cannabinoid receptor 2 (CB2) (panel B) on MC3T3 and RAW 264.7 cells. Results are expressed as mean ± SD (%) vs control (0% line) of 5 independent experiments, each performed in quadruplicate. * p<0.05 vs control; ** p<0.05 vs single agents.

Figure 3 shows the effect of PEA and Equisetum alone and in combination, on TRAP activity (panel A), bone resorption assay (panel B) and relative area count (panel C) on MC3T3 and RAW 264.7 in 3D culture. Results are expressed as mean ± SD (%) vs control (0% line) of 5 independent experiments, each performed in quadruplicate. * p<0.05 vs control; ** p<0.05 vs single agents.

Figure 4 shows the effect of PEA and Equisetum, alone and in combination, on the kinase activity of OPG (panel A), RANKL (panel B) and the ratio OPG/RANKL (panel C) on MC3T3 and RAW 264.7 in 3D culture. Results are expressed as mean ± SD (%) vs control (0% line) of 5 independent experiments, each performed in triplicate. * p<0.05 vs control; ** p<0.05 vs single agents.

Description of the invention

The Applicant, after extensive and intensive research and development activity, has surprisingly found that a combination of palmitoylethanolamide and Equisetum is capable of exerting anti-osteoporotic action and, acting through different mechanisms of action, provides powerful synergistic activity.

It is an object of the present invention a food, nutraceutical, and/or pharmaceutical combination comprising or alternatively consisting of palmitoylethanolamide and Equisetum (in short combination A1).

Preferably, in said combination A1 said Equisetum is Equisetum Arvense.

Preferably, in said combination A1, the PEN Equisetum weight ratio is in the range: from 10:100 (1:10) to 100:10 (10:1), preferably 80:40 (2:1); or from 10:50 (1:5) to 50:10 (5:1); or from 10:40 (1:4) to 40:10 (4:1), preferably 40:20; or from 10:30 (1:3) to 30:10 (3:1), or from 10:20 (1:2) to 20:10 (2:1); or

10:10 (1:1).

Preferably, in said combination A1 said PENEquisetum weight ratio is based, for example, considering Equisetum in powder form; more preferably a powder titrated at silica in an amount by weight from 1% to 20%, more preferably from 5% to 15%, even more preferably from 6% to 12%, even more preferably is a powder titrated at silica in an amount by weight of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, and/or 11 .5%.

In an embodiment, said PENEquisetum weight ratio in said combination A1 is from 40/20 (2:1) to 80/40 (2:1), said PENEquisetum weight ratio being based on Equisetum in powder form titrated at 7% in silica.

Preferably, in said combination A1, palmitoylethanolamide (PEA), present in combination or association with said Equisetum, is a palmitoylethanolamide having preferably an average fineness or size or average particle size ranging from 20 mesh to 120 mesh, more preferably ranging from 40 mesh to 100 mesh, even more preferably ranging from 60 mesh to 80 mesh. It is an object of the present invention a food, nutraceutical, and/or pharmaceutical composition comprising the combination A1, as described above, together with one or more excipients and/or physiologically acceptable food-grade or pharmaceutical grade vehicles (in short composition C1).

Preferably, said composition comprises (with respect to the total weight of composition C1) from 50 mg to 1500 mg by weight of PEA, preferably it comprises from 100 mg to 1200 mg of PEA, more preferably it comprises from 200 mg to 1000 mg of PEA, even more preferably it comprises from 300 mg to 800 mg of PEA, e.g., 400 mg, 500 mg, 600 mg, or 700 mg of PEA, with respect to the total weight of the composition, in association or combination with Equisetum in an amount by weight from 25 mg to 600 mg, preferably from 50 mg to 500 mg, more preferably from 100 mg to 400 mg, even more preferably from 150 mg to 300 mg, e.g., 200 mg, or 250 mg.

