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WO2025141142A1 - Nouveau procédé de récupération de composés limonoïdes - Google Patents

Nouveau procédé de récupération de composés limonoïdes Download PDF

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Publication number
WO2025141142A1
WO2025141142A1 PCT/EP2024/088540 EP2024088540W WO2025141142A1 WO 2025141142 A1 WO2025141142 A1 WO 2025141142A1 EP 2024088540 W EP2024088540 W EP 2024088540W WO 2025141142 A1 WO2025141142 A1 WO 2025141142A1
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Prior art keywords
plant
formula
cell
cells
compounds
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Blazej SLAZAK
Ulf GÖRANSSON
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Dicot Pharma AB
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Dicot Pharma AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • the present invention relates to a novel method for obtaining the limonoid compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof, cell products of the method and the use of said products and compounds.
  • Limonoids are compounds naturally produced by a variety of plants and trees. Limonoids consists of thousands of compounds of which some have shown to be pharmaceutically active compounds having an effect in the treatment and/or the prevention of sexual dysfunction such as erectile dysfunction and premature ejaculation (WO 2008/145966 A2 and WO 2013/110744 A2).
  • WO 2024/072288 Al relates to a process for the extraction of phragmalin derivatives from entandrophragma caudatum seed extracts and preparation of pharmaceutically active liminoids.
  • PCT/EP2024/073743 relates to compounds derived from phragmalin or part(s) thereof and new pharmaceutical compositions comprising said compounds as well as uses thereof in the treatment and/or prevention of sexual dysfunctions.
  • the document also describes a method for the preparation of the aforementioned compounds and compositions.
  • the present document is directed to an improved method for producing and recovering pharmaceutically active limonoid compounds or esters and/or derivatives thereof; products generated by this method and the use of these products.
  • the present document is directed to a method of obtaining at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof.
  • the method comprises culturing plant cells in a cell culture, wherein the cells produce the compound of Formula Ila and/or lib and/or one or more esters and/or derivatives thereof, and recovering said compounds from said cells and/or cell culture.
  • the plant cells are obtained or originate from an explant/s of the corresponding plant, said explant/s is cultured in a nutrient medium to provide friable callus material from which the plant cells are isolated.
  • the document also describes products of the method and/or cell culture.
  • the document also presents the use of said compound/s for producing other pharmaceutically active limonoid compound/s and its use in a medicament.
  • the present document is directed to a method of obtaining at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof:
  • R 1 is a hydrogen or a straight, branched or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with, for example, -OH, preferably R 1 is a methyl, wherein R 2 , R 3 and R 4 independently are selected from hydrogen, nicotinoyl or -C(O)R 5 ; where R 5 is alkyl, cycloalkyl, alkylene, aryl or heteroaryl such as nicotinyl, substituted or unsubstituted, having from one to six carbon atoms, one to five carbon atoms, more preferably one to four carbon atoms, alternatively R 2 , R 3 and R 4 independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso- pentanoyl or acyl, said method comprising :
  • step II recovering the compound of Formula Ila and/or lib and/or the one or more esters or derivatives thereof produced in step I.
  • the present document is also directed to the method wherein the method obtains at least one of the compounds of Formula la or lb:
  • R 1 is a straight, branched or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with e.g. OH or a halogen, such as F.
  • R 1 may be methyl.
  • derivatives we include the meaning of “structural analogs” (also known as “chemical analogs” or “analogs”), which are compounds having a structure similar to that of another compound, but differing from it in respect to a certain component.
  • the derivative may differ in one or more atoms, functional groups, or substructures, which are replaced with other atoms, groups, or substructures.
  • a structural analog can be imagined to be formed, at least theoretically, from the other compound.
  • the present document is also directed to the method wherein the plant cells are obtained or originate from an explant/s of the corresponding plant, said explant/s is cultured in a nutrient medium to provide friable callus material from which the plant cells are isolated.
  • the present document is also directed to the method where the explant/s of said plant is obtained from one part or multiple parts of said plant such as, but not limited to, callus, stem, leaves, roots, rhizome, fruit/s, flower/s, seeds, seedling/s, hypocotyl or a cotyledon, and/or a fragment/s thereof.
  • the present document is also directed to the method where the explant/s from said plant are cultured for 1-10 weeks or 2-8 weeks or 3-6 weeks or 2-5 weeks; are cultured at 15- 35°C or 17-33 °C or 19-31°C or 20-29°C or 22-27 °C or 25°C +/- 2°C, most preferably at 25°C +/- 2°C to obtain friable callus material from which plant cells suitable for inducing said plant cell culture can be isolated.
  • the present document is also directed to the method where the explant/s from said plant are cultured under controlled illumination.
  • the present document is also directed to the method where the nutrient media for culturing the plant cells and/or explant/s are supplemented with at least one or more plant hormone/s or growth regulator/s such as gibberellins, auxins, abscisins, ethylene, brassinosteroids, strigolactones and/or cytokinins, the plant hormone/s or growth regulator/s including but not limited to, indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), isopentenyladenine (IPA), N6-(A2- isopentenyl)adenin (iP), N6-(A2-isopentenyl)adenosine (iPR), salicylic acid (SA), polyamines such as spermidine, plant growth regulatory peptide hormones, nitric oxide (NO), strigolactone biosynthesis inhibitor (SBI), melatonin, 6-benzy
  • auxin plant hormone is at least one of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA) and the cytokinin plant hormones is any of kinetin (KIN) or thidiazuron (TDZ).
  • the present document is also directed to the method where two or more of said plant hormones or growth regulators are used in the method in any combination.
  • the present document is also directed to the method where the combination of plant hormones or growth regulators at least comprises: (i) 2,4-D and KIN; (ii) NAA and TDZ; or (iii) 2,4-D, KIN and IAA.
  • the present document is also directed to the method where at least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).
  • at least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).
  • the present document is also directed to the method where the concentration of each plant hormone/s or growth regulator/s is 0.01-10 mg/l in said nutrient media.
  • the present document is also directed to the method where the plant cells of the cell culture are cultured under controlled illumination.
  • the present document is also directed to the method where the plant cells of the cell culture are cultured at 15-35°C or 17-33 °C or 19-31°C or 20-29°C or 22-27 °C or 25°C +/- 2°C, most preferably at 25°C +/- 2°C.
  • the present document is also directed to the method where the plant cells of the cell culture are cultured for 1-10 weeks or 2-8 weeks or 3-6 weeks or 2-5 weeks.
  • the present document is also directed to the method wherein the plant cells in step I of the method are cultured as a suspension cell culture.
  • the present document is also directed to the method wherein said suspension cell culture is a continuous culture for growing cells in suspension.
  • the present document is also directed to the method where at least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, or at least one of the compounds of Formula la or lb, are recovered from the cell biomass fraction of the cell culture in step II of the method.
  • the present document is also directed to the method where at least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, or at least one of the compounds of Formula la or lb, are recovered from the non-cell biomass fraction of the cell culture in step II of the method.
  • the present document is also directed to the method where at least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, or at least one of the compounds of Formula la or lb, are recovered from the cell culture medium of the cell culture in step II of the method.
  • the present document is also directed to the method wherein said plant cells and/or said explant/s and/or fragments thereof originate from a plant selected from the Meliaceae family.
  • the present document is also directed to the method wherein said plant selected from the Meliaceae family is anyone of Xylocarpus granatum, Khaya senegalis, Xylocarpus moluccensis, Pseudocedrela kotschyi, Swietenia macrophylla, Neobeuga mahafalensis, Chukrasia tabularis or Entandrophragma caudatum.
  • the present document is also directed to the method wherein said plant selected from the Meliaceae family is from Swietenia macrophylla, Khaya senegalis, Neobeuga mahafalensis, Chukrasia tabularis or Entandrophragma caudatum; most preferably Entandrophragma caudatum.
  • the explant/s and/or fragments thereof do not originate from Neobeguea mahafalensis. In some embodiments, the explant/s and/or fragments thereof do not originate from Chukrasia tabularis.
  • the present document is also directed to an isolated plant cell, obtained from a method as disclosed herein.
  • the present document is also directed to an isolated plant cell line obtained or originating from the method.
  • the present document is also directed to an isolated plant cell batch obtained or originating from the method.
  • the present document is also directed to suspension cell culture obtained or originating from the isolated plant cell line or the isolated plant cell batch.
  • the plant cell produces, or is capable of producing, a compound of: (a) Formula Ila, Formula lib, and/or one or more esters or derivatives thereof; and/or (b) Formula la, Formula lb, and/or one or more esters or derivatives thereof.
  • the plant cell is a suspension cell culture.
