METHOD FOR PRODUCING CELASTROL AND PENTACYCLIC TRITERPENE DERIVATIVES

Abstract

The present invention relates to a method for producing a crude extract enriched in pentacyclic triterpenes, including celastrol, from cells of a plant from the Celastraceae family; an enriched crude extract that can be obtained using such a method; therapeutic applications of such an extract; and pharmaceutical and dermocosmetic compositions containing such an extract.

Claims

1. A process for producing a crude extract enriched in pentacyclic triterpenes comprising the following steps: (i) a phase of proliferation of cells of a plant of the Celastraceae family 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 monocarboxylic compound-type elicitor and at least one biotic elicitor, said biotic elicitor being an N-acetylaminoglucosamine, and (iii) preparation of a crude extract enriched in pentacyclic triterpenes from the cell culture obtained in step (ii).

2. The process for producing an enriched crude extract according to claim 1, wherein the biotic elicitor is chitin.

3. The process for producing an enriched crude extract according to claim 1, wherein the monocarboxylic compound-type elicitor is a methyl ester.

4. The process for producing an enriched crude extract according to claim 1, wherein the at least one monocarboxylic compound-type elicitor is methyl jasmonate and the at least one biotic elicitor is chitin.

5. 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.

6. The process for producing an enriched crude extract according to claim 5, wherein the at least one cell differentiation factor for plant cells is selected from a cytokine, a gibberellin and a mixture thereof.

7. The process for producing an enriched crude extract according to claim 5, wherein the at least one precursor of terpene synthesis is selected from the group consisting of sodium pyruvate, potassium pyrophosphate and a mixture thereof.

8. The process for producing an enriched crude extract according to claim 1, wherein the pentacyclic triterpene is selected from the group consisting of celastrol, tingenin A, tingenin B, pristimerin, tripterygone and mixtures thereof.

9. The process for producing an enriched crude extract according to claim 8, wherein the pentacyclic triterpene is celastrol.

10. The process for producing an enriched crude extract according to claim 1, wherein: (i) the plant of the Celastraceae family is Tripterygium wilfordii, (ii) the at least one monocarboxylic compound-type elicitor is methyl jasmonate and the at least one biotic elicitor is chitin, and (iii) the pentacyclic triterpene is celastrol.

11. The process for producing an enriched crude extract according to claim 1, further comprising a step (iv) of obtaining a purified extract of one or more pentacyclic triterpenes from the enriched crude extract obtained in step (iii).

12. An enriched crude extract obtainable by the process according to claim 1.

13-15. (canceled)

16. A pharmaceutical or dermocosmetic composition comprising an enriched crude extract according to claim 12.

17. The process for producing an enriched crude extract according to claim 3, wherein the monocarboxylic compound-type elicitor is methyl jasmonate.

18. The process for producing an enriched crude extract according to claim 6, wherein the at least one cell differentiation factor for plant cells is selected from benzylaminopurine (BAP), 6-?,?-dimethylallylaminopurine (2iP) and a mixture thereof.

19. A method for treating an inflammatory dermatosis inducing a TH17-type immune response comprising the administration to a person in need thereof of an effective amount of an enriched crude extract according to claim 12.

20. The method according to claim 19, wherein the inflammatory dermatosis is selected from the group consisting of psoriasis, atopic dermatitis and acne.

Description

FIGURES

[0112] FIG. 1: Curve of growth (solid line) of a Tripterygium wilfordii cell culture in g/L wet biomass (FW) as a function of time in days and of celastrol concentration (dotted line) in mg of celastrol per L cell suspension as a function of time in days.

[0113] FIG. 2: HPLC chromatogram (?=426 nm) of the enriched crude extract of Tripterygium wilfordii obtained at day 33.

[0114] FIG. 3: Percentage induction of IL-17A synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared with the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p-value<0.001).

[0115] FIG. 4: Percentage induction of INF-? synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared with the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p-value<0.001).

[0116] FIG. 5: Percentage induction of IL-22 synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared with the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p-value<0.001).

