HK40002512B - Method for producing celastrol and pentacyclic triterpene derivatives - Google Patents

Description

The present invention relates to a process for the production of a crude extract enriched with pentacyclic triterpenes, including Celastrol, from a suspended plant cell culture (PCC) of a plant of the Celastraceae family.

Tripterygium wilfordii is a medicinal plant, belonging to the Celastraceae family, used for centuries in Southeast Asia (including China) to treat inflammatory disorders, autoimmune diseases, and more recently cancer. Terpenes are among the most active components of the plant and are located mostly in the roots of the plant. Among them, a norfriedelane-type pentacyclic triterpene, Celastrol, is evaluated for its effectiveness in the treatment of obesity, rheumatoid arthritis, Crohn's disease, and prostate cancer.

The causes of these so-called autoimmune diseases are not yet fully understood but are thought to be related to a dysregulation of the immune system, particularly in cellular responses. Cellular responses can be classified into 3 categories: Th1, Th2, and recently Th17. Although psoriasis and atopic dermatitis have differences in clinical signs, they are said to have similarities, particularly in the expression of IL17 and IL22 characteristics of the ThNF17 response (Miyagaki et al. 2015). IL17 and IL22 are therefore thought to be interesting targets for treating these diseases.

In addition, the Th17 pathway is also known to be highly activated in lesions in patients with acne (Kelhala et al. 2014).

Celastrol is commonly obtained by plant extraction processes from plants of the Celastraceae family involving the use of solvents. These biosynthesis methods involve cycles of eight to ten years for a young plant sprout to reach the maturity necessary for slaughter. In addition, the use of plant protection products makes the process expensive: external contaminants (heavy metals) can accumulate and the extract generated contains multiple metabolites requiring several steps of purification of this plant extract in order to isolate the triterpene fraction.

Total chemical synthesis of Celastrol is possible (Camelio et al. 2015) but requires about 20 steps, which significantly increases the final cost of obtaining the product.

An alternative solution was to obtain Celastrol from cell cultures in suspension of stem cells generated from the roots or leaves of the plant. Recently, the Coppede team (Coppede et al. 2014) was able to obtain from a cell culture of M. ilicifolia a higher amount of Celastrol than that obtained by conventional extraction from the plant (0.304 mg of Celastrol per g of dry matter, maximum concentration obtained after 8 days of culture, 8.85 times better than the conventional extraction method). The team of Liu et al. (Liu et al. 2016) also showed that in a cell culture of M. ilicifolia (Jacasmon) without adding any other material: 50 μm of Celastrol in a culture of T. Jax (Jaca) once the cell culture of T. Jax is dry, the concentration of Celastrol is increased by 0.4 mg/g of Mefor (Jaca) compared to the concentration of Mefor (Jaca) without adding any other material.

These processes allow for improved cholesterol yields, but there is still a real need for economically viable processes that can produce cholesterol quickly in large quantities.

The present invention provides a method for the production of a raw extract enriched in Celastrol from plant cells of a plant of the Celastraceae family, Tripterygium wilfordii.

The inventors have developed a process to increase the yield of Celastrol in a surprising way in the culture of plant cells from a line of T. wilfordii by using and comparing various combinations of elicitors. The inventors have observed that cell division and production of Celastrol are not concomitant. Surprisingly, they are even incompatible. To solve this problem, the inventors have developed a process including a cell proliferation stage and an elicitation stage with a cocktail of eliciting agents that stops cell division.The yield in Celastrol of this cultured culture is then 22 times higher than that obtained by Liu et al. (op. cit.), the yield by the process of the invention is also 57 times higher than that obtained by Coppede et al. (op. cit.) without elicitation. The process of the invention also enables enrichment in Celastrol derivatives, in particular pentacyclic tripterpene type, such as Tingclinin A (also called Tingone or Maytenin),Tingenin B (also known as 22beta-hydroxytingenone), Pristimerin and Tripterygone.

In addition, the inventors showed surprisingly that a raw extract enriched with pentacyclic triterpenes obtained by this process inhibits with dose-specific effect the Th17-specific interleukins induced in human TCD4+ cells.

The present invention therefore concerns a process for the production of a crude extract enriched with pentacyclic triterpenes comprising the following steps: (i) a phase of cell proliferation of a plant of the Celastraceae family which is Tripterygium wilfordii in a proliferation medium, (ii) an induction phase by addition of an induction cocktail to the cell culture obtained in step (i), the said induction cocktail comprising at least one monocarboxylic compound type inducer which is methyl jasmonate and at least one biotic inducer which is chitine and (iii) the preparation of an extract enriched in crude pentacyte tripterpenes obtained from the cell culture obtained in step (ii).

In addition, the present invention concerns an extract which can be obtained by the process of the invention and its uses.

Pentacyclic triterpenes According to the invention, the pentacyclic triterpenes are those naturally produced by the cells of a plant in the family Celastraceae and include Celestrol (formula I), Tingenin A (also called Tingenone or Maytenine) (formula II), Tingenin B (also called 22beta-Hydroxy-tingenone) (formula III), Pristimerin (formula IV) and Tripterygone (formula V), including Celestrol, Tingenin A and Tingenin B, including Celestrol. - What?

Raw extract enriched with pentacyclic triterpenes according to the invention means an enriched raw extract containing at least 2 times, including at least 5 times, including at least 10 times, including between 10 and 100 times, the quantity of pentacyclic triterpenes obtained by a process without an excitation phase according to the invention.

Raw extract enriched with Celastrol according to the invention means an enriched raw extract containing a quantity of Celastrol at least 2 times, including at least 5 times, including at least 10 times, including between 10 and 60 times, including between 10 and 20 times that obtained by a process without an induction phase according to the invention.

plant of the Celastraceae family means all plants belonging to this family, and in particular the plants of the genus Tripterygium, Bhesa, Kokkona, Catha, Euonymus, Orthosphenia, Dicarpellum, Celastrus, Maytenus, Peritassa and Rzedowskia, in particular Tripterygium and Celastrus.

Cells of a plant of the Celastraceae family according to the invention, means cells of any part of the plant: seeds, roots, aerial parts, and especially aerial parts.

Aerial parts According to the invention, the parts of the plant above the ground, e.g. leaves, stems, petioles and/or inflorescences, including leaves.

cell proliferation phase of a Celastraceae plant means, according to the invention, a phase in which cells of a Celastraceae plant are suspended in a proliferation medium and under conditions suitable for their proliferation. These cells may be obtained from calcium prior to suspension. If necessary, the cell suspensions may be regularly re-seeded in order to maintain them in proliferation conditions.

The term cal in the present invention refers to a collection of undifferentiated cells, also called stem cells or meristematic cells.

The induction of calces may be obtained by any method known to the professional, the calces according to the invention may be obtained in particular by the following method.

The induction of callus from a tissue explant of a plant part, in particular an aerial part, of the Celastraceae family, which is Tripterygium wilfordii, is well known to the professional.

The induction of calcium may be achieved in particular by: the production of a plant tissue explant, e.g. a piece of leaf about 1 cm2 in size,the culture of the plant on a gelled growth medium according to the invention (e.g. by adding 4 to 12 g/L of agar, e.g. about 8 g/L of agar, to the growth medium according to the invention),incubation, in particular in the dark, at a temperature of about 25-30°C, e.g. about 27°C to 28°C.