  • plant cell we include the meaning of an isolated plant cell, an isolated plant cell line, an isolated plant cell batch, or a suspension cell culture (which may also be referred to as a suspension plant cell culture).
  • the plant cell may be induced into producing the compound under appropriate conditions.
  • the appropriate conditions may involve subjecting the plant cell to a method as described herein, such as by culturing the plant cell in a nutrient medium as a cell culture.
  • the present document is also directed to a plant cell culturing medium obtained or originating from the plant cell culture of a method as disclosed herein, from which the plant cells have been removed which comprises a non-cell biomass, derived from the plant cells.
  • the plant cell culturing medium comprises (or contains) a compound of: (a) Formula Ila, Formula lib, and/or one or more esters or derivatives thereof; and/or (b) Formula la, Formula lb, and/or one or more esters or derivatives thereof.
  • Any suitable method may be used to determine the presence or absence of such compounds, such as by mass spectrometry (e.g. LC-MS, as exemplified herein), HPLC-UV, HPLC-DAD, MS-MS, and/or NMR.
  • the culturing medium is sterile filtered and/or autoclaved.
  • the terms “culturing media” and “nutrient media” are used interchangeably herein.
  • the present document is also directed to a plant cell biomass comprising plant cells obtained from the friable callus material according to a method as disclosed herein, and/or a cell culture of a method as disclosed herein.
  • the plant cell biomass comprises plant cells that produce, or are capable of producing, a compound of: (a) Formula Ila, Formula lib, and/or one or more esters or derivatives thereof; and/or (b) Formula la, Formula lb, and/or one or more esters or derivatives thereof.
  • the biomass may be a suspension culture of plant cells.
  • the plant cell biomass comprises plant cells produce, or are capable of producing a compound of Formula la, wherein R 1 is methyl, and/or a compound of Formula lb, wherein R 1 is methyl.
  • the present document is also directed to an isolated explant/s or fragment/s thereof obtained or originating from a method as disclosed herein.
  • the isolated explant/s or fragment/s thereof comprises plant cells that produce, or are capable of producing, a compound of: (a) Formula Ila, Formula lib, and/or one or more esters or derivatives thereof; and/or (b) Formula la, Formula lb, and/or one or more esters or derivatives thereof.
  • the isolated explant/s or fragment/s thereof comprises plant cells that produce, or are capable of producing a compound of Formula la, wherein R1 is methyl, and/or a compound of Formula lb, wherein R 1 is methyl.
  • the present document is also directed to a compound as represented by Formula Ila or Formula lib or any esters or derivatives thereof, and/or a compound as represented by Formula la or lb.
  • the compound is obtained or obtainable by performing a method as disclosed herein; and/or recovered from a plant cell, a plant cell line, and/or a plant cell batch as disclosed herein; and/or recovered from a suspension cell culture, a cell culturing medium, and/or a non-cell biomass as disclosed herein; and/or recovered from a plant cell biomass as disclosed herein; and/or recovered from an explant/s as disclosed herein.
  • recovered from we include the meaning of “isolated from”, which enriches or purifies the concentration of the compound away from other material.
  • the present document is also directed to the method wherein the plant cells used for the cell culture in step I are obtained or originate from the isolated plant cell, plant cell line, or plant cell batch.
  • the present document is also directed to the use of the cell culturing medium or the noncell biomass, or the plant cell biomass as a medicament.
  • the invention describes a cell culturing medium, a non-cell biomass, and/or a plant cell biomass as described herein for use as a medicament or in medicine. In some embodiments, the invention describes the use of a cell culturing medium, a non- cell biomass, and/or a plant cell biomass as described herein for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disease, disorder or condition, optionally a metabolic disorder. In some embodiments, the invention describes a method of treatment with a cell culturing medium, a non-cell biomass, and/or a plant cell biomass as described herein in a subject in need thereof.
  • the present document is also directed to the use of a compound as represented by Formula Ila or Formula lib or any ester or derivative thereof, or a compound as represented by Formula la or lb, obtained or obtainable by the method, from the plant cell line and/or from the plant cell batch and/or from the suspension cell culture, from the cell culturing medium and/or non-cell biomass, or from the plant cell biomass; for producing a compound of Formula V, wherein R 1 preferably is methyl and R 2 is a straight, branched or cyclic optionally substituted alkyl group with 1 to 6 carbon atoms; or a compound of Formula VI.
  • R 1 preferably is methyl and R 2 is isopropyl.
  • the present document is also directed to the use of a compound of Formula V and/or a compound of Formula VI; optionally wherein the compound of Formula V or a compound of Formula VI is obtained from the use of a compound as represented by Formula Ila or Formula lib or any ester or derivative thereof, or a compound as represented by Formula la or lb, in a medicament.
  • Figure 1 shows a LC-MS graph of post-methanolysis extracts of Entandrophragma caudatum seeds (A) and a reference sample of purified SAE4a (B). The retention time for the limonoids SAE4a and SAE4 (Phragmalin) are highlighted.
  • Figure 2 shows a LC-MS graph of post-methanolysis extracts of Entandrophragma caudatum seeds (A) and two different callus samples: no 17 (B) and no 3 (C).
  • the retention time for the limonoids SAE4a and SAE4 (Phragmalin) are highlighted.
  • Figure 3 shows the average amounts (pg of compound/mg of post-methanolysis oil) of SAE 4a and SAE 4 in the post-methanolysis oil for tested calluses and seed samples. Bars indicate standard deviation.
  • Figure 4 shows the morphology of suspension cells grown in media no. Ill, with visible organelles and nuclei (n).
  • Figure 5 shows a LC-MS graph of post-methanolysis extract on cell biomass from cell suspension cultures comprising medium III.
  • a and B depict biomass extracts and C crude extracts.
  • Figure 6 shows a LC-MS graph of post-methanolysis extracts on cell culture medium from Entandrophragma caudatum cell suspension cultures comprising medium III.
  • Figure 7 shows photographic documentation of the cultures for three plates with 10 explants, which were prepared for each medium of Example 5.
  • Figure 8 shows photographic documentation of the cultures for three plates with 8 explants, which were prepared for each medium of Example 5.
  • Figure 9 shows LC-MS traces of the Entandofragma cantatum post-methanolysis seed extract (A) and purified SAE 4a (B) compound.
  • the peaks together with retention times (RT) are marked.
  • the numerals for the y-axis are from 0-100% intensity, and the numerals for the x-axis are from 0.80-6.80 minutes in 0.20 minute increments.
  • peaks are seen at 1.69, 2.09, 2.47, 3.07, 3.51, 3.92, 4.71, 5.10, 5.37, 5.55, and 6.06 minutes;
  • B peaks are seen at 1.59, 2.40, 3.04, 3.96 minutes; TOF MS ES+ BPI 2.43e7.
  • Figure 10 shows identification of a unique fragment signature to SAE 4 core molecule in MSe high-energy MSMS spectra LC-MS analysis of post- and premethanolysis extracts.
  • A postmethanolysis extract, the chromatographic peak corresponding to SAE 4 marked with an arrow.
  • B a chromatogram generated through mass filtering for a m/z 441.191 fragment, unique to SAE 4.
  • C LC-MSe chromatogram of the pre-methanolysis
  • E caudatum seed extract.
  • D a chromatogram generated through mass filtering for a m/z 441.191 fragment reveals compounds likely to share the core molecular structure with SAE 4.
  • the numerals for the y-axis are from 0-100% intensity, and the numerals for the x-axis are from 2.40-7.80 minutes in 0.20 minute increments.
  • Figure 11 shows the morphology of suspensions grown in media III (A, B) and X (C) with small, viable, dividing cells and small cell aggregates.
  • Figure 12 shows plant material of Entandrophragma caudatum after arrival on February 14th, 2024.1-9: explant plates; 10: suspension on filter; 11: T-flask containing cell suspension.
  • Figure 13 shows callus formation on E. caudatum explants (A), and isolated primary callus material on solid medium (B).
  • Figure 14 shows accumulated fresh weight of E. caudatum at end of growth cycles (vacuum-filtrated). Each dot represents the fresh weight amount accumulated at the end of a growth cycle, respectively.
  • Figure 15 shows development of cell morphology and colour of ent006 over time.
  • Figure 16 shows the growth index of ent006 over time.
  • One transfer cycle equals one week.
  • Figure 17 shows vacuum-filtrated biomass of the three individual flasks of ent006. Pictures show biomass from mature growth cycle s24 from 06.11.24. From left to right, the values indicated are 132.90 g, 132.87 g and 133.77 g, respectively.
  • Figure 18 shows microscopic images of E. caudatum.
  • A bright field microscopy
  • B fluorescent microscopy of cells stained by FDA
  • C overlay. Boxes indicate dead cells. Scale bar indicates 200 pm.