In an embodiment, said composition C1 comprises from 200 to 600 mg of PEA and from 100 to 300 mg of Equisetum, preferably said amount being based on Equisetum in powder form titrated at 7% in silica.

Preferably, said composition C1 is a composition suitable for oral administration.

Preferably, in said composition C1, palmitoylethanolamide (PEA), present in combination or association with said Equisetum, is a palmitoylethanolamide having preferably an average fineness or size or average particle size ranging from 20 mesh to 120 mesh, more preferably ranging from 40 mesh to 100 mesh, even more preferably ranging from 60 mesh to 80 mesh.

Another object of the present invention relates to a combination A1, as described above, for use in therapy, preferably in a method for the treatment and/or prevention of osteoporosis, of any origin, in a mammal, preferably in human beings.

Still another object of the present invention relates to a composition C1, as described above, for use in therapy, preferably in a method for the treatment and/or prevention of osteoporosis, of any origin, in a mammal, preferably in human beings.

Preferably, said combination A1, as described above, or said composition C1, as described above, are indicated for use in promoting bone building and limiting bone resorption, and/or in the prevention and/or treatment of bone fractures.

It is another object of the present invention a kit comprising at least one food, nutraceutical, and/or pharmaceutical composition comprising or, alternatively, consisting of PEA, together with one or more physiologically acceptable excipients or vehicles, and at least one food, nutraceutical, and/or pharmaceutical composition comprising or, alternatively, consisting of Equisetum, together with one or more physiologically acceptable excipients or vehicles.

Preferably, said kit comprises: a) at least one combination A1 or one food, nutraceutical, and/or pharmaceutical composition C1 comprising PEA, preferably together with one or more physiologically acceptable excipients or vehicles, and b) at least one combination A1 or one food, nutraceutical, and/or pharmaceutical composition C1 comprising Equisetum, preferably together with one or more physiologically acceptable excipients or vehicles. Thus, according to one of its aspects, it is an object of the invention a food, nutraceutical and/or pharmaceutical combination consisting of palmitoylethanolamide and Equisetum. Palmitoylethanolamide or N-(2-hydroxyethyl)hexadecanamide (here also just "PEA"), is the naturally occurring ethanolamide derivative of hexadecanoic acid and is found in particularly abundant amounts in some foods, including egg yolk and soybeans. At room temperature and pressure it occurs as a solid and is used in therapy primarily as an anti-inflammatory, neuroprotective, and pain-relieving agent.

Equisetum is a plant belonging to the family Equisetaceae and is commonly known as "horsetails." According to the present invention, the term "Equisetum" includes Equisetum Arvense. Equisetum is commonly used for its diuretic and mineralizing properties, particularly for bone tissue, due to its minerals and high silicon content. In the combination of the invention, Equisetum can be used in any of its forms, such as in powder form, as a fluid extract, mother tincture, or macerate; preferably the Equisetum used in the combination of the invention has a silica titer greater than 1%, more preferably 7%, for example, a silica titer around 10% and with a maximum titer at 15%.

PEA and Equisetum are also referred to here generically as the "active ingredients" or "components" of the combination of the invention.

According to a preferred embodiment, the combination of the invention consists of PEA and Equisetum in a PENEquisetum weight ratio ranging from 40/20 to 80/40, preferably 66/33, said ratio being based on Equisetum in powder form titrated in silica. PENEquisetum are present in the range of 200 mg/600 mg of PEA and 100 mg/300 mg of Equisetum in a PENEquisetum weight ratio of 2:1, preferably 300 mg/150 mg, twice a day, said ratio being based on Equisetum in powder form titrated at 7% in silica. These dosages are also considered safe by the Ministry of Health, which has authorized the use of PEA in dietary supplements, without establishing for the moment a possible maximum daily intake, and at the same time, there are not even recommended doses of silicon, a chemical element contained in silica, although it is conceivable that 20-30 mg/day may prevent connective tissue weakness.