Stage i: phase of cell proliferation

The expert who knows the cell cultures of a plant of the Celatraceae family will be able to easily determine the composition of the proliferation medium necessary for their proliferation. Preferably, this proliferation medium will allow the proliferation of cells in undifferentiated forms, i.e. totipotent forms.

The propagation medium of the invention may include: at least one macromolement, in particular selected from NH4NO3, KNO3, CaCl2.2H2O, MgSO4.7H2O, KH2PO4 and a mixture thereof, e.g. at a total macromolement concentration between 1000 and 9000 mg/L, e.g. between 3000 and 8000 mg/L of proliferation medium during the proliferation phase, the total macromolement concentration of the proliferation medium shall be in particular between 3000 and 5500 mg/L of proliferation medium; at least one macromolement, in particular selected from KI, H3BO3, MnSO4.4H2O, ZnSO4.H2O, Na2SO4.MoO4.2H2O, Cu4.5H2O, Cl2.6H2O, COTA7SO4.H2O, Na2H2O2 and a mixture of those:for example, a total concentration of micro-elements between 10 and 200 mg/L, including 50 and 150 mg/L of proliferation medium;at least one vitamin, including myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCI and a mixture thereof, for example, a total vitamin concentration of between 0,01 and 3 g/L, including 0,05 and 1 g/L of proliferation medium;at least one amino acid, including glycine, including a total amino acid concentration of between 0,15 and 5 mg/L, including 1 and 4 mg/L of proliferation medium during the proliferation phase:the amino acid concentration of the proliferation medium shall be in particular between 1 and 2,5 mg/L of proliferation medium;at least one carbon source, in particular sucrose, for example at a total carbon source concentration of 10 to 70 g/L of proliferation medium, for example at about 30 g/L;at least one plant hormone (also called plant growth hormone or plant growth factor or plant growth regulator) in particular chosen from one or more cytokinines, in particular kinetin and/or 6-furfuryopurine, one or more alkynes, in particular 2,4-dichlorophenoxyacetic acid (2,4D) and/or naphthalene acetic acid (NAA),During the proliferation phase the proliferation medium shall include at least one cytokinin and at least one auxin.

Plant growth hormones shall be added to the proliferation medium in a concentration and ratio which allows cells to proliferate in differentiated forms, in particular from kinetin, 6-furfurylaminopurine, 2,4-dichlorophenoxyacetic acid (2,4D), naphthalene acetic acid (NAA) and a mixture thereof; in particular from kinetin, 2,4-dichlorophenoxyacetic acid (2,4D), naphthalene acetic acid (NAA) and a mixture thereof.

The concentration of auxins according to the invention will be in the range of 0.001 to 10 mg/L of proliferation medium, e.g. between 0.1 and 3 mg/L of proliferation medium.

The concentration of cytokinines according to the invention will be in the range of 0.01 to 0.5 mg/L of proliferation medium, in particular between 0.05 and 0.15 mg/L.

In one embodiment, the auxin/ cytokinin ratio will be between 0.2 to 2.5/ 0.01 to 0.5, in particular between 1 to 2/ 0.05 to 0.2, in particular will be approximately 1.5/ 0.1.

In particular, the proliferation medium of the invention may comprise 1.5 mg/L of auxin, particularly 2,4-dichlorophenoxyacetic acid (2,4D) and naphthalene acetic acid (NAA), and 0.1 mg/L of cytokinin, particularly kinetin.

The growth medium shall be sterile and preferably at a pH close to neutral.

An example of a proliferation medium suitable for the proliferation of cells of a plant of the Celastraceae family according to the invention is described in particular by Murashige & Skoog (1962) or in the examples in this application.

For example, this growth medium may have the following composition (concentrations are expressed in terms of the volume of the non-cell growth medium): Macronutrients: NH4NO3 at 1650 mg/L, KNO3 at 1900 mg/L, CaCl2.2H2O at 440 mg/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 170 mg/L; Microelements: KI at 0.83 mg/L, H3BO3 at 6.2 mg/L, MnSO4.4H2O at 22.3 mg/L, ZnSO4.H2O at 6.6 mg/L, Na2MoO4.2H2O at 0.25 mg/L, Cubix 0.04 H2O at 0.25 mg/L, Co2Cl4.7H2O at 0.25 mg/L, CaCO2H4O at 27.7 mg/L; Na2L: Na2L: 27 mg/L; Nicotinamide or Nicotinamide; Acetylsacyl; Acetylsacyl; Nicotinamide or Nicotinamide; Acetylsacyl; Acetylsacyl; Acetyl; Thyclovir at 0.50 mg/L; Acetylsacyl; Thycyl; Thyclovir at 0.50 mg/L; Thycyl; Thyclovir at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl at 0.50 mg/L; Thycyl

Alternatively, the proliferation medium may have the following composition: (concentrations are expressed in terms of the volume of the non-cellular proliferation medium): Macronutrients: NH4NO3 at 1650 mg/L, KNO3 at 2500 mg/L, CaCl2.2H2O at 440 mg/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 130 mg/L; Microelements: at 0.41 mg/L, H3BO3 at 6.2 mg/L, MnSO4.4H2O at 22.3 mg/L, ZnSO4.H2O at 7.5 mg/L, Na2MoO4.2H2O at 0.25 mg/L, Cuyax 0,04.5H2O at 0.025 mg/L, CoCl2.6H2O at 370 mg/L, FeO4O4O4 at 19.7 mg/L; NaOH2 at 0.85 mg/L; Nicotinamide at 0.25 mg/L; Acetyl-acetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamines, Cycloacetamin

Alternatively, the proliferation medium according to the invention may have the following composition (concentrations are expressed in terms of the volume of cell-free proliferation medium): NH4NO3 at 20 mM, KNO3 at 19 mM, CaCl2.2H2O at 3 mM, MgSO4.7H2O at 1,50 mM, KH2PO4 at 1,2 mM, KI at 0,005 mM, H3BO3 at 0,1 mM, MnSO4.4H2O at 0,1 mM, ZnSO4.H2O at 0,04 mM, Na2MoO4.2H2O at 0,001 mM CuSO4.5H2O at 0,0001 mM, CoCl2.6H2O at 0,0001 mM, FeTA4.7H2O at 0,1 mM, Na2H2O2 at 0,1 mM, Na2MEDO2 at 0,005 mM, nicotinic acid, naphthalene, naphthalene, naphthalene, acetic acid, glycine, thiocyanide, glycine at 0,005 mM, glycine at 0,006 m, naphtinoacid at 0,00M, naphtinoacid at 0,00M, naphtinoacid at 0,00M, naphtinoacid at 0,00M, naphtinoacid at 0,00M, napht, naphtinoacid at 0,00 to 0,00M, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, napht, naph, napht, napht, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, naph, na

The seeding of the proliferation medium may be carried out by suspending calcium cells at a concentration of 20 to 300 g in 1 L of proliferation medium and preferably between 100 and 200 g in 1 L of proliferation medium, e.g. about 150 g in 1 L of proliferation medium.