  • the present invention is directed to a method of obtaining at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof.
  • the method comprises culturing plant cells in a nutrient medium in a cell culture, wherein the cells produce the compound of Formula Ila and/or lib, and recovering said compounds from said cells and/or cell culture.
  • the plant cells are obtained or originate from an explant/s of the corresponding plant, said explant/s is cultured in a nutrient medium to provide friable callus material from which the plant cells are isolated.
  • the document also describes products of the method and/or cell culture. The document also presents the use of said compound/s for producing other pharmaceutically active limonoid compound/s and its use in a medicament.
  • the wording callus as used herein describes a growing mass of unorganized plant parenchyma cells.
  • callus culture used herein describes a plant tissue culture comprising said unorganized growing and dividing mass of callus cells.
  • friable callus used herein is meant to describe a specific class of callus which can be used to generate for instance cell suspension cultures.
  • explant used herein is describing a part and/or fragment of a plant taken to be cultured and grown in nutrient medium.
  • Limonoids consist of thousands of compounds of which some have shown to have an effect in the treatment and/or the prevention of sexual dysfunction such as erectile dysfunction and premature ejaculation (WO 2008/145966 A2 and WO 2013/110744 A2).
  • these therapeutically active limonoid compounds and precursors to these active compounds can be recovered from plants or part of plants, and further processed to prepare a pharmaceutical composition suitable for the treatment and/or prevention of sexual disorders.
  • current processes are lengthy, complex and give a low yield, making production inefficient.
  • method(s) allowing to produce compounds that may be used as a medicament such as a medicament for treating or preventing sexual dysfunction or serve as a starting material for manufacturing such compounds.
  • the present invention seeks to protect a method of obtaining at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof:
  • R 1 is a hydrogen or a straight, branched or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with, for example, -OH, preferably R 1 is a methyl, wherein R 2 , R 3 and R 4 independently are selected from hydrogen, nicotinoyl or -C(O)R 5 ; where R 5 is alkyl, cycloalkyl, alkylene, aryl or heteroaryl such as nicotinyl, substituted or unsubstituted, having from one to six carbon atoms, one to five carbon atoms, more preferably one to four carbon atoms, alternatively R 2 , R 3 and R 4 independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso- pentanoyl or acyl, said method comprising :
  • step II recovering the compound of Formula Ila and/or lib and/or the one or more esters or derivatives thereof produced in step I.
  • the compound of Formula Ila can have any possible molecular structure by combination of the varying groups R 1 , R 2 , R 3 , R 4 and R 5 as described in this document.
  • R 1 can be a hydrogen or a straight, branched, or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with, for example, -OH, preferably R 1 is a methyl.
  • R 2 , R 3 and R 4 independently are selected from hydrogen, nicotinoyl or -C(O)R 5 ; where R 5 is alkyl, cycloalkyl, alkylene, aryl or heteroaryl such as nicotinyl, substituted or unsubstituted, having from one to six carbon atoms, one to five carbon atoms, more preferably one to four carbon atoms, alternatively R 2 , R 3 and R 4 independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso-pentanoyl or acyl.
  • the compound of Formula lib can have any possible molecular structure by combination of the varying groups R 1 , R 2 , R 3 and R 5 as described in this document.
  • R 1 can be a hydrogen or a straight, branched, or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with, for example, -OH, preferably R 1 is a methyl.
  • R 2 , R 3 are independently selected from hydrogen, nicotinoyl or -C(O)R 5 ; where R 5 is alkyl, cycloalkyl, alkylene, aryl or heteroaryl such as nicotinyl, substituted or unsubstituted, having from one to six carbon atoms, one to five carbon atoms, more preferably one to four carbon atoms, alternatively R2 and R3 independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso-pentanoyl or acyl.
  • the present invention seeks to protect the method wherein the method obtains at least one of the compounds of Formula la or lb:
  • R 1 is a straight, branched or cyclic alkyl group with 1 to 6 carbon atoms that is unsubstituted or substituted with e.g. OH or a halogen, such as F.
  • R 1 is a methyl or a trifluoromethyl group.
  • Some of the limonoid compounds produced by the cells cultured in the method are compounds of Formula la and lb, where the compound of Formula la may be a precursor to the compound of Formula lb.
  • the cultured cells can be stimulated to produce more of the compound of Formula lb.
  • Examples of altering the culturing conditions are for instance adding additives, such as, but not limited to, one or more plant hormone/s or growth regulator/s, to said nutrient medium; and/or by controlling the illumination of said cells being cultured; and/or by extending or shortening the cell culture time; and/or optimizing the temperature of the cell culture and/or optimizing the cell density in the cell culture.
  • the culturing of plant cells in step I of the method comprises culturing plant cells in a nutrient medium in a cell culture with the purpose to optimize the cells production of the compound of Formula Ila and/or lib and/or one or more esters or derivatives thereof.
  • the purpose of culturing the plant cells in step I of the method is to optimize the cells production of the compound of Formula la and/or lb.
  • cell culture is meant any kind of culture of cell/s such as but not limited to a primary or secondary cell culture or a cell line.
  • culturing is meant that the cells of the cell culture are proliferate for a limited, extended, or unlimited time when provided optimal conditions to do so.
  • the cells of the invention can have a limited lifespan such that they can only proliferate and be cultured for a limited/finite time and/or amount of cell cycles.
  • the cells of the cell culture can be a finite cell line.
  • the cells of the invention can have an unlimited lifespan, such as a continuous cell line, secondary cell culture and/or an immortalized cell line.
  • the cells of the invention cultured in step I of the method can be adherent cells, suspension cells and/or a mix thereof.
  • the cells of the cell culture in step I of the method are adherent cells.
  • the cells of the cell culture in step I of the method are suspension cells.
  • the cells of the cell culture in step I of the method are suspension cells cultured as a suspension cell culture.
  • said suspension cell culture is a continuous culture for growing cells in suspension.
  • the cells of the cell culture in step I of the method are a mix between adherent and suspension cells.
  • the cell culture of the method herein can be of batch or fed-batch type.
  • the batch type being a culture where cells, media, nutrients, and other additives (such as, but not limited to, hormones and/or growth regulators) are added to a suitable cell culturing container such as, but not limited to, flasks, bottles, roller-bottles, plates, bag/s or bioreactor/s, and are cultured under optimal cell culturing conditions to achieve optimal production and/or yield of compounds of Formula Ila and/or Formula lib and/or esters and/or derivates thereof, or at least one of the compounds of Formula la or lb as mentioned herein, without anything being added to or removed from the culture during the period of time said cell culture is ongoing (from the initiation of said culture until recovery of the cell culture).
  • the culture comprising the cultured cells and/or culturing medium and/or explant/s
  • This batch can then be divided up in separated fractions such as cell biomass, comprising the cells, and the culturing medium, comprising non-cell biomass and/or compounds/molecules excreted/released from the cultured cell/s, using standard methods.
  • fractions can then be further processed to recover the compounds of Formula Ila and/or Formula lib and/or esters and/or derivates thereof, or one of or both of the compounds of Formula la and lb.
  • the fed-batch type of culturing cells involves adding nutrients and other additives (such as, but not limited to, hormones and/or growth regulators), to an already started and active cell culture, at different timepoints or time intervals to optimize the continuous proliferation of the cells and/or the production and/or yield of the compounds of Formula Ila and/or Formula lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb, as mentioned herein.
  • This type of cell culturing is also known as continuous cell culture or perfusion cell culture.
  • the cells are maintained in a viable and/or productive state for continuous production of any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb, as mentioned herein.
  • the produced compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb, described herein can be recovered from the cell culture either after the cell culture is stopped and/or the cells of the culture are no longer proliferating and/or producing any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb.
  • said compounds and/or esters or derivatives thereof can be recovered during said cell culture is ongoing, i.e when the cells of the culture are proliferating and/or producing any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof and/or compounds of Formula la and/or lb.
  • the plant cells of the invention are cultured under optimal conditions for the purpose of sustaining the cell culture, allowing the cells of the culture to proliferate and produce a compound of Formula Ila and/or lib and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb, as described herein, giving as high yield as possible of said compounds and/or one or more esters and/or derivatives thereof.
  • Factors affecting these optimal conditions include, but is not limited to, culturing the cells of the invention in nutrient media adapted for the type of cells or the mix of type of cells being cultured, providing suitable growth regulators and/or hormones and optimal concentration of these growth regulators and/or hormones, for said above purpose, culturing the cells in an suitable temperature, moisture content, maintaining the cells at a cell density enabling said cell proliferation and/or production of said compounds, providing an optimal gaseous atmosphere and a suitable illumination (light and/or darkness exposure).