[0117] FIG. 6: Percentage induction of TNF-? synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared with the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p-value<0.001).

[0118] FIG. 7: Percentage induction of IL-6 synthesis by CD4+ T lymphocytes after incubation with samples 2, 3, 4 and 5 compared with the negative control incubated with sample 1 followed by stimulation with anti-CD3 and anti-CD28 antibodies (*** p-value<0.001).

EXAMPLES

Example 1

Cell Dedifferentiation Protocol

[0119] Calluses are obtained from Tripterygium wilfordii leaf explants.

[0120] Explants are sterilized with 70% ethanol, then with sodium hypochlorite with 2.5% active chlorine, then rinsed with sterile demineralized water. Optionally, washing with 7% hydrogen peroxide before rinsing with sterile demineralized water is possible.

[0121] Leaves are cut into pieces, for example into squares of about 8-10 mm on a side. Leaf explants are deposited on agar proliferation medium.

[0122] The composition of the proliferation medium is as follows:

[0123] Macroelements: NH.sub.4NO.sub.3 at 1650 mg/L, KNO.sub.3 at 1900 mg/L, CaCl.sub.2.2H.sub.2O at 440 mg/L, MgSO.sub.4.7H.sub.2O at 370 mg/L, KH.sub.2PO.sub.4 at 170 mg/L,

[0124] Microelements: KI at 0.83 mg/L, H.sub.3BO.sub.3 at 6.2 mg/L, MnSO.sub.4.4H.sub.2O at 22.3 mg/L, ZnSO.sub.4.H.sub.2O at 6.61 mg/L, Na.sub.2MoO.sub.4.2H.sub.2O at 0.25 mg/L, CuSO.sub.4.5H.sub.2O at 0.025 mg/L, CoCl.sub.2.6H.sub.2O at 0.025 mg/L, FeSO.sub.4.7H.sub.2O at 27.8 mg/L, Na.sub.2EDTA.2H.sub.2O at 37.3 mg/L,

[0125] Vitamins: myo-inositol at 100 mg/L, nicotinic acid at 0.5 mg/L, pyridoxine-HCl at 0.5 mg/L, thiamine-HCl at 0.5 mg/L,

[0126] Amino acid: glycine at 2 g/L,

[0127] Carbon source: sucrose at 30 g/L,

[0128] Plant hormones: kinetin at 0.1 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.5 mg/L, naphthalene acetic acid (NAA) at 1 mg/L.

[0129] The proliferation medium is gelled by adding agar at 8-12 g/L, its pH is 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.

[0130] Calluses are subcultured every month on the same agar medium. To that end, the calluses obtained are detached from the leaf explant and deposited on fresh agar proliferation medium.

Example 2

Formulation of Culture and Propagation Media (Proliferation Phase)

[0131] After a few months of subculturing, friable calluses are obtained and transferred to liquid proliferation medium.

[0132] The proliferation medium has, for example, the composition indicated below:

[0133] Macroelements: NH.sub.4NO.sub.3 at 1650 mg/L, KNO.sub.3 at 2500 mg/L, CaCl.sub.2.2H.sub.2O at 440 mg/L, MgSO.sub.4.7H.sub.2O at 370 mg/L, KH.sub.2PO.sub.4 at 130 mg/L,

[0134] Microelements: KI at 0.41 mg/L, H.sub.3BO.sub.3 at 6.2 mg/L, MnSO.sub.4.4H.sub.2O at 22.3 mg/L, ZnSO.sub.4.H.sub.2O at 7.5 mg/L, Na.sub.2MoO.sub.4.2H.sub.2O at 0.25 mg/L, CuSO.sub.4.5H.sub.2O at 0.025 mg/L, CoCl.sub.2.6H.sub.2O at 0.025 mg/L, FeSO.sub.4.7H.sub.2O at 19.85 mg/L, Na.sub.2EDTA.2H.sub.2O at 26.64 mg/L,

[0135] Vitamins: myo-inositol at 50 mg/L, nicotinic acid at 0.25 mg/L, pyridoxine-HCl at 0.25 mg/L, thiamine-HCl at 0.25 mg/L,

[0136] Carbon sources: sucrose at 30 g/L

[0137] Plant hormones: kinetin at 0.083 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.575 mg/L, naphthalene acetic acid (NAA) at 0.350 mg/L.