The proliferation phase will take place under conditions of multiplication of biomass. By conditions of multiplication of biomass according to the invention, we mean in particular the conditions of temperature, duration, agitation, and luminosity necessary for the proliferation of suspended cells. The professional who knows the cell cultures of a plant of the Celatraceae family will be able to easily determine the conditions of multiplication of biomass. In a mode of realization according to the invention, the stage of proliferation of biomass will be in the dark, at a temperature between 20 and 35°C, between 27 and 28°C, including about 27 or 28°C, especially under agitation between 200 and 100 rpm, including about 125 rpm (orbital of 22,5 mm) for about 10 to 30 days, including a culture of 15 to 15 days.

During this stage, the cells can be transplanted or propagated, for example every 7 to 15 days. Cell transplantation is well known to the professional, it may include dilution of part of the cell culture in a concentrated new medium. For example, 1/5th of the culture is resuspended in a volume of new medium corresponding to the volume of the initial culture. It allows the maintenance of the cell line in liquid medium in a state of proliferation.

Stage (ii) - selection phase

After the proliferation phase, the cells obtained in phase (i) are elicited by the addition of an elicitation cocktail. The proliferation medium to which the elicitation cocktail has been added will be named elicitation medium according to the present invention.

The elicitation phase is the phase in which cells are maintained in a physiological state that promotes the biosynthesis of secondary metabolites such as pentacyclic triterpenes.

The production of pentacyclic triterpenes, particularly celastrol, occurs during the excitation phase in the cytosol of the cell and may diffuse partially into the excitation medium.

Alternatively, the addition of the selection cocktail may be carried out when the cell concentration obtained during the selection phase is twice, or more than twice, the initial cell concentration in the selection medium. The addition of the selection cocktail may be carried out in particular when the cell concentration is above 200 g/L, e.g. between 200 and 400 g/L, or about 300 g/L (in quantity of cells per litre of selection medium). In a selection mode, the addition of the selection cocktail may be carried out in a culture in which the concentration of 150 g/L is reached at the beginning of the selection phase and the concentration at the end of the selection phase is between 200 g/L and 400 g/L.

Following the proliferation phase, the plant cells have consumed most or all of the elements contained in the proliferation medium, and in particular carbon sources such as sucrose, so it may be necessary to restore the composition of the medium before or at the same time as the addition of the elicitation cocktail.

In order to restore the composition of the medium, the cell culture obtained after the proliferation stage can be concentrated, for example by settling or filtration, and then new proliferation medium can be added to the cells obtained.

Alternatively, a concentrated mixture may be added to the cell culture to restore the concentrations of the elements of the proliferation medium, e.g. the concentrated mixture may be added just before, just after or at the same time as the elicitation cocktail, e.g. 1/5th of the cell culture may be replaced by an equivalent volume of the concentrated proliferation medium 5 times.

The concentration of elements in the proliferation medium is considered to be close to or equivalent to zero after 14, 15 or 16 days of proliferation phase, in particular the concentration of mineral elements (especially macro- and micro-elements) and carbonates (especially carbon sources) is considered to be close to or equivalent to zero after 14, 15 or 16 days of proliferation phase.

In one embodiment, the propagation medium of the invention at the beginning of the selection phase will include, inter alia: at least one macromolement, in particular selected from NH4NO3, KNO3, CaCl2.2H2O, MgSO4.7H2O, KH2PO4 and a mixture thereof, for example at a total macromolement concentration of 5000 to 8000 mg/L, preferably above 6000 mg/L of growth medium, preferably between 6000 and 8000 mg/L;at least one micromolement, in particular selected from KI, H3BO3, MnSO4.4H2O, ZnSO4.H2O, Na2MoO4.2H2O, CuSO4.5H2O, CoC12.6H2O, Fe4.SO7H2O, Na2EDTA.2H2O and a mixture thereof, for example at a total micromolement concentration of 10 to 200 mg/L,in particular between 50 and 150 mg/L of proliferation medium;at least one vitamin, in particular from myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and a mixture thereof, for example at a total vitamin concentration of up to 0,01 to 3 g/L, in particular between 0,05 and 1 g/L of proliferation medium;at least one amino acid, in particular glycine, for example at a concentration in the proliferation medium of between 3 and 4 mg/L of proliferation medium;at least one carbon source, in particular sucrose, for example at a total carbon source concentration of 10 to 70 g/L of proliferation medium,

In one embodiment the proliferation medium at the beginning of the elicitation phase will not include, but will include a negligible amount, notably less than 0,001 g/ml, of cytokinin and auxin.

The proliferation medium during the elicitation phase will be advantageously sterile and preferably at a pH close to neutral.

The proliferation medium during the initiation phase may be composed in particular of: Macromolecules: NH4NO3 at 2.8 g/L, KNO3 at 3 g/L, CaCl2.2H2O at 0.45 g/L, MgSO4.7H2O at 74 mg/L, KH2P04 at 34 mg/L; Microelements: KI at 0.16 mg/L, H3BO3 at 6.2 mg/L, MnSO4.4H2O at 18.5 mg/L, ZnSO4.H2O at 6.6 mg/L, Na2MoO4.2H2O at 0.25 mg/L, CuSO4.5H2O at 0.025 mg/L, Co2.6H2O 0.025 mg/L, COTA4.7H2O at 28 mg/L, NaLEDO2H2O: 37 mg/L; Nicotinamide at 1 mg/L; Acetamines at 1 mg/L; Acetaminophenol at 1 mg/L; Nicotinamide at 1 mg/L; Acetaminophenol at 1 mg/L; Thycoacetaminol at 1 mg/L; Acetaminophenol at 1 mg/L; Nicotinamide at 1 mg/L; Acetaminophenol at 1 mg/L; Thycloid at 1 mg/L; Thycloid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; Thyric acid at 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L; 1 mg/L;

Elicitation cocktail according to the invention, means a cocktail to stop cell division. This elicitation cocktail includes at least one monocarboxylic compound type elicitor and at least one biotic elicitor. The elicitation cocktail is introduced, for example, using concentrated mother solutions in the culture medium.

The monocarboxylic compound type elicitor according to the invention, is more particularly to be understood as methyl jasmonate (MeJa), or a mixture thereof with salicylic acid and/or 5-chlorosalicylic acid.

Biotic elicitor according to the invention, more specifically chitin is meant. Chitin is a linear polymer of repeating pattern: beta-1,4 N-acetyl D-glucosamine. The biotic elicitor will be added in particular to obtain a final concentration of between 0.05 and 50 g/L of eliciting medium, e.g. 0.1 to 10 g/L, e.g. 0.5 to 7 g/L, in particular 1 to 5 g/L of eliciting medium.

In one embodiment, the elicitation cocktail includes methyl jasmonate, in particular at a final concentration in the elicitation medium between 0,002 and 0,005 g/L, and chitin, in particular at a final concentration between 1 and 4 g/L of elicitation medium.

In one embodiment, the induction cocktail of the invention will additionally include at least one cell differentiation factor of plant cells and/or at least one precursor of the terpene synthesis pathway.

The cell differentiation factor of plant cells according to the invention may be selected from the group preferably consisting of a cytokinin, in particular benzyl aminopurine (BAP), abscisic acid, kinetin, thidiazuron, 6-γ,γ-dimethyllallylaminopurine (2iP or isopentenyladenin) or zeatin, a gibberelline and a mixture thereof, in particular BAP and/or 2iP, in particular 2iP.