  • the nutrient media for culturing the plant cells and/or explant/s are supplemented with at least one or more plant hormone/s and/or growth regulator/s.
  • suitable hormone/s and/or growth regulator/s of the invention are exemplified with, but should not be viewed as an exhaustive list, gibberellins, auxins, abscisins, ethylene, brassinosteroids, strigolactones and/or cytokinins, the plant hormone/s or growth regulator/s including but not limited to, indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), isopentenyladenine (IPA), N6-(A2-isopentenyl)adenin (iP), N6-(A2-isopentenyl)adenosine (iPR), salicylic acid (SA), polyamines such as spermidine, plant growth regulatory
  • IAA indole
  • the auxin plant hormone is at least one of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) or indole- 3-acetic acid (IAA) and the cytokinin plant hormones is any of kinetin (KIN) or thidiazuron (TDZ).
  • two or more of said plant hormones and/or growth regulators are used in the method in any combination.
  • the combination of plant hormones or growth regulators at least comprises: (i) 2,4-D and KIN; (ii) NAA and TDZ; or (iii) 2,4- D, KIN and IAA.
  • At least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).
  • the concentration of said plant hormones and/or growth regulators need to be kept at an optimal concentration throughout the culturing of said plant cells.
  • concentration of said plant hormones and/or growth regulators need to be kept at an optimal concentration throughout the culturing of said plant cells.
  • Many different factors affect what is an optimal concentration in a cell culture. For example, what plant hormone or growth regulator is used, what cells are cultured, the origin of these cells, the composition of the nutrient media and the type of culture are factors which affect what concentration is optimal. Therefore, optimal concentrations often must be determined by thorough testing before starting production.
  • the concentration of each plant hormone/s and/or growth regulator/s is 0.01-10 mg/l in said nutrient media, such as 0.03- 10 mg/l, 0.05-10 mg/l, 0.1-10 mg/l, 0.5-10 mg/l, 1-10 mg/l, or 2-5 mg/l in said nutrient media.
  • the access to light can affect the proliferation of the cultured cells and/or the yield of compounds produced by said plant cells.
  • the effect of light stimulation varies. Some plant cells show optimal proliferation and compound production when cultured in darkness as other plant cells need regular photo stimulation for optimal proliferation and compound production.
  • the plant cells of the cell culture are cultured under controlled illumination.
  • illumination is meant the exposure to light and with controlled illumination herein is meant that the cultured cells exposure to light is controlled and adapted to the specific needs of the cultured plant cell/s. This need can vary and depend on what plant cell/s (plant origin), what type of plant cells (stem, leaves, root, rhizome, fruits, seeds etc.) that are cultured but also the setup of said cell culture.
  • the cells exposure to light/photoperiod is optimally tested before the start of culturing the cells for production of compound/s.
  • photoperiod is meant the total time a plant is exposed to light/illumination per 24h.
  • the plant cells of the method of the invention can be cultured with a photoperiod from 0- 24h.
  • the plant cells of the cell culture are cultured in darkness with a Oh photoperiod. In one embodiment of the method of the invention the plant cells of the cell culture are cultured with a lh, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, llh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h photoperiod.
  • the plant cells of the cell culture are cultured with a l-24h photoperiod, such as a 2-22h, a 4-20h, a 6-18h, or an 8-16h photoperiod.
  • a l-24h photoperiod such as a 2-22h, a 4-20h, a 6-18h, or an 8-16h photoperiod.
  • the cells of the culture are first cultured in darkness for an optimal amount of time before being cultured in any of the above-described photoperiods.
  • the cells of the culture are first cultured in a photoperiod as described herein before being cultured in darkness for an optimal amount of time.
  • the optimal amount of time for culturing cells in darkness depends on the cells and need to be tested. Therefore, the time cells are cultured in darkness can vary between one day to several weeks.
  • any photoperiod, from l-23h, can be spread out over the total 24h of a day.
  • said plant cells and/or explant/s as described herein can be illuminated in one or more intervals, over a total of 24h, each interval comprising a period of light and a period of darkness.
  • These intervals of light/dark periods can be adjusted to optimize the proliferation of the cell/s and/or the explant/s of the invention and/or the optimization of the production and yield of the compounds of Formula Ila and/or lib, and/or Formula la and/or lb as described herein.
  • the culturing time of the cells herein can vary from a few days to one or several weeks, such as 1 week-10 weeks. It is considered obvious that said photoperiods relate to the total amount of light exposure said cell/s receive per 24h during these days or 1-10 weeks of culturing.
  • Cell cultures need to be maintained at an optimal temperature for the cultured cells to proliferate. Depending on the origin of the cells (plant species, animal species, microbial species etc) and what type of cell it is the optimal temperature can vary. Plant cells can therefore be cultured at a variety of temperatures.
  • the plant cells of the cell culture are cultured at 15-35°C, such as at 17-33°C, at 19-31°C, at 20-29°C, or at 22-27°C.
  • the plant cells of the cell culture are cultured at 25°C +/- 2°C, most preferably at 25°C +/- 2°C.
  • the duration of a plant cell culture varies depending on tissue or cell sample.
  • the culturing time can vary as some cultures take longer due to that the cells need to adapt to the in vitro conditions, establish in the nutrient medium and start proliferating. Also, as some cells take longer to establish and start proliferating extra time might also be needed for the production to start of compound/s produced by said plant cells.
  • a culturing time is also determined by how long a cell culture can be kept active producing compound/s of interest. Therefore, in one embodiment of the method of the invention the plant cells of the cell culture are cultured for 1-10 weeks, such as at for 2-8 weeks, for 3-6 weeks, or for 2-5 weeks.
  • the cells of the invention can be cultured in any suitable medium for culturing plant cells.
  • a suitable medium comprises additives to support the growth and/or proliferation of the cells cultured and promote the production of the compound/s of Formula Ila and/or lib, and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb.
  • Additives are exemplified with, but not limited to, nutrients, plant hormone/s or growth regulator/s.
  • cells of the invention can be cultured in MS basal media complemented with plant hormones and/or growth regulator/s of the invention.
  • cell culturing conditions can be optimized to achieve maximum yield of any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb, as mentioned herein.
  • the compound of Formula Ila and/or lib and/or the one or more esters or derivatives thereof, and/or compounds of Formula la and/or lb, produced by the plant cells in the cell culture of step I in the method are recovered in step II of the method using any suitable method known to the skilled person.
  • “recovered” is meant that the compound of Formula Ila and/or lib and/or the one or more esters or derivatives thereof, and/or compounds of Formula la and/or lb, are extracted and isolated from the cultured cells of step I and/or the cell culture medium in which the cells have been cultured and/or the noncell biomass of said culture, through suitable extraction methods.
  • the produced compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb, described herein can be recovered from the cell culture either after the cell culture is stopped and/or the cells of the culture are no longer proliferating and/or producing any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb.
  • said compounds and/or esters or derivatives thereof can be recovered during said cell culture is ongoing, when the cells of the culture are either proliferating or is alive but in a non-proliferating state and/or is producing any of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb.
  • the recovery of the compound of Formula Ila and/or lib and/or esters and/or derivates thereof, and/or compounds of Formula la and/or lb can be performed with any suitable method comprising first harvesting the cultured cells and/or the cell culture media in which the cells have been cultured and/or the non-cell biomass of said culture and then performing a suitable extraction method on the harvested material, such as organic solvent extraction, followed by methanolysis.
  • said suitable extraction method comprises methanol extraction, followed by methanolysis.
  • At least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb, are recovered from the cell biomass fraction of the cell culture in step II of the method.
  • At least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb, are recovered from the non-cell biomass fraction of the cell culture in step II of the method.
  • At least one of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb, are recovered from the cell culture medium of the cell culture in step II of the method.
  • the plant cells of step I of the method can be cells obtained and/or originating from an earlier cell culture, such as a primary or secondary cell culture or a cell line as described above.
  • the plant cells of step I of the method can be cells obtained and/or originating from an explant of the same and/or corresponding plant.
  • Said explant is first cultured in a nutrient medium to provide friable callus material from which a plant cell/s are isolated.
  • This isolated cell/s are then cultured in a nutrient medium in a cell culture of step I of the method.
  • Said cultures comprising the explant/s can be optimized using the culturing conditions as described herein.
  • the plant cells are obtained or originate from an explant/s of the corresponding plant, said explant/s is cultured in a nutrient medium to provide friable callus material from which the plant cells are isolated.
  • the explant/s of said plant is obtained from one part or multiple parts of said plant such as, but not limited to, callus, stem, leaves, roots, rhizome, fruit/s, flower/s, seeds, seedling/s, hypocotyl or a cotyledon, and/or a fragment/s thereof.