It is evident that when an Equisetum having a silica titer different than 7% is used in the combination of the invention, the ratio can be calculated from the above ratio according to the silica titer. The same calculation should be made when using an Equisetum in liquid form; in this case, an amount of Equisetum, such as at the rate of drops of mother tincture or macerate, should be calculated to provide an equal amount of silica.

The combination of the invention is well tolerated, nontoxic, and therefore can be used in therapy.

According to another of its aspects, it is an object of the invention a food, nutraceutical, and/or pharmaceutical composition that comprises the combination of the invention together with one or more physiologically acceptable excipients and/or vehicles.

According to another of its aspects, it is an object of the invention the combination and composition of the invention for use in therapy and in particular in the treatment and/or prevention of osteoporosis, of any origin, in a mammal.

Said mammal is preferably the human being, for example, but not limited to, a perimenopausal and menopausal woman.

Said osteoporosis can also be osteoporosis caused by corticosteroid therapies or other origin. In general, the combination and composition of the invention are useful in promoting bone building and limiting bone resorption.

The combination and composition of the invention are also useful in the treatment and prevention of fractures.

If desired or necessary, the composition of the invention may also contain additional active ingredients, preferably of natural origin, useful for the prevention and treatment of the bone diseases described herein. By way of example, the composition may also include minerals, e.g., calcium, and vitamins, e.g., vitamin D and the B group vitamins.

As will be shown in the Experimental Section that follows, it was observed that both PEA and Equisetum, separately, show anti-osteoporotic action but it was surprisingly found that their combination exerts a powerful synergistic action against bone resorption while promoting bone formation.

It is evident, therefore, that the combination and composition of the invention provide an effective natural therapeutic response to treat and prevent bone resorption-related diseases, including osteoporosis, and are a viable alternative to conventional anti-osteoporotic drugs.

According to a preferred embodiment, the compositions of the invention are oral or buccal, solid, semisolid, gel, or liquid compositions and, as said, contain physiologically acceptable excipients and/or conventional vehicles.

Compositions of the invention may be in the form of tablets, hard capsules, soft capsules, granules, fine granules, powders, tablets, syrups, emulsions, suspensions and solutions suitable for oral administration. Other suitable food, nutraceutical and/or pharmaceutical forms may also be used.

Such compositions can be taken alone or with water or, especially when in the form of powders or granules, can be mixed with other foods, such as with yoghurt, creams, gels, and the like.

Alternatively, the compositions of the invention can be in ready-to-use beverage form, such as in the form of drinking sachets, of the "stick pack" type, in this case preferably in cream or gel form.

The types of food, nutraceutical and/or pharmaceutical additives used in preparing the compositions of the invention, the content ratios of the additives to the active ingredients, and the methods for preparing the food, nutraceutical and/or pharmaceutical composition may be appropriately selected by the person skilled in the art.

For conventional vehicles and excipients, organic or inorganic substances, or solid or liquid substances may be used as excipients and vehicles, provided they are edible, physiologically acceptable, and compatible with all other components of the composition.

As said, the person skilled in the art is perfectly capable of selecting the most suitable vehicles and excipients for the preparation of the composition.

As examples, organic or inorganic substances, or solid or liquid substances may be used as excipients and vehicles, provided they are edible and pharmaceutically acceptable. Examples of excipients used in the preparation of solid food, nutraceutical, and/or pharmaceutical compositions include, for example, lactose, sucrose, starch, talc, cellulose, dextrins, kaolin, calcium carbonate, stearic acid or magnesium stearate, lactose, polyethylene glycol, mannitol, sorbitol, chelating agents, anti-caking agents, sweetening agents, preservative agents, and flavoring agents. For the preparation of liquid compositions for oral administration, a conventional inert diluent such as water or an oil, such as a vegetable oil, may be used. The liquid composition may contain, in addition to the inert diluent, auxiliaries such as wetting agents, suspending agents, sweeteners, flavorings, colorants, and preservatives. The liquid composition may be included in capsules of an absorbable material, such as gelatin.