The precursor to the synthesis of terpenes according to the invention may be chosen from the group preferably comprising sodium pyruvate; potassium pyrophosphate; mevalonic acid; geraniol; farnesol; isopentenyl, dimethyllallyl, including their pyrophosphate forms; sodium acetate; pyruvic acid and their mixtures, including geraniol, farnesol, sodium pyruvate, potassium pyrophosphate and their mixtures, such as sodium pyruvate and/or potassium pyrophosphate.

In particular, BAP may be used at a final concentration in the eliciting medium between 0,01 and 5 mg/L, e.g. between 0,5 and 5 mg/L of eliciting medium.

In particular, 5-chlorosalicylic acid (5-Chloro SA) may be used at a final concentration in the elicitation medium between 0,1 and 15 mg/L.

Salicylic acid may be used in particular at a final concentration in the elicitation medium between 0,1 and 100 mg/L, e.g. between 20 and 60 mg/L, e.g. around 45 mg/L.

In particular, farnesol may be used at a final concentration in the elicitation medium of 1 to 100 mg/L, e.g. 15 to 30 mg/L, e.g. about 30 mg/L.

In particular, geraniol may be used at a final concentration in the elicitation medium of 1 to 100 mg/L, e.g. 20 to 30 mg/L.

In particular, sodium pyruvate may be used at a final concentration in the elicitation medium of 100 to 5000 mg/L, e.g. 500 to 2000 mg/L.

Potassium pyrophosphate may be used in particular at a final concentration in the elicitation medium of 1 to 2000 mg/L, e.g. 100 to 1000 mg/L of elicitation medium.

In particular, 2iP may be used at a final concentration in the elicitation medium of 0,005 to 10 mg/L, e.g. 0,01 to 3 mg/L, e.g. 0,1 to 2 mg/L.

In one embodiment according to the invention, the elicitation cocktail includes methyl jasmonate, chitin, sodium pyruvate, potassium pyrophosphate or a mixture thereof.

In one embodiment of the invention, the induction cocktail includes methyl jasmonate, chitin, sodium pyruvate, potassium pyrophosphate, and possibly PAH and/or 2iP.

In one embodiment of the invention, the elicitation cocktail includes or consists of (concentrations given in brackets correspond to the concentration in the elicitation medium, the initial cocktail being more or less concentrated depending on the intended dilution) sodium pyruvate (500 to 2000 mg/L), potassium pyrophosphate (100 to 1000 mg/L), 2iP (0.1 to 0.2 mg/L), methyl jasmonate (0.002 to 0.005 g/L) and chitine (1 to 4 g/L).

In another embodiment, the emulsion cocktail includes or consists of (concentrations given in brackets correspond to the concentration in the emulsion medium, the initial cocktail may be more or less concentrated depending on the intended dilution) benzyl aminopurine (BAP) (from 0,5 to 5 mg/L, including 0,5 to 3 mg/L); 5-chloro salicylic acid (5-Chloro SA) (from 2 to 6 mg/L, including 3-5 mg/L, including about 3 or about 5 mg/L); acetyl salicylic acid (ASA) and/or salicylic acid (SAJA) (from 20 to 60 mg/L, including 30 to 30 mg/L, including 30 to 30 mg/L); jasmine (FUF) and methanol (FUF) (from 20 to 40 mg/L) (from 1 to 30 mg/L) and jasmine (from 1 to 40 g/L) (from 20 to 30 mg/L) (from 30 to 30 to 40 g/L); jasmine (from 20 to 40 g/L) (from 30 to 40 to 40 g/L); jasmine (from 20 to 40 to 40 g/L) (from 20 to 40 to 40 g/L) (from 20 to 40 to 40 g/L).

In another embodiment, the emulsion cocktail includes or consists of (concentrations given in brackets correspond to the concentration in the emulsion medium, the initial cocktail being more or less concentrated depending on the intended dilution) sodium pyruvate (500 to 2000 mg/L), potassium pyrophosphate (100 to 1000 mg/L), 2iP (0.1 to 1 mg/L), methyl jasmonate (MeJA) (0.002 to 0.005 g/L, including 10 to 40 mg/L) and chitin (1 to 4 g/L).

In another embodiment the elicitation cocktail includes or consists of (concentrations given in brackets correspond to the concentration in the elicitation medium, the cocktail may be more or less concentrated depending on the intended dilution) sodium pyruvate (approximately 1,5 g/L), potassium pyrophosphate (approximately 0,4 g/L), 2iP (approximately 0,4 mg/L), methyl jasmonate (MeJA) (approximately 0,03 mg/L), chitin (approximately 2 g/L).

During the elicitation phase, after addition of the elicitation cocktail, the culture is kept agitated, in particular at 50 to 200 rpm, in particular at about 125 rpm, for a period of 3 to 30 days, in particular 10 to 25 days, in particular 12 to 15 days, in particular at a temperature of 20 to 35 °C, in particular about 27 °C and preferably with a dissolved oxygen content in the culture medium of 2 to 40%, preferably about 16%. A supply of sterile air with sufficient oxygen enrichment may be provided if necessary, in particular in the dead bioreactor volume or during diffusion into the medium.

Step (iii) - preparation of the crude extract enriched with pentacyclic tripterpenes

After the elicitation phase, the process of the invention includes a step to prepare a crude extract enriched with pentacyclic tripterpenes, including Celastrol.

The enriched raw extract may be obtained by separation of biomass and culture medium, by direct filtration (0-50 μm), by centrifugation or by cell settling.

In one embodiment, the raw extract enriched according to the invention may consist of the surfactant of the culture thus recovered.

The raw extract enriched according to the invention can also be obtained after lysing the biomass. For example, the cells contained in the recovered biomass can be lysed by a physical method (sonication or grinding) or chemical (acid lysing) and then this lysate will be subjected to extraction by solvent. The organic phase, including triterpenes from the cytosol, is then recovered, notably by decantation or centrifugation. The solvent will be an ester type solvent, and more specifically an alkyl acetate, the alkyl being more particularly linear or branched to 1 to 6 carbon atoms, notably biethyl acetate or acetylated acetate.

Preferably, the raw extract enriched according to the invention will correspond to the organic phase thus recovered.

Alternatively, the raw extract enriched according to the invention may be obtained after evaporation of the solvent and its substitution in particular by a medium suitable for the field of use of the extract (cosmetics, pharmaceuticals) and in particular vegetable oils.

The raw enriched extract of the invention is not purified.

When the enriched crude extract is purified to give a purified extract, the process of the invention further includes a step (iv) to obtain a purified extract of one or more pentacyclic triterpenes from the enriched crude extract obtained in step (iii).

The purified extract of the invention will then contain more than 90%, including more than 95%, including more than 98% of one or more pentacyclic triterpenes of the invention.

The purification of the enriched extract according to the invention may be achieved in particular by a phase separation of the pentacyclic triterpene (s) including Celastrol, Tingenin A and Tingenin B, in particular by HPLC (High Performance Liquid Chromatography) fractionation, in particular the 426 nm peaks including Celastrol, Tingenin A and Tingenin B output at 10,5 min, 8,2 min and 6,5 min respectively. In particular, the purification of Celastrol may be performed as described in the examples of this application.

Another subject of the description but not of the invention relates to a pharmaceutical composition, such as a dermatological or dermocosmetic composition, comprising a raw enriched extract which can be obtained by the process of the invention and one or more pharmaceutically or dermocosmetically acceptable excipients.