  • the explant/s of said plant is obtained from fresh seeds that first has been germinated.
  • the purpose of culturing said explants in/on nutrient medium is to provide formation of friable callus from which plant cells, suitable for further culturing in a cell culture of the method, can be isolated.
  • culturing conditions can vary. Examples of parameters affecting the culturing of explants are for instance cell medium composition, medium additives such as plant hormone/s and/or growth regulators, culture temperature, illumination, and culture time.
  • explants of the invention can be cultured on a solid nutrient medium, such as a nutrient medium comprising a suitable amount of agarose in addition to the other medium components.
  • a solid nutrient medium such as a nutrient medium comprising a suitable amount of agarose in addition to the other medium components.
  • explants can be cultured in a liquid nutrient medium, completely or partially submerged in the medium. In another embodiment of the invention explants can be cultured first on a solid nutrient medium and then in a liquid nutrient medium.
  • the explant/s from said plant are cultured for 1-10 weeks or 2-8 weeks or 3-6 weeks or 2-5 weeks to obtain friable callus material from which plant cells suitable for inducing said plant cell culture can be isolated.
  • the explant/s from said plant are cultured at 15-35°C or 17-33°C or 19-31°C or 20-29°C or 22-27°C or 25°C +/- 2°C, most preferably at 25°C +/- 2°C to obtain friable callus material from which plant cells suitable for inducing said plant cell culture can be isolated.
  • the explant/s from said plant are cultured under controlled illumination.
  • Explants of the invention can be cultured in a nutrient medium comprising plant hormone/s and/or growth regulators.
  • the plant hormone/s and/or growth regulators are added to the medium to improve growth and/or proliferation of the cells of the explant and to stimulate the formation of a friable callus comprising cell/s which can be isolated said cell/s are suitable for and used for inducing a cell culture.
  • the explant/s of the invention can be cultured in any suitable medium for culturing explant/s.
  • a suitable medium comprises additives to support the growth and/or proliferation of the cells of the explant/s cultured and promote the production of the compound/s of Formula Ila or lib, and/or one or more esters and/or derivatives thereof, and/or compounds of Formula la and/or lb.
  • Additives are exemplified with, but not limited to, nutrients, plant hormone/s and/or growth regulator/s.
  • explant/s of the invention can be cultured in MS basal media complemented with, sucrose, plant hormones and/or growth regulator/s of the invention.
  • a solid nutrient medium agarose is also added to the culturing medium to solidify said medium allowing the explant/s to be cultured on the nutrient medium to provide a friable callus material from which the plant cells are isolated.
  • the nutrient media for culturing the explant/s are supplemented with at least one or more plant hormone/s or growth regulator/s.
  • suitable plant hormone/s and/or growth regulator/s of the invention are exemplified with, but should not be viewed as an exhaustive list, gibberellins, auxins, abscisins, ethylene, brassinosteroids, strigolactones and/or cytokinins, the plant hormone/s or growth regulator/s including but not limited to, indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), isopentenyladenine (IPA), N6-(A2-isopentenyl)adenin (iP), N6-(A2-isopentenyl)adenosine (iPR), salicylic acid (SA), polyamines such as spermidine, plant growth regulatory peptide hormones,
  • the auxin plant hormone is at least one of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) or indole- 3-acetic acid (IAA) and the cytokinin plant hormones is any of kinetin (KIN) or thidiazuron (TDZ).
  • two or more of said plant hormones and/or growth regulators are used in the method in any combination.
  • the combination of plant hormones or growth regulators at least comprises: (i) 2,4-D and KIN; (ii) NAA and TDZ; or (iii) 2,4- D, KIN and IAA.
  • At least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).
  • Plant hormone and growth regulator concentrations Plant hormone and growth regulator concentrations:
  • the concentration of said plant hormones and/or growth regulators need to be kept at an optimal concentration throughout the culturing of said plant cells.
  • concentration of said plant hormones and/or growth regulators need to be kept at an optimal concentration throughout the culturing of said plant cells.
  • Many different factors affect what is an optimal concentration in a cell culture. For example, what plant hormone or growth regulator is used, what cells are cultured, the origin of these cells, the composition of the nutrient media and the type of culture are factors which affect what concentration is optimal. Therefore, optimal concentrations often must be determined by thorough testing before starting production.
  • the concentration of each plant hormone/s and/or growth regulator/s is 0.01-10 mg/l in said nutrient media, such as 0.03- 10 mg/l, 0.05-10 mg/l, 0.1-10 mg/l, 0.5-10 mg/l, 1-10 mg/l, or 2-5 mg/l in said nutrient media.
  • the access to light can affect the growth and/or proliferation of the cells therein and/or stimulate the formation of a friable callus comprising cell/s which can be used for inducing a cell culture.
  • the effect of light stimulation varies depending on what type of explant that is cultured.
  • the growth and/or proliferation of the cells therein and/or the stimulation of the formation of a friable callus is optimal in darkness as other explants need regular photo stimulation/illumination for optimal proliferation and/or stimulation of formation of a friable callus.
  • the explant/s from said plant are cultured under controlled illumination.
  • illumination is meant the exposure to light and with controlled illumination herein is meant that the cultured explant/s exposure to light is controlled and adapted to the specific needs of the cultured explant/s. This need can vary and depend on plant origin, what type of explants (e.g. callus, stem, leaves, roots, rhizome, fruit/s, flower/s, seeds, seedling/s, hypocotyl, or a cotyledon, and/or a fragment/s thereof) that are cultured but also the setup of said cell culture.
  • explants e.g. callus, stem, leaves, roots, rhizome, fruit/s, flower/s, seeds, seedling/s, hypocotyl, or a cotyledon, and/or a fragment/s thereof
  • the explant/s exposure to light/photoperiod is optimally tested before the start of culturing the cells for production of compound/s.
  • the explant/s of the method of the invention can be cultured with a photoperiod from 0- 24h. In one embodiment of the method of the invention the explant/s are cultured in darkness with a Oh photoperiod.
  • the explant/s are cultured with a lh, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, Uh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h photoperiod.
  • the explant/s are cultured with a l-24h photoperiod, such as a 2-22h, a 4-20h, a 6-18h, or an 8-16h photoperiod.
  • the explant/s of the culture are first cultured in darkness for an optimal amount of time before being cultured in any of the above-described photoperiods.
  • the explant/s of the culture are first cultured in a photoperiod as described herein before being cultured in darkness for an optimal amount of time.
  • the optimal amount of time for culturing explant/s in darkness depends on the explant/s and need to be tested. Therefore, the time explants are cultured in darkness can vary between one day to several weeks.
  • any photoperiod, from l-23h, can be spread out over the total 24h of a day.
  • said explant/s as described herein can be illuminated in one or more intervals, over a total of 24h, each interval comprising a period of light and a period of darkness.
  • These intervals of light/dark periods can be adjusted to optimize the proliferation of the cell/s and/or the explant/s of the invention and/or the optimization of the production and yield of the compounds of Formula Ila and/or lib, and/or Formula la and/or lb as described herein.
  • the culturing time of the explants herein can vary such as from 1 week-10 weeks. It is considered obvious that said photoperiods relate to the total amount of light exposure said explant/s receive per 24h during these 1-10 weeks of culturing.
  • the plant cell/s and/or the explant/s of the invention comprises cells that comprises and/or are capable of producing at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof, and/or one of the compounds of Formula la or lb, as described herein.
  • These cell/s and/or explants can originate from different plants of different plant families or originate from different plants of the same plant family. With originate from herein is meant that the plant cell/s and/or the explant/s of the invention at some point in time have been isolated from the plant. Thus, the cell/s can have been isolated from said plant at an earlier point in time and kept alive for future use or the cell/s have been isolated more recently to be used subsequently.
  • said plant cells and/or said explant/s and/or fragments thereof originate from a plant selected from the Meliaceae family.
  • said plant selected from the Meliaceae family is anyone of Xylocarpus granatum, Khaya senegalis, Xylocarpus moluccensis, Pseudocedrela kotschyi, Swietenia macrophylla, Neobeuga mahafalensis, Chukrasia tabularis or Entandrophragma caudatum.
  • said plant selected from the Meliaceae family is from Swietenia macrophylla, Khaya senegalis, Neobeuga mahafalensis, Chukrasia tabularis or Entandrophragma caudatum; most preferably Entandrophragma caudatum.
  • said plant selected from the Meliaceae family is from Swietenia macrophylla or Entandrophragma caudatum.
  • said plant selected from the Meliaceae family is Entandrophragma caudatum.