Sweeteners may be one or more natural sugars, optionally reduced, such as sucrose, dextrose, xylitol, mannitol, or sorbitol, or a synthetic product such as sodium saccharin, aspartame, acesulfame k, or sucralose. Acidifying agents may also be added.

Flavoring agents are food-acceptable, nutraceutical and/or pharmaceutical flavors and tastes of synthetic oils or natural oils, the latter extracted from plants, flowers, fruits and combinations thereof, such as cinnamon, mint, anise, and citrus leaves, bitter almonds, citrus fruits, especially orange and/or lemon oil, tilia, vanilla, chocolate and grapefruit oils. Chocolate, vanilla or eucalyptus flavorings and fruit essences, especially apple, pear, peach, strawberry, apricot, orange, lemon and grape, can also be used advantageously.

According to a preferred embodiment, the composition of the invention comprises from 200 to 600 mg of PEA and from 100 to 300 mg of Equisetum, said amount being based on Equisetum in powder form titrated at 7% in silica, preferably 300/150 mg twice a day. As the ratio varies, the dosage of administration also varies: 200/100 mg up to 3 times a day, 600/300 mg once a day.

It is evident that when an Equisetum having a silica titer different than 7% is used in the combination of the invention, the ratio can be calculated from the above ratio according to the silica titer. The same calculation should be made when using an Equisetum in liquid form; in this case, an amount of Equisetum, such as at the rate of drops of mother tincture or macerate, should be dosed to provide an equal amount of silica.

The combination of the invention, preferably in the form of a composition as defined above, may be administered once or more times a day, preferably two or three times a day. The dosage and daily dose may of course vary according to the age, sex and health status of the subject to be treated as well as the severity of the disease and the type of therapy, preventive or curative.

According to another of its aspects, it is an object of the invention a kit that comprises at least one food, nutraceutical and/or pharmaceutical composition including PEA together with one or more physiologically acceptable excipients or vehicles; and at least one food, nutraceutical and/or pharmaceutical composition including Equisetum, as defined above, together with one or more physiologically acceptable excipients or vehicles.

The two compositions can be in any food, nutraceutical, and/or pharmaceutical form, independently of each other, and are compositions suitable for oral administration.

According to one embodiment, the kit of the invention comprises from 5 to 60 compositions of PEA and from 5 to 60 compositions of Equisetum, e.g., from 10 to 30 compositions of each of the two components. The two formulations can be taken simultaneously or sequentially, advantageously they are administered simultaneously or at least within a short period of time so that they can exert a synergistic effect.

According to one embodiment, the kit of the invention also includes an instruction leaflet. According to another of its aspects, it is an object of the invention a method for the prevention and/or treatment of osteoporosis and, more generally, of diseases that benefit from the reconstruction of bone and/or the reduction of its resorption, which comprises administering to a subject in need there of an effective dose of the combination or, preferably, the composition of the invention as defined above.

As will be shown in the Experimental Section, the effects of PEA and Equisetum, alone and in combination, on osteoblast and osteoclast functions were examined. A new role was also revealed for endocannabinoids, which may regulate bone homeostasis by increasing osteoblast maturation and decreasing osteoclast functions by acting on CB1 and CB2 receptors. Therefore, the direct influence of PEA and Equisetum, alone and in combination, on osteoclast function was examined by analyzing resorption activity. The results showed that osteoclasts show increased sensitivity toward the association of PEA and Equisetum, which significantly reduces their functions. In addition, the effects of PEA and Equisetum, alone and in combination, in regulating the RANK/OPG axis, an important pathway involved in osteoclastogenesis, were further evaluated. These results provide for the first time evidence that the combination of PEA and Equisetum plays an important role in maintaining bone homeostasis. The synergistic action of the two active ingredients of the invention has therefore been demonstrated in the Experimental Section below, for illustrative purposes and in no way limiting.