The pharmaceutically or dermocosmetically acceptable excipients may be any excipient known to the artisan. The composition shall include a topical composition, in particular in the form of a cream, lotion, gel, ointment, emulsion, microemulsion, spray, etc.

The pharmaceutical composition, such as dermatological or dermocosmetic, may in particular contain additives and aids to formulation, such as emulsifiers, thickeners, gelling agents, water fixers, spreading agents, stabilizers, colours, fragrances and preservatives.

Another item described herein is a raw enriched extract obtained by the process of the invention for use as a medicinal product, in particular in the treatment or prevention of inflammatory dermatosis inducing a TH17-mediated cell response.

The professional will be able to easily identify inflammatory dermatoses inducing a TH17-mediated cell response, in particular by measuring the level of IL-22, IL-17 and/or TNF-α expression in an injured area compared to a control sample.

In particular, inflammatory dermatosis may be selected from the group preferably comprising psoriasis, atopic dermatitis and acne.

The present invention is illustrated by the figures and non-limiting examples detailed below.

The figures:

FIGURE 1 : Growth curve (continuous trait) of a Trypterigium wilfordii cell culture in g/L of wet biomass (FW) as a function of time in days and concentration of Cholestrol (dotted trait) in mg of Cholestrol per L of cell suspension as a function of time in days.FIGURE 2 : HPLC chromatogram (λ =426 nm) of Tripterigium wilfordii enriched extract obtained at J33.FIGURE 3 : Percentage of induction of IL-17A synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared to the negative incubated with sample 1 followed by stimulation with anti-CD antibodies and p*** (p-0, p-0, p-0)FIGURE 4 : Percentage of induction of INF-γ synthesis by CD4+ T cells after incubation with samples 2, 3, 4 and 5 compared to negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p<0.001).FIGURE 5 : Percentage of induction of IL-22 synthesis by CD4+ T cells after incubation with samples 2, 3, 4 and 5 compared to negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p<0.001).FIGURE 6: Percentage of induction of TNF-α synthesis by T cells after incubation with CD4 + 2, 001).3, 4 and 5 compared to the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p<0.001).FIGURE 7: Percentage of induction of IL-6 synthesis by CD4+ T cells after incubation with samples 2, 3, 4 and 5 compared to the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p<0.001).

Examples: The following is the list of the methods used for the determination of the concentration of the test chemical:

Cals are obtained from leaf expanses of Tripterygium wilfordii.

The explants are sterilised with 70% ethanol, then with sodium hypochlorite with 2.5% active chlorine, and then rinsed with sterile demineralized water.

The leaves are cut into pieces, e.g. squares of about 8-10 mm on each side.

The composition of the proliferation medium is as follows: The following is a list of the active substances which may be used in the preparation of the active substance: macro-elements: NH4NO3 at 1650 mg/L, KNO3 at 1900 mg/L, CaCl2.2H2O at 440 mg/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 170 mg/L, Micro-elements: Kl at 0.83 mg/L, H3BO3 at 6.2 mg/L, MnSO4.4H2O at 22.3 mg/L, ZnSO4.H2O at 6.61 mg/L, Na2MoO4.2H2O at 0.25 mg/L, CuSO4.5H2O at 0.025 mg/L, CoCl2.6H2O at 0.025 mg/L, FeSO4.7H2O at 27.8 mg/L, Na2EDTA.2H2O at 37.3 mg/L, Vitamins: myo-amines at 100 mg/L, Nicotinic acid at 0.5 mg/L, Na-acetylsacyl-acetamines at 0.4 mg/L, Nicotinic acid at 0.5 mg/L, acetic acid at 0.4 mg/L, glyclorophosate at 0.5 mg/L, glyclorophosate at 0.4 mg/L, acetic acid at 0.4 mg/L, glyphosate (0.5 mg/L) at 0.4 mg/L, acetic acid at 0.4 mg/L, glyphosphoric acid at 0.4 mg/L, glyphosate (0.5 mg/L) at 0.4 mg/L, acetic acid at 0.4 mg/L, glyphosate (0.5 mg/L) at 0.4 mg/L, acetic acid at 0.4 mg/L, glyphosphoric acid (0.4 mg/L, glycosyl-acetylsacloride (0.4 mg/L) at 0.4 mg/L, glycosyl-acetyl-acetyl-acethanol at 0.4 mg/L, glycoside (0.4 mg/L) at 0.4 mg/L, glycosyl-acetyl-acetyl-acethanol at 0.4 mg/L, glycoside (0.4 mg/L) at 0.4

The growth medium is gelled by addition of agar at 8-12 g/L, pH adjusted to pH 6 ± 0.5 (with KOH, 1M) before autoclaving for 20 min at 121°C. Petri dishes containing the explants are incubated in the dark at 27-28°C.

The cals are transplanted every month on the same glazed medium, the resulting cals are removed from the leaf growth and deposited on new gels of the proliferation medium.

Example 2: Formulation of culture and propagation media (proliferation phase)

After a few months of transplanting, brittle calluses are obtained and transferred to the liquid proliferation medium.

The proliferation medium has, for example, the following composition: The following is a list of the active substances which may be used in the preparation of the product: macro-elements: NH4NO3 at 1650 mg/L, KNO3 at 2500 mg/L, CaCl2.2H2O at 440 mg/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 130 mg/L, Micro-elements: KI at 0.41 mg/L, H3BO3 at 6.2 mg/L, MnSO4.4H2O at 22.3 mg/L, ZnSO4.H2O at 7.5 mg/L, Na2MoO4.2H2O at 0.25 mg/L, CuSO4.5H2O at 0.025 mg/L, CoCl2.6H2O at 0.025 mg/L, FeSO4.7H2O at 19.85 mg/L, Na2EDH2O2 at 26.64 mg/L, Vitalines: myo-amines at 50 mg/L, Nicotinic acid at 0.25 mg/L, Nicotinic acid at 0.25 mg/L, Cyclorophenol at 0.25 mg/L, Pyrophenic acid at 0.25 mg/L, Thioclorophenic acid at 0.25 mg/L, Acetyl-Acetyl at 0.25 mg/L, Cyclorophenol at 0.25 mg/L, Nicotinic acid at 0.25 mg/L, Cyclorophenic acid at 0.25 mg/L, Cyclorophenol at 0.25 mg/L, Cyclorophenol at 0.25 mg/L, Cyclorophenol at 0.25 mg/L, Cyclorophenol at 0.25 mg/L at 0.25 mg/L, Cyclorophenol at 0.25 mg/L at 0.25 mg/L, Cyclorophenol at 0.25 mg/L at 0.25 mg/L, Cyclorophenol at 0.25 mg/L at 0.25 mg/L, Cyclorophenol at 0.25 mg/L 0.25 mg/L:

The pH of the medium shall be adjusted to pH 6 ± 0,5 (by addition of KOH, 1M) before an appropriate sterilisation treatment, e.g. autoclave at 121°C for a minimum of 20 minutes or sterilising filtration over 0,2 μm.

Cell suspension is obtained by depositing about 40 g of brittle callus in a 200 ml Erlenmeyer containing the propagation medium, incubating for one week on a stirring table at 100 RPM (rotation per minute) in the dark at 27-28°C. The cell surplus is collected by pipette leaving the residual callus clusters.