  • the method of the invention enables a faster and more efficient way of obtaining at least one of the compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof.
  • the method of the invention enables a faster and more efficient way of obtaining at least one of the compounds of Formula la or lb.
  • These products comprising said isolated plant cell, isolated plant cell line, isolated plant cell batch, suspension culture, plant cell culturing medium, plant cell biomass, isolated explant/s, are all products of an artificial method, not occurring in nature, executed in a laboratory setting comprising a combination of innovative plant cell proliferative stimuli (e.g plant hormones and/or growth regulators, controlled illumination, controlled temperature etc.) enabling artificial proliferation and increased production of the compounds as presented in this invention. Said stimuli can also be used to alter the relative ratio of the compounds produced in the cell/s of the invention.
  • innovative plant cell proliferative stimuli e.g plant hormones and/or growth regulators, controlled illumination, controlled temperature etc.
  • Said stimuli can also be used to alter the relative ratio of the compounds produced in the cell/s of the invention.
  • the invention also seeks to protect an isolated plant cell, obtained from the method.
  • the invention seeks to protect an isolated plant cell line obtained or originating from the method.
  • the invention seeks to protect an isolated plant cell batch obtained or originating from the method.
  • the invention seeks to protect suspension cell culture obtained or originating from the isolated plant cell line or the isolated plant cell batch. In another embodiment the invention seeks to protect a plant cell culturing medium obtained or originating from the plant cell culture of the method, from which the plant cells have been removed, which comprises non-cell biomass, derived from the plant cells.
  • non-cell biomass any biomass, other than the cells, obtained or originating from the cell culture of the method.
  • non-cell biomass is, any of the compounds of Formula Ila or lib and/or one or more esters and/or derivatives thereof and/or one of the compounds of Formula la or lb as presented herein.
  • non-cell biomass examples include, but not limited to, cellular fragments, intracellular structures, organelles, nuclei, vesicles, microsomes, exosomes, lipid structures, proteins, peptides, polysaccharides, compounds and/or molecules.
  • the invention seeks to protect a plant cell culturing medium obtained or originating from the plant cell culture of the method, from which the plant cells have been removed.
  • the invention seeks to protect a plant cell biomass comprising plant cells obtained from the friable callus material according to the method and/or the cell culture of the method.
  • the invention seeks to protect an isolated explant/s or fragment/s thereof obtained or originating from the method.
  • the invention seeks to protect a compound as represented by Formula Ila or Formula lib or any esters or derivatives thereof obtained or obtainable by performing the method; and/or recovered from the plant cell, the plant cell line, the plant cell batch; and/or recovered from the suspension cell culture, the cell culturing medium, the non-cell biomass; and/or recovered from the plant cell biomass; and/or the explant/s.
  • the plant cell/s of the invention used for a new cell culture, and for step I of the method herein can be obtained from an isolated plant cell, plant cell line, and/or plant cell batch, established and retrieved from a separate previous cell culture of the method herein.
  • said plant cell/s can also be obtained from an explant/s of the method herein.
  • the invention seeks to protect the method where the plant cells used for the cell culture in step I are obtained or originate from the isolated plant cell, plant cell line, or plant cell batch.
  • Some limonoids produced by a variety of plants and/or trees have shown to be pharmaceutically active compounds having an effect in the treatment and/or the prevention of sexual dysfunction such as erectile dysfunction and premature ejaculation (WO 2008/145966 A2 and WO 2013/110744 A2).
  • the invention seeks to protect the use of the cell culturing medium, the non-cell biomass, or the plant cell biomass as a medicament.
  • the invention seeks to protect the use of a compound as represented by Formula Ila or Formula lib or any ester or derivative thereof, or a compound as represented by Formula la or lb, obtained or obtainable by the method, from the plant cell line and/or from the plant cell batch and/or from the suspension cell culture, from the culturing medium and/or non-cell biomass, and/or from the plant cell biomass; for producing a compound of Formula V, wherein R 1 preferably is methyl and R 2 is a straight, branched or cyclic optionally substituted alkyl group with 1 to 6 carbon atoms; or a compound of Formula VI:
  • a compound of Formula V wherein R 1 is methyl and R 2 is isopropyl.
  • R 1 preferably is methyl and R 2 is isopropyl.
  • the compound of Formula VI is a monohydrate of the compound of Formula V, wherein R 1 is methyl and R 2 is isopropyl.
  • the invention seeks to protect the use of a compound of Formula V or a compound of Formula VI, obtained from the use as described here above, in a medicament.
  • the compounds of described herein may be depicted as a stereoisomer.
  • phragmalin may be depicted as
  • the described invention herein presents a novel method for obtaining at least the compounds of Formula Ila and/or lib, and/or one or more esters and/or derivatives thereof, and/or the compounds of Formula la and/or lb, by culturing plant cells, capable of producing these compounds, in a suitable nutrient medium to facilitate the synthesis of these compounds in the cultured cells before recovering said compounds.
  • said cells are retrieved from friable calli material produced by first culturing explant/s taken from plants of the Meliaceae family.
  • explant/s and/or cells of the invention are cultured in the presence of plant hormones and/or growth regulators enabling the explant/s and the cells of the method to produce compounds of Formula Ila or lib, and/or one or more esters and/or derivatives thereof, and/or the compounds of Formula la and/or lb.
  • This invention solves the problems of current methods used for retrieving limonoid compounds from plants comprising harvesting vast amounts of biomaterial such as plants, trees and seeds and extracting the compounds from this biomaterial in complex and lengthy processes often resulting in a low yield of said compounds.
  • the method presented herein can be scaled up in a convenient way to enable large scale production of said compounds allowing a more efficient and cost-effective way of production. This will in turn enable further processes for production of pharmaceutically active limonoids needed in the treatment and/or prevention of disease and/or dysfunctions, such as, but not limited to sexual dysfunctions.
  • SAE4a the compound of Formula la, wherein R 1 is methyl
  • SAE-4a the compound of Formula lb, wherein R 1 is methyl
  • SAE4a the compound of Formula III
  • SAE4a may be referred to as a compound of Formula IV.
  • Plant material Seeds from the plant Entandrophragma caudatum and Chukrasia tabularis were used for germination. The seeds used had the following specifications.
  • the seeds were prepared by removal of the wing and the skin and surface sterilized by 1 min treatment with 70% ethanol, followed by 15 min in 50% ACE commercial bleach and 3 times washing with deionized water. All seeds were sowed onto % MS basal media solidified with 8 g/L agarose (Sigma), pH 5.7, autoclaved, in plastic transparent boxes sterilized with UV. The seeds were kept in darkness for the first week and then in 16h/8h day/night photoperiod, 25°C ⁇ 2°C. These treatments resulted in >90% germination and no infections.
  • Entandrophragma caudatum and Chukrasia tabularis seedlings germinated in sterile conditions were used to initiate the culture.
  • Cultures were maintained on MS basal media supplemented with 30 g/L sucrose, different combinations of plant growth regulators (Table 1) and solidified with 8 g/L agarose. The cultures were maintained in 16h/8h day/night photoperiod, 25°C ⁇ 2°C.
  • Table 1 The combinations of plant growth regulators used in Entandrophragma caudatum and Chukrasia tabularis in vitro callus cultures trials.
  • Table 2 Entandrophragma caudatum cotyledon callus cultures overview. Three plates with 10 explants each (30 explants overall) were prepared for each medium.
  • the callus samples were harvested, divided into three replicates and freeze-dried. Three replicates from each of the calli were prepared and labeled with an Arabic number corresponding to the Roman number ID of the media and the replicates A, B, and C (e.g. 1A, IB, 1C are three replicates for the callus obtained on medium I). For Entandrophragma caudatum the calli from media V-VIII were small, with signs of necrosis, and were not analyzed any further.
  • Post methanolysis samples for LC-MS and HPLC-UV analysis were prepared by dissolving the post-methanolysis oil in methanol (1 mg of post-methanolysis oil dry weight/ml).
  • the post-methanolysis reaction mixture was cooled to 30°C, and acetic acid was added to stop the reaction (2 mmol per g of the initial oil dry weight). The mixture was stirred for 30min at 30°C, followed by adding water and stirring for 30 minutes at 30°C. Subsequently the methanol was evaporated, and the remaining residue was extracted with ethyl acetate. The organic phase was separated, evaporated to dryness (post methanolysis oil) and weighted. Overall, 10 methanolysis reactions were carried over. The extraction and methanolysis conditions are outlined in Table 8.
  • the samples were analyzed using the UPLC-MS system: Waters nanoAcquity (all methanol extracts) or ACQUITY Premier UPLC (all samples after methanolysis) -Xevo G2-XS QTof Quadrupole Time-of-Flight MS, Waters.