Experimental Section

Cell cultures

An MC3T3-E1 osteoblast cell line purchased from ATCC was cultured in Dulbecco's modified Eagle's medium (DMEM, Merck Life Science, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Merck Life Science, Milan, Italy), L-glutamine 2 mM (Merck Life Science, Milan, Italy) and 1% penicillin-streptomycin (Merck Life Science, Milan, Italy) at 37 °C and 5% CO2 [Park E, Kim J, Kim MC, et al. Anti-Osteoporotic Effects of Kukoamine B Isolated from Lycii Radicis Cortex Extract on Osteoblast and Osteoclast Cells and Ovariectomized Osteoporosis Model Mice. Int J Mol Sci. 2019;20(11):2784. Published 2019 Jun 6. doi:10.3390/ijms20112784], RAW 264.7 monocytes purchased from ATCC were cultured in Dulbecco's modified Eagle's medium (DMEM, Merck Life Science, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Merck Life Science, Milan, Italy), L-glutamine 2 mM (Merck Life Science, Milan, Italy) and 1% penicillin-streptomycin (Merck Life Science, Milan, Italy) at 37 °C and 5% CO2; cells were differentiated into osteoclasts using RANKL + 10 ng/mL MCSF. Culture media were replenished every 2-3 days [Jarrar H, Qetin Altindal D, GUmU§derelioGlu M. The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF. Turk J Biol. 2020 Dec 14;44(6):427-436. doi: 10.3906/biy-2007-85. PMID: 33402869; PMCID: PMC7759193.] Preparation of active ingredients

PEA 100 piM and Equisetum (titrated at 7% in silica) 120piM were dissolved in linseed oil (100%) to obtain a 1000x mother to be used at 1x final concentration in both cell types. To test the efficacy of this new combination, the effects were compared to the individual active ingredients.

Staining for tartrate-resistant acid phosphatase (TRAP) and TRAP activity assay

Mature osteoclasts were washed with DPBS and fixed in 10% formaldehyde for 10 min. After fixation, the cells were stained using a TRAP staining kit (Merck-life Sciences, Italy) according to the manufacturer's instructions. The cells were washed with deionized water, and positive TRAP-multinucleated cells (>3 nuclei) were counted under an inverted microscope (Olympus, Tokyo, Japan). To quantify TRAP activity, 50pl of supernatant was mixed with 150pl of tartrate acid solution and after 30 min, 50pl of NaOH 0.5 M was added, then it was measured at 405 nm using an ELISA reader (Tekan). [Kim JH, Kim EY, Lee B, Min JH, Song DU, Lim JM, Eom JW, Yeom M, Jung HS, Sohn Y. The effects of Lycii Radicis Cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells. Int J Mol Med. 2016 Mar;37(3):649-58. doi: 10.3892/ijmm.2016.2477. Epub 2016 Feb 1. PMID: 26848104; PMCID: PMC4771095.].

Alkaline phosphatase (ALP) activity test.

After treatment, cells were washed with PBS and lysed with lysis buffer containing Tris-HCL 50nM (pH 7.4) and 1% Triton X-100 to collect supernatants and determine ALP activity and protein concentration. ALP activity was determined with 4 mg/mL of 4-nitrophenyl phosphate (4NPP) in 2-amino-2-methyl-1 -propanol 0.2 M with MgCI2 4 mM as substrate for 30 min at 37°C. The reaction was stopped by NaOH 0.1 M, and the yellow solution was measured at 405 nm. Protein concentration was measured using the Pierce BOA Protein Assay Kit (BOA, ThermoFisher Scientific, Waltham, MA, USA). ALP activity was normalized to protein concentration and shown in terms of pmole/min/mg protein normalizes to the untreated control sample [Yodthong T, Kedjarune-Leggat U, Smythe C, Sukprasirt P, Aroonkesorn A, Wititsuwannakul R, Pitakpornpreecha T. Enhancing Activity of Pleurotus sajor-caju (Fr.) Sing p-1,3-Glucanoligosaccharide (Ps-GOS) on Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells through the Involvement of BMP-2/Runx2/MAPK/Wnt/p-Catenin Signaling Pathway. Biomolecules. 2020 Jan 27;10(2):190. doi: 10.3390/biom10020190. PMID: 32012654; PMCID: PMC7072289.].