The Erlenmeyers' rate of filling is 40 per cent (80 mL) and the rate of inoculation by cell suspension transfer is 20-25 per cent by volume, or about 160 g/L of fresh biomass. The culture is thus carried out for 15 days in the dark at 27-28°C under orbital agitation of 110-120 RPM. At this stage the biomass is present at a concentration of about 300 g/L of fresh biomass per litre of suspension.

Example 3: Production of triterpenes in Erlenmeyer (election and preparation of the enriched raw extract). Selection phase:

After 15 days of culture, 1/5th of the cell culture is removed from the Erlenmeyer and 20 mL of a concentrated proliferation medium is added to the Erlenmeyer. The composition of the concentrated medium is as follows: Macronutrients: NH4NO3 at 13.9 g/L, KNO3 at 15.2 g/L, CaCl2.2H2O at 2.2 g/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 170 mg/L; Micro-nutrients: Vitamin KI at 0.83 mg/L, H3BO3 at 31.2 mg/L, MnSO4.4H2O at 91.5 mg/L, ZnSO4.H2O at 33.05 g/L, Na2O4.2H2O at 1.25 mg/L, CuL: CaCl5.5H2O at 0.125 mg/L; Nicotinamide at 0.6 mg/L; Co2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2O2

The elicitation cocktail is then added to the Erlenmeyer in the proliferation medium using parent solutions made in dimethyl sulfoxide. The composition of the elicitation cocktail allows the following concentrations to be obtained in the elicitation medium (+ cells): sodium pyruvate 1,5 g/L, potassium pyrophosphate 0,440 g/L, 2iP 0,0004 g/L, methyl jasmonate 0,036 g/L and chitine 2 g/L.

The production of Celastrol and its derivatives is carried out for 12 days in the dark at 27-28°C under 120 rpm orbital agitation.

Harvesting of biomass for extraction:

When the culture is stopped, the medium is filtered to recover all the biomass that contains the majority of the Celestrol.

After separation of the biomass by 0-50 μm filtration, 2 volumes of ester-type solvent and more particularly alkyl acetate, especially ethyl acetate (also called isopropyl acetate), are mixed to 1 weight of biomass. This mixture is subjected to sonication extraction to lysis the cells and make the elements of the cytosol available.

The concentration of Celastrol in the organic phase is measured, the concentration of Celastrol per litre of suspension is estimated at 553 mg, which corresponds to a weight of 0.0166 g of Celastrol per gram of dry weight of cells.

Example 4: Production of Triterpenes (Celastrol and derivatives) in single-use WAVE pocket bioreactors

The example illustrated is described for WAVE reactors, e.g. Sartorius brand, for volumes of 5 L or 10 L or 2 X 5 L, but the method can be adapted and applied to larger volumes and to materials from other manufacturers.

The binary system described above for traditional laboratory glass or industrial stainless steel bioreactors is applied in the same way with the 2 WAVE pockets.

Proliferation:

The Wave A bioreactor (5 L), placed on its support, is filled with the proliferation medium by in-line sterilizing filtration and inflated with air.

A pre-culture of Trypterygium wilforddi is carried out for 15 days in Erlenmeyer as described in Example 2. The bioreactor proliferation medium is then seeded by this pre-culture at a concentration of 160 g/L (pocket A).

The bioreactor shall be incubated under the following conditions: The test is performed at a temperature of 27°C. The test is performed at a temperature of 27°C.

During this proliferation phase, cell growth is measured daily (Figure 1).

Elicitation and production of triterpenes (celastrol and derivatives):

The medium of the medium A is supplemented by 1000 mL of concentrated medium in demineralized water with the following composition: macro-methane: NH4NO3 at 13.9 g/L, KNO3 at 15.2 g/L, CaCl2.2H2O at 2.2 g/L, MgSO4.7H2O at 370 mg/L, KH2PO4 at 170 mg/L; Micro-methane: KI at 0.83 mg/L, H3BO3 at 31.2 mg/L, MnSO4.4H2O at 91.5 g/L, ZnSO4.05 carbonoxylate at 33.05 mg/L, Na2SO4.2H2OL: Na2SO4.25 g/L; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicotinamide; Nicot

The contents of the A-bag are thus selected by agitation at 27°C.

The culture in the elicitation phase is followed by cell growth and cell cholesterol concentration measurements in the culture for 16 days (Figure 1).

It can be seen that the concentration of Celastrol in pocket A increases steadily up to J32 and is at its maximum at 553 mg per litre of elicitation medium after 15 days of incubation.

The rate of production of Celastrol is approximately 46 mg/ L of cellular suspension per day for 15 days after elicitation up to J32.

The kinetics of Celestrol production begin to change shortly after almost all of the available sucrose is consumed (Figure 1).

Example 5: Production of a Celastrol enriched extract by solid/liquid extraction of biomass from cell suspension from Tripterygium wilfordii (TW08)

At 15 days after ellicitation (32 days after inoculation), the majority of the biomass is recovered by filtration of the cell suspension with a nylon (20 - 50 μM) filter (TW08).

From 5 L of suspension, approximately 1925 g of biomass is recovered. This biomass is extracted with ethyl acetate (also isopropyl acetate) in a ratio of 2:1 (Vol.: Weight) to biomass weight (here 3850 mL of solvent for 1925 g of biomass). The biomass/solvent mixture is then subjected to physical extraction by sonication. The organic phase is then recovered after maceration under agitation.

The concentration of Celastrol and derivatives in the organic phase is measured by HPLC (Quantification of Celastrol, Tingenin derivatives) (see Figure 2) Experimental conditions: Waters Atlantis column dC18 4.6 x 150 mm - 5μ equipped with a guard column Atlantis dC18 5 μm 4.6 x 20 mm Gd Column - 5μm. Mobile phases: (A) Ammonium acetate 0.1 mM pH 4.0 (B) Ammonium acetate 0.1 mM 4.0 in acetonitrile pH 99.8%. Gradient: 25 %A) /75 %B) - 20 min 0 %A) %A/100% (B) - 24.5 min 0 (A/100%) - 25 min (B) - 25 %A/75 (B) - 25 min (B) - 25 %A/75 (B) - 30 min (B) - 25 min (B) - 25 min (B) (B) - 25 min (B) Ting (Ting) - 42 (B) - 1 (B), (B) - 6 (B) (B) (B) (Ting) - 25 min (B) (B) (B) - 25 min (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (B) (

Example 6: Cleaning of Celestrol from Tripterygium wilfordii culture

From the extract obtained in example 5, ethyl acetate (or isopropyl acetate) is evaporated at reduced pressure to obtain a dry extract. Part of the extract thus obtained is first purified by medium pressure liquid chromatography (CLMP) on silica (40 g, 125 x 25 mm, 30 μm) with an elution gradient of CH2Cl2/MeOH (100/100/0 to 0). All the resulting fractions are purified by thin-layer chromatography (CCM - Cyclic Phase: Silica 60 Å; High-mobile Phase: Toluene / Acetyl Toluene / Acetic Acid 70 Å; Absolute Phase: 33 33 HP) and the fractions containing the acetyl linearity and its gradient of Toluene and Toluene (A) are collected at a concentration of approximately 250 g/mL. In order to obtain a secondary concentration of Toluene and Toluene (A) on a single crystalline culture, the products can be obtained from a concentration of approximately 250 g/mL (A) and a concentration of 100 g/mL (A) of Toluene and Toluene (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A

In parallel, part of the resulting dry extract is taken up in a buffer to give extract R003034 which will be evaluated for anti-Th17 pharmacological activity.