  • the UPLC system operated with a C18 column and the gradient of 5%-95% ACN in water, 0.1% FA.
  • the mass spectra were collected in the MSe positive ion mode, in a window of 100 m/z - 1000 m/z. All post-methanolysis samples from the A series of Entandrophragma caudatum (Table 5) were analyzed with LC-MS.
  • Fig. 1 shows LC-MS traces of the Entandrophragma caudatum post-methanolysis seed extract (A) and purified SAE 4a (B) compound. The peaks together with retention times (RT) are marked. Note that “phragmalin” in this figure is also referred to as SAE 4 herein. Note that seed extract (A) is referred to as raw A* in Table 5.
  • Mass chromatograms for each of the compounds were generated using the compound characteristics (Table 9) and mass filtering tool.
  • Phragmalin/SAE4 (compound of Formula la, wherein R 1 is methyl) was detected in seeds of Khaya senegalis, Entandrophragma caudatum, Cedrela montana, Chukrasia tabularis and Swietenia macrophylla seeds with the highest relative abundance in seeds of Chukrasia tabularis. Seeds of Entandrophragma caudatum was the only tested seed which also comprises SAE4a (compound of Formula lb, wherein R 1 is methyl) (Table 10).
  • Phragmalin/SAE4 (compound of Formula la, wherein R 1 is methyl) was present also in some callus samples of Chukrasia tabularis with the highest abundance, comparable to content in the seeds, in callus grown on media III, V and VI.
  • the quantitative analysis was performed using HPLC-UV and an external standard calibration curve. All analyses were performed with LC-20 Shimadzu HPLC system equipped with a PDA detector and a C18 Kinetex 100 A, 250 x 4.6 mm column. The samples were separated in a 5%-95% gradient of ACN, and 0.05% TEA. The callus and seed post- methanolysis samples were dissolved in methanol (dilution rate normalized to the dry weight of the post-methanolysis oil to obtain the concentration of 1 mg of post- methanolysis oil/1 ml of methanol).
  • SEA 4a and SEA 4 standards in methanol were prepared to generate the external standard calibration curve: 1 mg/ml, 0.2 mg/ml, 0.04 mg/ml, 0.008 mg/ml.
  • the volumes used for injection to HPLC were 20 ml for the standard samples and 100 ml for the callus and seed samples.
  • peaks in the standard samples and peaks in the callus and seed samples were integrated, and area under the curve (AUC) values were calculated.
  • Standard curves were prepared, and the quantities were expressed as mg of compound/mg of post-methanolysis oil (Fig. 3). The samples with AUC different by an order of magnitude or more from the other replicates were considered outliers and discarded.
  • Fig. 3 shows the average amounts (pg/mg of post-methanolysis oil) of SAE 4a (compound of Formula lb, wherein R 1 is methyl) and SAE 4 (compound of Formula la, wherein R 1 is methyl) in the post-methanolysis oil across all the Entandrophragma caudatum calluses and seed samples (Table 5). Bars indicate standard deviation.
  • compositions were chosen for the cell suspension cultures. These compositions comprised the growth regulators III, IV, and X and concentrations, as presented in Table 1.
  • the three-week-old callus of Entandrophragma caudatum produced using the methods described in Section 2 was used.
  • the callus from 5 explants was fragmented and added to 60 ml of liquid media of the same composition as the one used to produce the callus, in 100 ml E-flasks.
  • Three flasks for each media were prepared. Subsequently, the flasks were placed on an orbital shaker (150 rpm) in a culture chamber (16h/8h day/night photoperiod, 25°C ⁇ 2°C).
  • the larger pieces of the callus were drained, and the media was replaced: the suspension was allowed to sediment and the old media was carefully decanted and fresh liquid media was added to the sediment (cell). During that time the suspensions were sampled for microscopy evaluation which showed the presence of viable, dividing cells (Fig. 4). The resulting suspensions were placed back in the culture cabinet and shaken for 3 weeks.
  • Fig. 4 shows the example morphology of suspension cells grown in media III, with visible organelles and nuclei (n).
  • the cell biomass was harvested by filtration through filter paper under a vacuum of the pooled content of the three E-flasks prepared for each media (180 ml of the suspension). The biomass and media in which the cells were grown were collected separately. The biomass was freeze-dried.
  • the post-methanolysis oil samples were prepared in 1 mg/ml concentration in methanol and analyzed with LC-MS.
  • the analysis showed the presence of SAE 4 (compound of Formula la, wherein R 1 is methyl) and SAE 4a (compound of Formula lb, wherein R 1 is methyl) in all three samples.
  • Figure 5 shows an example of the presence of these compounds detected in extracts from cell biomass grown in media III.
  • Fig. 5 shows the example LC-MS traces for the post-methanolysis extract from the cell biomass grown in media III.
  • C crude LC-MS trace of the extract.
  • Table 12 The combinations of plant growth regulators in media tested for the presence of SAE-4 and SAE-4a. Approximately 50 ml of the medium from cell cultures described herein were probed from each culture. The premethanolysis oil was obtained by faze extraction/partition with 10 ml of ethyl acetate. The organic phase was collected, dried and the resulting premethanolysis oil was weighted. The methanolysis reaction and subsequent isolation of the postmethanolysis oil were done according to the methods described herein. The LC-MS analysis was performed using the same methods as described herein.
  • SAE-4 and SAE-4a were detected in cultures comprising media III.
  • SAE-4 and SAE-4a was also detected in cultures comprising media IV (not shown).
  • Cultures comprising media X did not show any detectable SAE4 or SAE4a.
  • Fig. 6 shows the example LC-MS traces of the Entandrophragma caudatum postmethanolysis medium III extract: raw chromatogram (A) and chromatogram generated with mass filtering tool for SAE 4a (B) and SAE 4 (C) compounds. A peak for an unknown compound with identical molecular weight as SAE 4a but different retention time marked with *.
  • Plant in vitro culture has been proven in many cases as a viable option for sustainable production of medicinal products which are not feasible or difficult to obtain through chemical synthesis.
  • Entandrophragma caudatum callus culture induction Media supplemented with different combinations of 2,4-D, NAA, IAA, KIN, BAP or TDZ plant growth regulators were prepared in the course of Exp. 1 and Exp. 2, one type of explant (fragments of cotyledons) 3 plates per media with 10 or 8 explants each, respectively.
  • Example 5 Replication of callus induction experiments from Entandrophragma caudatum The experiments were performed on two separate occasions: November 2023 - March 2024 (Exp. 1) and April-July 2024 (Exp. 2). Exp. 1 and 2 followed the procedures developed during the Dicot I Project. Extraction followed by full methanolysis before LC-MS analysis was performed for all the samples from Exp. 1, whereas samples from Exp. 2 were only extracted (methanolysis performed on selected samples only), and LC-MS data from raw methanol extracts was gathered for further analysis.
  • the seed germination procedures developed previously were used.
  • the seeds were prepared by removal of the wing and the skin, and surface sterilized by 1 min treatment with 70% ethanol, followed by 15 min in 50% ACE commercial bleach and 3 times washing with deionized water. All seeds were sowed onto % MS basal media solidified with 8 g/L agarose (Sigma), pH 5.7, autoclaved, in plastic transparent boxes sterilized with UV. The seeds were kept in darkness for the first week and then in 16h/8h day/night photoperiod, 25°C ⁇ 2°C.
  • the age of the seeds appeared to be important for the germination rates.
  • Entandrophragma caudatum seedlings germinated in sterile conditions were used to initiate the culture.
  • the fragments of cotyledons taken from two- to three-week-old seedlings were used as explants.
  • Cultures were maintained on MS basal media supplemented with 30 g/L sucrose, different combinations of plant growth regulators (Tables 14 and 15), and solidified with 8 g/L agarose. The cultures were maintained in 16h/8h day/night photoperiod, 25°C ⁇ 2°C.
  • the callus samples were harvested, divided into three replicates, freeze-dried, and shipped from Krakow to Uppsala. Three replicates from each of the calli were prepared and labeled with an Arabic number corresponding to the Roman number ID of the media and the replicates A, B, and C (e.g. 1A, IB, 1C are three replicates for the callus obtained on medium I).
  • each replicate/sample was grounded to a powder in a mortar and weighted. Subsequently, the samples were extracted for 24 h with methanol on a shaking table at room temperature (100 mg of initial DW of callus in 10 ml methanol) then the extract was centrifuged, and the supernatant was collected. The supernatant constituted raw methanol extracts collected for LC-MS analysis.
  • the methanolysis reaction was carried out under reflux at 64°C for 24h.