[0138] The pH of the medium is adjusted to pH 6?0.5 (by adding KOH, 1M) before an appropriate sterilization treatment, for example autoclaving at 121? C. for a minimum duration of 20 minutes or by 0.2 ?m sterilizing filtration.

[0139] Cell suspension is achieved by depositing about 40 g of friable callus in a 200 ml Erlenmeyer flask containing the propagation medium, incubation for one week on a shaker table at 100 rpm (rotations per minute) in the dark at 27-28? C. The cell supernatant is collected by pipette, leaving the residual callus clusters. The cell suspension obtained is cultured for 15 days then propagated by dilution to ?.sup.th in fresh medium every 15 days.

[0140] The Erlenmeyer flasks are filled to 40% (80 mL) and the inoculation rate by cell suspension transfer is 20-25% of the volume, or about 160 g/L of fresh biomass. The culture is then conducted for 15 days in the dark at 27-28? C. with orbital shaking at 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 an Erlenmeyer Flask (Phase of Elicitation and of Preparation of the Enriched Crude Extract)

[0141] Elicitation Phase:

[0142] After 15 days of culture, ?.sup.th of the cell culture is removed from the Erlenmeyer flask and 20 mL of concentrated proliferation medium is added to the Erlenmeyer flask. The composition of the concentrated medium is as follows: Macroelements: NH.sub.4NO.sub.3 at 13.9 g/L, KNO.sub.3 at 15.2 g/L, CaCl.sub.2.2H.sub.2O at 2.2 g/L, MgSO.sub.4.7H.sub.2O at 370 mg/L, KH.sub.2PO.sub.4 at 170 mg/L; Microelements: KI at 0.83 mg/L, H.sub.3BO.sub.3 at 31.2 mg/L, MnSO.sub.4.4H.sub.2O at 91.5 mg/L, ZnSO.sub.4.H.sub.2O at 33.05 mg/L, Na.sub.2MoO.sub.4.2H.sub.2O at 1.25 mg/L, CuSO.sub.4.5H.sub.2O at 0.125 mg/L, CoCl.sub.2.6H.sub.2O at 0.125 mg/L, FeSO.sub.4.7H.sub.2O at 139 mg/L, Na.sub.2EDTA.2H.sub.2O at 186.5 mg/L; Vitamins: myo-inositol at 1250 mg/L, nicotinic acid at 8.5 mg/L, pyridoxine-HCl at 5 mg/L, thiamine-HCl at 5 mg/L; Amino acid: glycine at 20 mg/L; Carbon source: sucrose at 150 g/L (dissolved in demineralized water).

[0143] The elicitation cocktail is then added to the proliferation medium in the Erlenmeyer flask using stock solutions prepared in dimethylsulphoxide. The composition of the elicitor cocktail provides the following concentrations in the elicitation medium (+cells): 1.5 g/L sodium pyruvate, 0.440 g/L potassium pyrophosphate, 0.0004 g/L 2iP, 0.036 g/L methyl jasmonate and 2 g/L chitin.

[0144] The production of celastrol and derivatives thereof is conducted for 12 days in the dark at 27-28? C. with orbital shaking at 120 rpm.

[0145] Harvesting of the Biomass for Extraction:

[0146] When the culture stops, the medium is filtered to recover the totality of the biomass containing the majority of celastrol.

[0147] After separation of the biomass by 0-50 ?m filtration, 2 volumes of ester-type solvent and more particularly alkyl acetate, notably ethyl acetate (or isopropyl acetate), are mixed with 1 weight of biomass. This mixture is extracted by sonication to lyse the cells and make the cytosol components available. The organic phase comprising the triterpenic fraction is recovered after centrifugation of the mixture.

[0148] The celastrol concentration in the organic phase is measured. The celastrol concentration per litre of suspension is estimated at 553 mg, which corresponds to a weight of 0.0166 g of celastrol per gram of dry cell weight.