Alizarin red staining for mineralization

To examine mineralization, MC3T3-E1 cells were placed in differentiation medium at a concentration of 1 x 10 ⁵ cells/well in 6-well plates. After 21 days, the medium was removed and stained with Alzarin red. The cells were washed with PBS three times and then fixed with 75% ethyl alcohol for 30 min at 4°C. The cells were then washed with distilled water three times and stained with 1% Alzarin red solution (pH 4.2) for 30 min at 37°C. Unbound dye was removed by washing several times with distilled water. The mineralized nodules were observed using a low-magnification microscope and were photographed. To quantify matrix mineralization 1mL of cetylpyridinium chloride 100 mM was added to each well and incubated for 1 hour to dissolve and release the calcium-bound dye. The absorbance was measured at 570nm.

Creation of a co-culture system for bone resorption.

A new co-culture system was established using Transwell® inserts (Corning Incorporated Number 3450, NY, USA). Differentiated RAW 264.7 cells were embedded at a density of 5x10 ⁵ cells/cm ² in the lower compartment of each insert. MC3T3-E1 cells were incubated at a density of 5x10 ⁴ cells/ cm ² in the upper compartment of the inserts. After each cell was cultured for 24 hours, Transwell inserts with MC3T3-E1 cells were combined in the bone resorption assay plate with RAW 264.7 cells. This co-culture system was maintained in DMEM supplemented with 10% fetal foal serum (FCS) with penicillin-streptomycin-neomycin (PSN) antibiotic mixture (5 mg each of penicillin and streptomycin and 10 mg/mL of neomycin, GIBCO 15640, Life Technologies, Inc.) in 5% CO2 at 37°C. After culture for 5 days, subsequent experiments were performed [Ishikawa S, Tamaki M, Ogawa Y, Kaneki K, Zhang M, Sunagawa M, Hisamitsu T. Inductive Effect of Palmatine on Apoptosis in RAW 264.7 Cells. Evid Based Complement Alternat Med. 2016; 2016:7262054. doi: 10.1155/2016/7262054. Epub 2016 May 31. PMID: 27340419; PMCID: PMC4906184.].

Quantification of osteoclastic activity: analysis of calcium phosphate and bone resorption

Osteoclast bone resorption activity was evaluated using a bone resorption assay kit (PG Research, Tokyo, Japan) under the same culture conditions described above. The bottom of the inserts was composed of polyester materials with a pore size of 0.4 zm that allows only small soluble factors to pass through [Ishikawa S, Tamaki M, Ogawa Y, Kaneki K, Zhang M, Sunagawa M, Hisamitsu T. Inductive Effect of Palmatine on Apoptosis in RAW 264.7 Cells. Evid Based Complement Alternat Med. 2016; 2016:7262054. doi: 10.1155/2016/7262054. Epub 2016 May 31. PMID: 27340419; PMCID: PMC4906184.].

Co-cultured cells were incubated on bone resorption assay plates and fluorescein-labeled CaP-coated 24-well plates without change of medium under light-shielded conditions. After 5 days, 100 L of the cell culture supernatant were transferred to a 96-well plate for fluorescence measurement, mixed with 50 zL of bone resorption assay buffer added to each well, and then mixed using a plate shaker. Fluorescein-labeled chondroitin sulfate fluorescence intensity was measured using a fluorescence plate reader (Tekan) with excitation and emission wavelengths of 485 and 535 nm.