  • the post-methanolysis reaction mixture was cooled to 30°C, and 1 pl of acetic acid was added to stop the reaction.
  • the mixture was stirred for several minutes and transferred to a 50 ml Falcon tube. Subsequently, 25 ml of water and 10 ml of ethyl acetate were added, the mixture was shaken and then centrifuged at 3000 rpm for phase separation. The upper ethyl acetate layer was collected and evaporated to dryness and constituted the post-methanolysis oil.
  • the post-methanolysis oil was dissolved in 0.5 ml of methanol for LC-MS analysis.
  • the samples were analyzed using the UPLC-MS system: Waters nanoAcquity (all methanol extracts) or ACQUITY Premier UPLC (all samples after methanolysis) -Xevo G2-XS QTof Quadrupole Time-of-Flight MS, Waters.
  • the UPLC system operated with a C18 column and a gradient of 5%-95% ACN in water, 0.1% FA.
  • the mass spectra were collected in the SONAR and MSe acquisition method, in positive ion mode, and a window of 100 m/z - 1000 m/z. All methanol extracts were saved for future LC-MS analyses. All post- methanolysis samples from Exp.
  • Raw extracts from E. caudatum contain a plethora of phragmalin limonoids. Those limonoids have a common core molecular structure, which is "decorated” with different side chains through ester bonds. Furthermore, the limonoids are precursors of Dicot reference compounds SAE 4 and SAE 4a, produced by methanolysis, a chemical transformation breaking all ester bonds. Compounds sharing the same core structure can be quantified in the post-methanolysis samples. However, the methanolysis process is labor-intensive, time-consuming, and not applicable to small sample sizes, limiting the number and type of samples that can be tested over time.
  • the two media compositions were chosen for the trials: III, and X.
  • Two-week and three- weeks-old callus tissue grown on solid MS and Gamborg B5 media were used to initiate suspension cultures.
  • the three-week-old callus produced using the methods described in Section 2 was used.
  • the callus from 20 explants was fragmented and added to 50 ml of liquid media of the same composition as the one used to produce the callus in 250 ml E- flasks.
  • Three flasks for each media were prepared. Subsequently, the flasks were placed on an orbital shaker (150 rpm) in a culture chamber (16h/8h day/night photoperiod and in darkness, 25°C ⁇ 2°C).
  • Gamborg B5 induced the fast-growing suspension cultures and occurred more suitable for the cultures than MS, is the difference in composition between the two media (Table 19). The most important difference is most probably in the macro elements in the nitrogen source. Gamborg B5 contains much less ammonia ions in the form of (NF SC instead of NH4NO3 like in the MS media.
  • the explants were transferred on solid Gill medium, the material from the suspension plate was transferred into a liquid cultivation medium and the suspension from the T-flask was transferred into a 125 ml Erlenmeyer flask.
  • the plates with the explants were cultivated at 25°C in the dark.
  • the two suspensions were cultivated on a rotary shaker at 120 rpm at 25°C in the dark.
  • a basal salt mixture is used for the preparation of culture medium Gill.
  • This mixture derives from PhytoTech Labs (Kansas, USA) and is Gamberg 8-5 Basal Medium (G398).
  • G398 Gamberg 8-5 Basal Medium
  • 3.21 g of this basal salt mixture is dissolved in ⁇ 800 ml deionized water. After dissolution, 30 g sucrose are added and are dissolved, too. Afterwards, 2 ml 2,4-D (stock solution 1 mg/ml solved in KOH) is added, and the medium is filled up to 1000 ml. Finally, the pH is adjusted to 5.6 with KOH.
  • the medium can be either sterile filtered or autoclaved. The shelf life of the medium is three weeks.
  • the primary callus material formed on the explants (Fig. 13A) was isolated from the explant and further cultivated on solidified GUI medium (Fig. 13B). Once enough callus material was formed, suspension cultures were initiated from this material. Three suspension cultures that could be initiated which were named ent006, ent007 and ent008. While suspension ent007 and ent008 exhibited no cell growth after 4 weeks, suspension ent006 showed good cell growth.
  • the suspension ent006 was initiated on 10th May 2024 from primary callus material with ⁇ 1.5 g of biomass in 25 ml cultivation medium. The development of the culture is shown in Figure 14.
  • the biomass of Entandrophragma arrived after two days of transit in good condition.
  • the suspension culture named ent006 was initiated from primary callus material with ⁇ 1.5 g of fresh weight biomass in 25 ml cultivation medium.
  • the cell line was continuously maintained in GUI cultivation medium.
  • a basal salt mixture was used to prepare culture medium GUI. This mixture derives from PhytoTech Labs (Kansas, USA) and is Gamberg B-5 Basal Medium (G398).
  • This basal salt mixture was dissolved in ⁇ 800 ml deionized water. After dissolution, 30 g sucrose was added and dissolved, too. Afterwards, 2 ml 2,4-D (stock solution 1 mg/ml solved in KOH) was added, and the medium is filled up to 1000 ml. Finally, the pH is adjusted to 5.6 with KOH.
  • the medium can be either sterile filtered or autoclaved. The shelf life of the medium is three weeks.
  • the suspension culture ent006 is currently maintained in three individual 3,000 ml Fernbach flasks containing 1,000 ml cultivation medium GUI.
  • the culture is transferred weekly into a fresh cultivation medium and cultivated at 25°C at 120 rpm in the dark. Prior to each transfer into fresh medium the biomass is separated from the old cultivation medium via vacuum-filtration.
  • the growth data are shown in Figure 16.
  • Growth index is the quotient from end to start fresh weight (a growth index of 2 is the doubling of biomass within 7 days of cultivation). After a short adaption phase of two weeks, cell growth increased to a growth index of approx. 2. At transfer cycle 9, upscale of biomass was initiated and three flasks of ent006 were established. The average of the last 10 growth cycles is a growth index of 2.4 indicating a good cell growth under the current cultivation conditions. However, in a potential development project, growth media optimization might lead to even higher growth rates. The last data point (s25) corresponds to November 13 th .
  • Figure 17 shows three individual pictures of vacuum-filtrated biomass from Erlenmeyer flasks.
  • the vacuum-filtrated biomass from ent006 appeared light yellow and friable containing a few larger cell clusters (grey spots in the Buechner beaker). All flasks have been inoculated with 60 g of fresh weight biomass and end up with roughly 130 g within 1 week of cultivation.
  • the cell culture is characterised by smaller cell cluster and cell chains of 15-20 cells. This suspension showed a very high cell viability of approx, over 90% as only a few dead cells (non-green) are visible. This is indicated by the blue boxes in the overlay (Fig. 18C). The suspensions in both other Fernbach flasks were characterised as well. Also, those showed the same high cell viability of over 90%.

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Abstract

La présente invention concerne un nouveau procédé d'obtention des composés limonoïdes de formule IIa ou IIb et/ou un ou plusieurs esters et/ou dérivés de ceux-ci, par culture de cellules végétales dans une culture cellulaire et récupération desdits composés à partir desdites cellules et/ou culture cellulaire. Dans un mode de réalisation du procédé, les cellules végétales sont obtenues ou proviennent d'un ou plusieurs explants de la plante correspondante. La présente invention concerne également des produits cellulaires du procédé et l'utilisation desdits produits et composés.
PCT/EP2024/088540 2023-12-29 2024-12-27 Nouveau procédé de récupération de composés limonoïdes Pending WO2025141142A1 (fr)

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WO2013110744A2 (fr) 2012-01-25 2013-08-01 Dicotyledon Ab Composés inédits, leur fabrication et leurs utilisations (ii)
WO2017203454A1 (fr) * 2016-05-25 2017-11-30 Universidad Nacional De Colombia Procédé d'obtention d'un extrait d'azadirachta indica
WO2024072288A1 (fr) 2022-09-30 2024-04-04 Dicot Ab Procédé d'extraction de dérivés de phragmaline à partir de graines d'entandrophragma caudatum et préparation de limonoïdes pharmaceutiquement actifs

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WO2013110744A2 (fr) 2012-01-25 2013-08-01 Dicotyledon Ab Composés inédits, leur fabrication et leurs utilisations (ii)
US20150011616A1 (en) * 2012-01-25 2015-01-08 Dicotyledon Ab Phragamalin limonoids for the treatment of sexual dysfunction
WO2017203454A1 (fr) * 2016-05-25 2017-11-30 Universidad Nacional De Colombia Procédé d'obtention d'un extrait d'azadirachta indica
WO2024072288A1 (fr) 2022-09-30 2024-04-04 Dicot Ab Procédé d'extraction de dérivés de phragmaline à partir de graines d'entandrophragma caudatum et préparation de limonoïdes pharmaceutiquement actifs

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