Example 4

Production of Triterpenes (Celastrol and Derivatives) in Wave-Type Single-Use Bioreactor Bags

[0149] The example illustrated is described for wave-type reactors, for example, Sartorius brand, for volumes of 5 L or 10 L or 2?5 L, but the method can be adapted and applied to larger volumes and to equipment from other manufacturers.

[0150] The binary system previously described for traditional laboratory bioreactors made of glass or industrial bioreactors made of stainless steel is applied in the same way with the 2 wave bags.

[0151] Proliferation:

[0152] Wave bioreactor A (5 L), placed on its platform, is filled with the proliferation medium by inline sterilizing filtration and inflated with air.

[0153] A Tripterygium wilfordii pre-culture is prepared for 15 days in an Erlenmeyer flask as described in example 2. The proliferation medium of the bioreactor is then seeded with this preculture at a concentration of 160 g/L (bag A).

[0154] The bioreactor is incubated according to the following conditions: [0155] rocking angle: 5-8?; [0156] rocking frequency: 16-30 rpm; [0157] aeration rate: 0.1-0.5 L/min of air enriched to 50% pure oxygen; [0158] temperature: 27? C.; [0159] duration: 17 days.

[0160] During this proliferation phase, cell growth is measured daily (FIG. 1).

[0161] Elicitation and Production of Triterpenes (Celastrol and Derivatives):

[0162] A volume of about 1000 ml of culture from bag A (5 L) is transferred to bag B placed next to bag A on the same platform. The bag A medium is supplemented with 1000 mL of concentrated medium in demineralized water having the following composition: Macroelements: NH.sub.4NO.sub.3 at 13.9 g/L, KNO.sub.3 at 15.2 g/L, CaCl.sub.2.2H.sub.2O at 2.2 g/L, MgSO.sub.4.7H.sub.2O at 370 mg/L, KH.sub.2PO.sub.4 at 170 mg/L; Microelements: KI at 0.83 mg/L, H.sub.3BO.sub.3, at 31.2 mg/L, MnSO.sub.4.4H.sub.2O at 91.5 mg/L, ZnSO.sub.4.H.sub.2O at 33.05 mg/L, Na.sub.2MoO.sub.4.2H.sub.2O at 1.25 mg/L, CuSO.sub.4.5H.sub.2O at 0.125 mg/L, CoCl.sub.2.6H.sub.2O at 0.125 mg/L, FeSO.sub.4.7H.sub.2O at 139 mg/L, Na.sub.2EDTA.2H.sub.2O at 186.5 mg/L; Vitamins: myo-inositol at 1250 mg/L, nicotinic acid at 8.5 mg/L, pyridoxine-HCl at 5 mg/L, thiamine-HCl at 5 mg/L; Amino acid: glycine at 20 mg/L; Carbon source: sucrose at 150 g/L; to which is added the following elicitation cocktail: 7.5 g/L sodium pyruvate, 2.2 g/L potassium pyrophosphate, 0.002 g/L 2iP, 1.8 g/L methyl jasmonate and 10 g/L chitin.

[0163] The contents of bag A are then elicited with shaking at a temperature of 27? C.

[0164] Culture in elicitation phase is followed by measurement of cell growth and celastrol concentration in the culture for 16 days (FIG. 1).

[0165] It can be seen that the celastrol concentration in bag A increases steadily up to day 32 and is at its maximum at 553 mg per litre of elicitation medium after 15 days of incubation.

[0166] The celastrol production rate is about 46 mg/L of cell suspension per day for 15 days after elicitation up to day 32.

[0167] The kinetics of celastrol production begin to shift shortly after almost all available sucrose has been consumed (FIG. 1).

Example 5

Production of an Extract Enriched in Celastrol by Solid/Liquid Extraction of Biomass from a Tripterygium wilfordii Cell Suspension (TW08)

[0168] At 15 days after elicitation (32 days after inoculation), most of the biomass is recovered by filtration of the cell suspension with a nylon filter (20-50 ?M) (TW08).