Example 7: Anti-Th17 activity on human CD4+ cells from the Celastrol enriched CCV extract.

In inflammatory dermatoses (especially psoriasis), CD4+ T cells are known to overexpress IL17, IL6, IL22, and IFN gamma and TNF alpha interleukins. In atopic dermatitis, IL17 is overexpressed in a certain phase of the disease. In addition, the Th17 pathway is strongly activated in acne. The inhibitory power of an enriched extract according to the invention on overexpression of these molecules has been tested.

Human CD4+ cells were isolated from the mononuclear cells of 2 donors on Ficoll Paque plus® according to the manufacturer's (GE Healthcare) recommended protocol. CD4+ lymphocytes are isolated by positive sorting by the Miltenyi Biotec (CD4) kit and an LS column and are resuspended in a RPMI (Sigma-Aldrich: containing L-glutamine and 10% fetal bovine serum) culture medium supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin. The resuspended lymphocytes are broken into microplate wells. Different extracts or controls are then added to the wells: The test chemical is administered as a single dose to the patient, with the following results: 1. negative control (the same volume of reagent-free buffer is added); 2. extract R003034 at 0.06 mg/ml (Celastrol titrated at 9 ng/ml) (final well concentration); 3. extract R003034 at 0.2 mg/ml (Celastrol titrated at 30 ng/ml) (final well concentration); 4. extract R003034 at 0.6 mg/ml (Celastrol titrated at 90 ng/ml) (final well concentration); 5. 2 μM xanthate (positive control) (final well concentration).

After 2 hours of incubation at room temperature, CD4+ lymphocytes are activated at 37°C for 20 hours with anti-CD3 and anti-CD28 antibodies at a final concentration of 300 ng/ml and 400 ng/ml respectively.

The cytokines IL-17A, INF gamma, IL-22, TNF alpha and IL-6 were quantified in the surgeon of each tube by the Multiplex Immunoassays method (Jager et al. 2003). The results obtained are from 2 experiments with lymphocytes from 2 different donors. The results are presented in Figures 3 to 7 as a percentage of induction relative to the negative control (1). positive control 5 (dexamethasone (2 μM)) inhibits overexpression of all cytokines : IL-17A, INF gamma, IL-22, TNF alpha and IL-6; the extract of the invention (2, 3 and 4) strongly inhibits overexpression of cytokines IL-17A, INF gamma, IL-22, TNF alpha and IL-6. Inhibition of overexpression of IL-17A and TNF alpha is dose-dependent. From the lowest dose, titrated at 9 ng/ml in Celestrol, there is an inhibition of overexpression of IL-17A of 75%, an inhibition of overexpression of TNF alpha of 80% and almost total inhibition of overexpression of INF-gamma, IL-22 and IL-6.

This demonstrates that R003034 extract has very strong anti-Th17 activity, comparable if not superior to dexamethasone (2 μM), which gives it remarkable activity in the treatment of Th17-dependent pathologies such as psoriasis, acne and atopic dermatitis.

Example 8: Comparison of the cellastrol yields obtained from plant cell cultures of Tripterygium wilfordii.

Ten erlenmeyer cultures (volume = 50 mL) were carried out in parallel from the same cell line of Tripterygium wilfordii, in duplicate under the same conditions of agitation, aeration and temperature. At the end of the growth phase, with the maximum density of the suspension, the amount of fresh biomass weighed was equivalent (average weight of 340 g of fresh biomass/L) in each erlenmeyer. At this point, the elicitors were added to the culture medium of the ten erlenmeyer at the concentrations shown in the table below, in which the yield is expressed in Cestrel/kg of fresh biomass. - What?

Erlen	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10
MeJa (µM)	0	0	64	36	36	64	64	64	92	92
Chitine (g/L)	0	2	0	2	4,2	1,3	2,5	3,7	3,1	4,2
Rendement	4 ,5	55	738	1730	2178	1506	1724	2025	1986	1878

The dosage of Celastrol suspension was performed by HPLC in accordance with example 5 with a calibration curve using control products.

Suspensions in erlenmeyers rated E1, E2, E3 were elicited with a control solution with no elicitor for E1, with chitin alone (2 g/L) for E2 and with MeJa alone (64 μM) for E3. The MeJa concentration in E3 is equivalent to that described by Liu et al. 2016, i.e. 64 μM.

Suspensions in erlenmeyers rated E4 to E10 were elicited at three different concentrations in MeJa (36, 64 and 92 μM) and five different concentrations in Chitin (1.3 , 2.5, 3.1 and 4.2 g/L).

It was found that the Celastrol yields obtained for E6, E7 and E8 were increased by 204%, 233% and 274% respectively compared to E3, with the MeJa concentration being the same in these four erlenes.

Example 9: Inhibitory activity with respect to NFκB

The transcription factor NPκB controls the expression of a large number of genes involved in regulating the inflammatory response. In the inactive state, NPκB is sequestered in the cytoplasm by the IKB protein. Some pro-inflammatory stimuli such as TNF alpha and IL-1 beta lead to activation of NPκB, i.e. its nuclear translocation. Once in the nucleus, NPκB will induce the transcription of pro-inflammatory genes coding for cytokines, chemokines, adhesion molecules, growth factors and inducible enzymes.

The anti-inflammatory activities of extracts obtained from the suspensions contained in the erlenmeyers E1, E2, E3 and E6 of example 8 were evaluated in vitro on human HaCat keratinocytes using the method described by Albanesi el al. The dry weight extracts were collected in a buffer and introduced into a HaCat cell culture solution at equivalent final concentrations (ng/mL) for each extract, and incubated for 1 h. NPκB expression was then induced by stimulation with TNF alpha ng (0.3 ng/mL). - What?

	TNF alpha	Dex	Extrait issu de E1		Extrait issu de E2		Extrait issu de E3		Extrait issu de E6
Conc.	0,3	2 µM	0,19	0,58	0,19	0,58	0,19	0,58	0,19	0,58
% inh.	0	42,5	-1	-9	-7	2	3	1	20	68
sem	4	6	2	3	3	3	2	3	3	4

Dex : Dexaméthasone ; Conc. : concentration (ng/mL) ; %inh : % d'inhibition de l'expression de NPκB par rapport à l'induction de TNF alpha ; sem : erreur standard moyenne.

Dexamethasone 2μM, used as a positive control, inhibits NFκB expression by more than 40%.

The extract according to the present invention (derived from E6, with the pair of MeJa and chitin elicitors) evaluated at concentrations of 0,19 and 0,58 ng/mL inhibits NFκB expression by 20% and 70%, respectively.

The extract prepared by Liu et al. (from E3, with MeJa as the sole elicitor) evaluated at the same concentrations did not induce any significant inhibition of NFκB expression. Similarly, extracts from E2 (with chitin as the sole elicitor) and the E1 control have no effect on NFκB expression.

Only the extract from culture elicited according to the present invention strongly inhibits NFκB, and in a concentration-dependent manner.