The remaining plates were washed with PBS and treated with 5% sodium hypochlorite for 5 min. After washing the plates with tap water and drying them, five different regions in each well were photographed under a microscope (Olympus Co.) and the cavity areas were measured with Imaged software.

Protein activity by ELISA

OPG, OPN and RANKL activity was measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Samples were measured at 450nm using a microplate reader (Tekan). Results were calculated by generating a standard curve (0-10pg/mL) and expressed as pg/mL [Chen ST, Kang L, Wang CZ, Huang PJ, Huang HT, Lin SY, Chou SH, Lu CC, Shen PC, Lin YS, Chen CH. (-)- Epigallocatechin-3-Gallate Decreases Osteoclastogenesis via Modulation of RANKL and Osteoprotegrin. Molecules. 2019 Jan 3;24(1):156. doi: 10.3390/molecules24010156. PMID: 30609798; PMCID: PMC6337469.].

Western Blot

After stimulation, cells were washed with PBS and lysed on ice with Complete Tablet Buffer (Roche, CN 11836145001) supplemented with sodium orthovanadate 2 mM (Na3VO4, Sigma-Aldrich, Milan, Italy, CN S6508), sodium fluoride 0.1 M (Sigma-Aldrich, Milan, Italy, CN 450022), 1 :1000 phenylmethan-sulfonyl fluoride (PMSF, Sigma-Aldrich, Milan, Italy, CN P7626), and 1 :100 mixture of protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy CN S8820). After centrifugation (12,000 x g at 4°C) for 15 min, the protein-containing supernatants (the protein samples) were collected. The protein concentrations of the samples were measured with a BCA protein assay kit (Invitrogen, Milan, Italy) according to the manufacturer's instructions. In total, 35 pg of protein from each lysate was added to Laemmly Buffer (1g SDS, TRIS-HCI 1.5M pH 6.8, Beta-mercaptoethanol, glycerol, bromine pieno blue) 5X, boiled to denature the proteins, resuspended in 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy) that were incubated overnight at 4°C with a specific primary antibody: anti-CB1 (1 :250, Santa Cruz, CA, USA), anti-CB2 (1 :250, Santa Cruz, CA, USA).

Protein expressions were normalized and verified by detection of anti-p-actin (1 :5000, Sigma-Aldrich, CN A5441). Results are expressed as mean ± SD (% compared with control).

Statistical analysis

At least five independent experiments were performed for each experimental protocol; results were expressed as mean ± SD of independent experiments performed on at least three technical replicates using one-way ANOVA followed by Bonferroni correction for statistical analysis. Values of p < 0.05 were considered statistically significant.

Results

Analysis of the effect of the combination of PEA and Equisetum, on the balance between osteoblast and osteoclast (Figure 1)

Alkaline phosphatase (ALP) is an essential enzyme during the early stage of osteoblastic differentiation; in fact, it is considered a marker of early osteogenic differentiation, whereas mineralization is the process observed during a later stage of osteoblast differentiation. To confirm that MC3T3-E1 cells differentiated into mature osteoblasts, ALP activity analysis and staining with Alzarin red was performed. Our initial data revealed that all substances (panel A and B), alone and in combination, could increase osteoblast activity compared with control (p<0.05). In particular, the combination of PEA and Equisetum is able to amplify the beneficial effects on matrix synthesis and mineralization compared with control and single agents (p<0.05). These results demonstrate the ability of the combination to improve osteoblast function probably due to their increased bioavailability. In addition, further experiments were conducted to explore the effects of PEA and Equisetum, alone and in combination on osteoclast activity. As shown in Panel C, the results obtained confirmed that the combination of PEA and Equisetum can reduce osteoclast activity compared with control and single agents (p<0.05); in particular, the combination of PEA and Equisetum is able to amplify the reduction of TRAP activity. These data confirmed the hypothesis formulated in which the association of PEA and Equisetum acts synergistically to maintain osteoblast/osteoclast balance.