[0169] From 5 L of suspension, about 1925 g of biomass is recovered. This biomass is extracted with ethyl acetate (or isopropyl acetate) in a proportion of 2:1 (Vol:Weight) based on the weight of biomass (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 with agitation. The addition of solvent (followed by maceration with agitation and recovery of the organic phase) is repeated 2 times.

[0170] The concentration of celastrol and derivatives in the organic phase is measured by HPLC assay (quantification of celastrol, tingenin derivatives) (see FIG. 2) Experimental conditions: Waters Atlantis dC18 column 4.6?150 mm-5? equipped with an Atlantis dC18 guard column 5 ?m 4.6?20 mm Gd Column-5?. Mobile phases: (A) Ammonium acetate, 0.1 mM pH 4.0; (B) Ammonium acetate, 0.1 mM pH 4.0 in 99.8% acetonitrile. Gradient: initial 25% (A)/75% (B)20 min 0% (A)/100% (B)24.5 min 0% (A)/100% (B)25 min 25% (A)/75% (B)30 min 25% (A)/75% (B). Flow rate: 1 mL/min. Detection at 426 nm: retention peaks (min): tingenin B (6.5), tingenin A (8.2), celastrol (10.5).

Example 6

Purification of Celastrol from the Tripterygium wilfordii Culture

[0171] From the extract obtained in example 5, ethyl acetate (or isopropyl acetate) is evaporated under reduced pressure to obtain a dry extract. Part of the extract thus obtained is purified first by mediumpressure liquid chromatography (MPLC) on silica (40 g, 125?25 mm, 30 ?m) with a CH.sub.2Cl.sub.2/MeOH elution gradient (100/0 to 0/100). All fractions obtained are analysed by thin-layer chromatography (TLCStationary phase: 60 ? silica; Mobile phase: 70:33:3 toluene/ethyl acetate/acetic acid) and the fractions mainly containing celastrol and its tingenin derivatives (A and B) are collected. In a second step, the fraction containing the products of interest (celastrol and derivatives) is purified by high-performance liquid chromatography (HPLC) in reversed-phase mode (LiChrospher 100RP18, 250?25 mm, 5 ?M) with a water/acetonitrile/0.1% formic acid linear gradient (80/20 to 0/100). The celastrol peak is collected and concentrated under reduced pressure; about 2.5 to 3 g of celastrol is obtained from 5 L of initial culture suspension. Optionally, it may be crystallized to have absolute purity.

[0172] In parallel, part of the dry extract obtained 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 of the Celastrol-Enriched PCC Extract

[0173] In inflammatory dermatoses (notably in psoriasis), it is known that CD4+ T cells overexpress interleukins IL17, IL6 and IL22, as well as IFN-gamma and TNF-alpha. In the case of atopic dermatitis, IL17 is overexpressed in a certain phase of the disease. Furthermore, the Th17 pathway is strongly activated in acne. The inhibitory power of an enriched extract according to the invention on the overexpression of these molecules was tested.

[0174] Human CD4+ cells were isolated from blood mononuclear cells of 2 donors on Ficoll Paque Plus? according to the protocol recommended by the manufacturer (GE Healthcare). CD4+ lymphocytes are isolated by positive sorting using the Miltenyi Biotec kit (CD4) and an LS column and are resuspended in RPMI culture medium (Sigma-Aldrich: containing L-glutamine and 10% foetal calf serum) supplemented with 100 ?g/ml streptomycin and 100 U/ml penicillin. Suspended lymphocytes are distributed in microplate wells. Various extracts or controls are then added to the wells:

[0175] 1. negative control (the same volume of buffer without reagent is added);

[0176] 2. extract R003034 at 0.06 mg/ml (celastrol titre 9 ng/ml) (final concentration in the well);

[0177] 3. extract R003034 at 0.2 mg/ml (celastrol titre 30 ng/ml) (final concentration in the well);

[0178] 4. extract R003034 at 0.6 mg/ml (celastrol titre 90 ng/ml) (final concentration in the well);

[0179] 5. 2 ?M dexamethasone (positive control) (final concentration in the well).