Example 10: Antimicrobial activity in relation to propionibacterium from an extract prepared in accordance with the method of the present invention compared to other compounds

The activity is evaluated on Propionibacterium acnes strain (CIP 53117T), with an inoculation suspension at 108 CFU/mL. The maintenance medium is Columbia gellosis + 5% sheep blood, with incubation for 24 h at 36 ± 1 °C under anaerobic conditions. The test medium is Mueller Hinton broth + 10% fetal veal serum (FVS) and Columbia gellosis + 5% sheep blood, with incubation for 24 h at 36 ± 1 °C under anaerobic conditions. The determination of minimum inhibitory concentrations (MICs) is performed by micromethode in liquid culture medium. 100 μL of each liquid medium are deposited in a 96-well micropoly culture medium.100 μL of the test product is deposited in the first well of a line. Dilutions by a ratio of 2 are then made from wells 1 to 10. Propionibacterium acnes test suspensions are prepared extemporaneously in Tryptone salt. 100 μL is deposited in each well of a second microplate, except for column 11. The entire first microplate is inoculated, using a Denley multipoint seeder, from the second microplate.Two benchmarks were tested in parallel: the determination of minimum bactericidal concentrations (BMC) is carried out by grafting micro-plates of CMI onto a gel medium using the Denley multipoint seeder; after incubation, the BMC is defined as the highest dilution with no visible growth; all tests are performed in duplicate.

The compounds tested are: The extract prepared in accordance with the method of the present invention in pentylene glycol, (mother solution = 2 % / 2 % pentylene glycol) Tingenin A in DMSO, (mother solution = 150 μg/mL / 10 % DMSO) Tingenin B in DMSO, (mother solution = 150 μg/mL / 10 % DMSO) The references are amoxicillin, DMSO (mother solution = 100 %), and pentylene glycol (mother solution = 2 %)

The results obtained are summarised in the table below, which shows the values of CMI and CMB for the 4 samples and the two excipients. - What?

Composés	CMI (µg/mL)	CMB (µg/mL)
Célastrol	0,59	1,17
DMSO	5	5
Extrait selon le procédé	0,25*	0,25*
Pentylène glycol	>1	>1
Tingénine A	2,34	2,34
Tingénine B	1,17	1,17

* : soit 0,375 µg/mL en équivalent célastrol.

In conclusion, although celastrol has quite interesting antibacterial activity on Propionibacterium acnes, it appears that the extract prepared according to the method of the invention has the highest level of antibacterial activity on Propionibacterium acnes.

Example 11: Antimicrobial activity against Staphylococcus aureus of an extract prepared in accordance with the method of the present invention compared to other compounds

S. aureus is known to colonize lesions in patients with atopic dermatitis, and is also present in patients with psoriasis (Tomi et al. 2005).

The antibacterial activity against S. aureus of the extract obtained by the process of the present invention was evaluated and the experiment was performed as described in Example 10 with the pathogenic strain S. aureus CIP 4.83, the maintenance medium Gélose Trypticase-Soja, the CMI/CMB test medium Mueller broth and Hinton gélose and incubation at 36°C for 24 h.

The results obtained are summarized in the table below: it is found that although Celastrol has a high antibacterial activity, the anti-S. aureus activity of the extract obtained by the process of the present invention titrated as Celastrol equivalent is even better. - What?

Composés	CMI (µg/mL)	CMB (µg/mL)
Célastrol	0,29	0,59
DMSO	>5	>5
Extrait selon le procédé	0,125*	0,25**
Pentylène glycol	>1	>1
Tingénine A	1,17 - 2,34	2,34
Tingénine B	0,59	1,17

* 0,1875 µg/mL en équivalent célastrol ; ** 0.375 µg/mL en équivalent célastrol.

In conclusion, this extract not only has interesting anti-Th17 pharmacological activity, but surprisingly strong anti-S. aureus activity, which makes it very attractive as an active in the topical treatment of atopic dermatitis and psoriasis.

Example 12: Cream for topical application

glycérine	8 à 10 %
copolymère d'hydroxyacrylate	1,8 à 2 %
gomme xanthane	0,1 à 0,3 %
cétéareth 33 et alcool cétéarylique	4 à 6 %
stéarate de glycéryle	1,5 à 2 %
palmitate d'éthylhexyle	12 à 15 %
	0,1 à 2,0 %
acide glycolique	2 à 4 %
Lactamide MEA	3 à 6 %
Laureth-9	1 à 3 %
sulfonate d'huile de schiste sodique	1 à 3 %
extrait de racine de chicorée	1 à 3 %
Eau complétée à	100 %

The extract of Tripterygium wilfordii is prepared according to the present invention, its concentration being expressed in dry extract weight in a solvent compatible with the formulation, such as olive oil, pentylene glycol or myritol 318 or others.

This example of formulation is by no means exhaustive and may be adapted according to the treatment.

For example, for the treatment of the scalp, a shampoo can be formulated using the knowledge of the man of art.

The Commission shall be assisted by the European Parliament.

The following is a list of the most commonly used chemical compounds in the food industry: Camelio et al. JACS 2015, 137:11864-67Coppede et al. Plant Cell Tiss. Organ Cuit. 2014, 118:33-43Jager et al. Clin. Diagn. Lab. Immunol. 2003, 10(1):133-9Kelhala et al. PLOS One 2014, 9(8):e105238Liu et al. J. Asian Nat. Prod. Res. 2016, 19:1-10Lowes et al. Annu. Rev. Immunol. 2014, 32:227Miyagaki et al. J. Derm. Science 2015, 78:89Murashige & Skoog Physiol. Plant. 1962, 15: 473-497Albanesi el al. Curr. Drug Targets Inflamm. Allergy 2005, 4(3):329-334Tomi and al. J. Am. Acad. Dermatol. 2005, 53:67-72.

Claims (6)

1. A process for producing a crude extract enriched in a pentacyclic triterpene, which is celastrol, comprising the following steps:

   (i) a phase of proliferation of cells of a plant of the Celastraceae family, which is Tripterygium wilfordii, in a proliferation medium,

   (ii) a phase of elicitation by adding an elicitation cocktail to the cell culture obtained in step (i), said elicitation cocktail comprising at least one elicitor of monocarboxylic compound type, which is methyl jasmonate, and at least one biotic elicitor, which is chitin, and

   (iii) preparation of a crude extract enriched in pentacyclic tripterpenes from the cell culture obtained in step (ii).
2. The process for producing an enriched crude extract according to claim 1, wherein the elicitation cocktail further comprises at least one cell differentiation factor for plant cells and at least one precursor of the terpene synthesis pathway.
3. The process for producing an enriched crude extract according to claim 2, wherein the at least one cell differentiation factor for plant cells is selected from a cytokine, a gibberellin and a mixture thereof.
4. The process for producing an enriched crude extract according to claim 3, wherein the at least one cell differentiation factor for plant cells is selected from benzylaminopurine (BAP), 6-γ,γ-dimethylallylaminopurine (2iP) and a mixture thereof.
5. The process for producing a crude extract enriched according to any one of claims 2 to 4, wherein the at least one precursor of terpene synthesis is selected from the group consisting of sodium pyruvate, potassium pyrophosphate and a mixture thereof.
6. The process for producing an enriched crude extract according to any one of claims 1 to 5, further comprising a step (iv) of obtaining a purified extract of the pentacyclic triterpene from the enriched crude extract obtained in step (iii).