Analysis of PEA and Equisetum on endocannabinoid receptors (Figure 2)

To investigate the possible involvement of the endocannabinoid system and related molecules during the differentiation process of bone-forming cells, it is considered very important to test the activities of endocannabinoid receptors (CB1 and CB2). In particular, CB1 and CB2 have well-known effects on osteoclast and osteoblast cells, suggesting their role on the balance of the bone microenvironment. In addition, single substances can activate both receptors as shown in Figure 2. In addition, PEA and Equisetum, alone and in combination, can regulate CB1 and CB2 signaling by enhancing osteoblast maturation and reducing osteoclast activation.

These results indicate that CB1 and CB2 play an important role in regulating bone mass formation and reducing bone loss.

Creation of a 3D culture system for bone resorption (Figure 3) RAW 264.7 cells are known to differentiate into osteoclastic cells during days of culture. Therefore, the differentiation of RAW 264.7 cells into osteoclasts was examined by TRAP quantification in 3D culture in which RAW 264.7 cells and MC3T3-E1 were bound. As shown in panel A, single agents and Equ/sefum-associated PEA were able to decrease TRAP activity compared with control (p<0.05) and single agents with a greater effect on this parameter than previously observed (Figure 1, panel C), when osteoclasts were not linked with osteoblasts. These data confirm the efficacy of the combination of PEA and Equisetum on osteoclast function.

In addition, further experiments were conducted to explore the effects of previously tested substances, alone or in combination, to verify their role on osteoclast resorption activity. The bone resorption capacity of RAW 264.7 cells was examined using a bone resorption assay kit in 3D culture of RAW 264.7 and MC3T3-E1 cells. Bone resorption activity was assessed by measuring the fluorescence intensity of the conditioned medium. As shown in panel B, fluorescence intensity decreased significantly with all substances, alone and in combination, enhancing osteoblast activity and decreasing osteoclast function compared with control (p<0.05). Specifically, PEA and Equisetum were able to decrease osteoclast activity compared with control and single agents (p<0.05) confirming the ability of PEA and Equisetum to inhibit osteoclast activity by suppressing osteoclastogenesis and bone resorption. These were confirmed by the erosion area analysis (panel C), where it can be observed that both PEA and Equisetum alone reduced the erosive capacity related to osteoclast activity (p<0.05) compared to the control, and this effect is amplified by the simultaneous stimulation with PEh+Equisetum both compared to the control (p<0.05) and to the single agents (p<0.05), thus supporting the presence of a postive synergistic effect in suppressing osteoclastic activity.

Creation of a 3D culture system for OPG/RANK (Figure 4)

The most important pathway in osteoclastogenesis is the RANK/OPG axis, which plays a central role in the regulation of osteoclast proliferation and differentiation. OPG and RANK activities were increased with all tested agents compared with control (p<0.05); in particular, the combination of PEA and Equisetum may enhance the activities of these two proteins by implementing osteoblast-related OPG activity while reducing RANKL-related osteoclast activity. The RANK/OPG ratio decreased with PEA and Equisetum alone compared with control (p<0.05), and this effect was amplified when these two agents were added together, confirming a synergistic effect produced by PEA and Equisetum. Therefore, PEA and Equisetum confirm their role in counteracting osteoblast senescence through the RANK/OPG axis to maintain osteoblast/osteoclast balance. These data also confirm that PEA and Equisetum can modulate the bone microenvironment by reducing osteoclast activity and maintaining bone architecture.

Examples of compositions

Example 1

A composition is prepared in capsule form comprising:

Table 1

Example 2

A composition is prepared in tablet form comprising: Table 2

Example 3

A composition is prepared in capsule form comprising:

Table 3

Example 4

A composition is prepared in capsule form comprising:

Table 4

12

RECTIFIED SHEET (RULE 91 ) ISA/EP