[0180] After 2 hours of incubation at room temperature, the 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. Anti-CD3 and anti-CD28 antibodies are known to induce a Th17-type response.

[0181] Cytokines IL-17A, INF-gamma, IL-22, TNF-alpha and IL-6 were quantified in the supernatant of each tube by the multiplex immunoassay method (Jager et at 2003). The results obtained are based on 2 experiments with lymphocytes from 2 different donors. The results are presented in FIGS. 3 to 7 as a percentage of induction compared with the negative control (1). It can be seen that: [0182] positive control 5 (2 ?M dexamethasone) inhibits the overexpression of all the cytokines: IL-17A, INF-gamma, IL-22, TNF-alpha and IL-6; [0183] the extract according to the invention (2, 3 and 4) strongly inhibits the overexpression of cytokines IL-17A, INF-gamma, 1L-22, TNF-alpha and IL-6. The inhibition of IL-17A and TNF-alpha overexpression is dose-dependent. At the lowest dose, celastrol titre of 9 ng/ml, there is a 75% inhibition of IL-17A overexpression, an 80% inhibition of TNF-alpha overexpression, and an almost complete inhibition of INF-gamma, IL-22 and IL-6 overexpression.

[0184] This shows that extract R003034 has a very strong anti-Th17 activity, comparable or even superior to dexamethasone (2 ?M), which gives it a remarkable activity in the treatment of Th17-dependent diseases such as psoriasis, acne and atopic dermatitis.

Example 8

Comparison of Celastrol Yields Obtained from Tripterygium wilfordii Plant Cell Cultures

[0185] Ten cultures in Erlenmeyer flasks (volume=50 mL) were carried out in parallel, from the same Tripterygium wilfordii cell line, in duplicate under the same shaking, aeration and temperature conditions. At the end of the growth phase, the density of the suspension being maximal, the amount of fresh biomass weighed was equivalent (average weight of 340 g of fresh biomass/L) in each of the Erlenmeyer flasks. At that moment, the elicitors were added to the culture medium of the ten Erlenmeyer flasks at the concentrations indicated in the table below, in which the yield is expressed in mg of celastrol/kg of fresh biomass.

TABLE-US-00001 Reference examples Examples involving the use of a cocktail of 2 elicitors Erlenmeyer flask E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 MeJa (?M) 0 0 64 36 36 64 64 64 92 92 Chitin (g/L) 0 2 0 2 4.2 1.3 2.5 3.7 3.1 4.2 Yield 4.5 55 738 1730 2178 1506 1724 2025 1986 1878

[0186] The assay of suspended celastrol was performed by HPLC according to example 5, with a calibration curve prepared with control products.

[0187] The suspensions contained in Erlenmeyer flasks E1, E2, E3 were elicited with a control solution without any elicitor for E1, with chitin alone (2 g/L) for E2 and with MeJa alone (64 ?M) for E3. The MeJa concentration in E3 is equivalent to that described by Liu et al. 2016, i.e. 64 ?M.

[0188] The suspensions contained in Erlenmeyer flasks E4 to E10 were elicited at three different concentrations of MeJa (36, 64 and 92 ?M) and five different concentrations of chitin (1.3; 2; 2.5; 3.1 and 4.2 g/L).

[0189] It is noted that the celastrol yields obtained for E6, E7 and E8 are increased by 204%, 233% and 274%, respectively, compared with E3, the MeJa concentration being the same in these four Erlenmeyer flasks. An even better yield is observed for E5, in which the MeJa concentration is different.

Example 9

Inhibitory Activity Against NF?B

[0190] The transcription factor NF?B controls the expression of a large number of genes involved in regulation of the inflammatory response. In the inactive state, NF?B is sequestered in the cytoplasm by the IKB protein. Certain pro-inflammatory stimuli such as TNF-alpha and IL-1-beta lead to the activation of NF?B, i.e. its nuclear translocation. Once in the nucleus, NF?B will induce the transcription of pro-inflammatory genes encoding cytokines, chemokines, adhesion molecules, growth factors and inducible enzymes.

[0191] The anti-inflammatory activities of the extracts obtained from the suspensions contained in Erlenmeyer flasks E1, E2, E3 and E6 of example 8 were evaluated in vitro on human HaCaT keratinocytes according to the method described by Albanesi et al. The extracts, weighed in dry weight, were taken up in a buffer and introduced into a HaCaT cell culture solution in equivalent final concentrations (ng/mL) for each extract and incubated for 1 hour. NF?B expression is then induced by stimulation with TNF-alpha (0.3 ng/mL).

TABLE-US-00002 Extract Extract Extract Extract TNF- derived derived derived derived alpha Dex from E1 from E2 from E3 from E6 Conc. 0.3 2 ?M 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: Dexamethasone; Conc.: concentration (ng/mL); % inh: % inhibition of NF?B expression with respect to induction of TNF-alpha; sem: standard error of the mean.

[0192] Dexamethasone at 2 ?M, used as positive control, inhibits NF?B expression by more than 40%.

[0193] The extract according to the present invention (obtained from E6, with the pair of elicitors MeJa and chitin) evaluated at concentrations of 0.19 and 0.58 ng/mL inhibits NF?B expression by 20% and 70%, respectively.

[0194] The extract prepared according to Liu et al (from E3, with MeJa as sole elicitor) evaluated at the same concentrations does not induce any appreciable inhibition of NF?B expression. Similarly, extracts derived from E2 (with chitin as sole elicitor) and from the control E1 have no effect on NF?B expression.

[0195] Only the extract derived from elicited culture according to the present invention strongly inhibits NF?B, and in a concentration-dependent manner.

Example 10

Antimicrobial Activity Against Propionibacterium acnes of an Extract Prepared According to the Process of the Present Invention Compared with that of Other Compounds

[0196] Activity is evaluated on Propionibacterium acnes (CIP 53117T), with an inoculation suspension at 10.sup.8 CFU/mL. The maintenance medium is Columbia agar+5% sheep blood, with incubation for 24 hours at 36?1? C. under anaerobic conditions. The test medium is composed of Mueller Hinton broth+10% foetal calf serum (FCS) and Columbia agar+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 micro-method in liquid medium. 100 ?L of liquid culture medium is deposited in each well of a sterile 96-well microplate. 100 ?L of the product to be tested is deposited in the first well of a row. One-half dilutions are then made from wells 1 to 10. Propionibacterium acnes test suspensions are prepared extemporaneously in tryptone salt. 100 ?L is deposited in each well of a second microplate, except column 11. All of the first microplate is inoculated, using a Denley multipoint inoculator, from the second microplate. After incubation, MICs are defined as the highest dilution with no visible growth. Columns 11 and 12 serve as negative and positive growth control, respectively. Two references were tested in parallel. The determination of minimum bactericidal concentrations (MBCs) is performed by subculturing MIC microplates on agar medium using the Denley multipoint inoculator. After incubation, MBC is defined as the highest dilution with no visible growth. All tests are performed in duplicate.

[0197] The compounds tested are:

[0198] purified celastrol in DMSO (stock solution=150 ?g/mL/10% DMSO)

[0199] the extract prepared according to the process of the present invention in pentylene glycol (stock solution=2%/2% pentylene glycol)

[0200] tingenin A in DMSO (stock solution=150 ?g/mL/10% DMSO)

[0201] tingenin B in DMSO (stock solution=150 ?g/mL/10% DMSO)

[0202] the references are amoxicillin, DMSO (stock solution=100%), and pentylene glycol (stock solution=2%)

[0203] The results obtained are summarized in the table below, which shows the MIC and MBC values for the 4 samples and both excipients. The maximum test concentration corresponds to the concentration of the stock solution/2.

TABLE-US-00003 Compounds MIC (?g/mL) MBC (?g/mL) Celastrol 0.59 1.17 DMSO 5 5 Extract according to the process 0.25* 0.25* Pentylene glycol >1 >1 Tingenin A 2.34 2.34 Tingenin B 1.17 1.17 *or 0.375 ?g/mL in celastrol equivalent.

[0204] In conclusion, celastrol has a quite attractive antibacterial activity on Propionibacterium acnes, but it appears that it is the extract prepared according to the process of the invention that has the highest level of antibacterial activity on Propionibacterium acnes. Tingenin B has higher antibacterial activity than that observed with tingenin A.

Example 11

Antimicrobial Activity Against Staphylococcus aureus of an Extract Prepared According to the Process of the Present Invention Compared with that of Other Compounds

[0205] It is known that S. aureus is a pathogen which colonizes the lesions of patients with atopic dermatitis and that it is also present in patients with psoriasis (Tomi et al. 2005).

[0206] The antibacterial activity against S. aureus of the extract obtained according to the process of the present invention was evaluated. The experiment was performed as described in example 10, with the pathogenic strain S. aureus CIP 4.83, the maintenance medium Trypticase Soya Agar, the test medium MIC/MBC broth and Mueller Hinton agar and incubation at 36? C. for 24 h.

[0207] The results obtained are summarized in the table below. It can be seen that while celastrol has a high antibacterial activity, the anti-S. aureus activity of the extract obtained according to the process of the present invention titrated in celastrol equivalent is even better. Furthermore, the tingenin B molecule has a better activity than tingenin A.

TABLE-US-00004 Compounds MIC (?g/mL) MBC (?g/mL) Celastrol 0.29 0.59 DMSO >5 >5 Extract according to the process 0.125* 0.25** Pentylene glycol >1 >1 Tingenin A 1.17-2.34 2.34 Tingenin B 0.59 1.17 *0.1875 ?g/mL in celastrol equivalent; **0.375 ?g/mL in celastrol equivalent.

[0208] In conclusion, this extract has not only an attractive anti-Th17 pharmacological activity, but surprisingly a strong anti-S. aureus activity, which makes it very attractive as active agent in the topical treatment of atopic dermatitis and psoriasis.

Example 12

Cream for Topical Application

[0209]

TABLE-US-00005 Ingredients Mass percentage glycerine 8 to 10% hydroxyacrylate copolymer 1.8 to 2%.sup. xanthan gum 0.1 to 0.3% ceteareth 33 and cetearyl alcohol 4 to 6% glyceryl stearate 1.5 to 2%.sup. ethylhexyl palmitate 12 to 15% Tripterygium wilfordii extract (in 0.1 to 2.0% solution; 10 to 600 mg/L) glycolic acid 2 to 4% Lactamide MEA 3 to 6% Laureth-9 1 to 3% sodium shale oil sulphonate 1 to 3% chicory root extract 1 to 3% Water made up to .sup.100%

[0210] The Tripterygium wilfordii extract is prepared according to the present invention, its concentration being expressed as weight of dry extract in a solvent compatible with the formulation, such as olive oil, pentylene glycol or myritol 318 or others.

[0211] This exemplary formulation is by no means limiting and may be adapted according to the treatment.

[0212] Thus, for treatment of the scalp, a shampoo may be formulated by calling upon on the knowledge of the person skilled in the art.

REFERENCES

[0213] Camelio et al. JACS 2015, 137; 11864-67 [0214] Coppede et al. Plant Cell Tiss. Organ Cult. 2014, 118:33-43 [0215] Jager et al. Clin. Diagn. Lab. Immunol. 2003, 10(1):133-9 [0216] Kelhala et al. PLOS One 2014, 9(8):e105238 [0217] Liu et al. J. Asian Nat. Prod. Res. 2016, 19:1-10 [0218] Lowes et al. Annu. Rev. Immunol. 2014, 32:227 [0219] Miyagaki et al. J. Derm. Science 2015, 78:89 [0220] Murashige & Skoog Physiol. 1962, 15: 473-497 [0221] Albanesi et al. Curr. Drug Targets Inflamm. Allergy 2005, 4(3):329-334 [0222] Tomi et al. J. Am. Acad. Dermatol. 2005, 53(1):67-72.