A novel method for recovering limonoid compounds

Abstract

The present invention relates to a novel method for obtaining the limonoid compounds of Formula IIa or IIb, and / or one or more esters and / or derivatives thereof, by culturing plant cells in a cell culture and recovering said compounds from said cells and / or cell culture. In one embodiment of the method the plant cells are obtained or originate from an explant / s of the corresponding plant. The present invention also relates to cell products of the method and the use of said products and compounds.

Description

[0001] A novel method for recovering limonoid compounds

[0002] Technical field:

[0003] 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.

[0004] Background:

[0005] 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).

[0006] Today, these pharmaceutically active compounds, and / or precursors of these active compounds, are extracted from the plants in lengthy, complex, and inefficient processes.

[0007] Thus, to meet future demands for using these therapeutically active limonoid compounds in pharmaceutical compositions, to treat and / or prevent sexual dysfunction, new and more efficient methods are needed to produce these active compounds.

[0008] 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.

[0009] 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.

[0010] It is an object of the present disclosure to provide a method allowing to produce compounds that may be used as a medicament such as a medicament for treating or preventing sexual dysfunction and / or serve as a starting material for manufacturing such compounds. It is also an object of the present disclosure to provide aspects and / or advantages not provided by hitherto known techniques.

[0011] Summary:

[0012] 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.

[0013] 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. In one embodiment of the method 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:

[0014] Formula Ila Formula lib wherein R1is 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 R1is a methyl, wherein R2, R3and R4independently are selected from hydrogen, nicotinoyl or -C(O)R5; where R5is 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, R3and R4independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso- pentanoyl or acyl, said method comprising :

[0015] I. culturing plant cells in a nutrient medium in a cell culture, wherein the cells produce the compound of Formula Ila and / or lib and / or one or more esters or derivatives thereof;

[0016] II. recovering the compound of Formula Ila and / or lib and / or the one or more esters or derivatives thereof produced in step I.

[0017] The present document is also directed to the method wherein the method obtains at least one of the compounds of Formula la or lb:

[0018] Formula la Formula lb wherein R1is 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. For instance, R1may be methyl.

[0019] By "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. For example, 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The present document is also directed to the method where the explant / s from said plant are cultured under controlled illumination.

[0024] 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-benzylaminopurine, kinetin (KIN) or thidiazuron (TDZ).

[0025] The present document is also directed to the method where 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).

[0026] 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.

[0027] 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.

[0028] 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).

[0029] 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.

[0030] The present document is also directed to the method where the plant cells of the cell culture are cultured under controlled illumination.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The present document is also directed to the method wherein said suspension cell culture is a continuous culture for growing cells in suspension.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In some embodiments, 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.

[0041] 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.

[0042] Thus, in some embodiments, 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. In some embodiments, the plant cell is a suspension cell culture.

[0043] By "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).

[0044] By "capable of producing", we include the meaning that the plant cell may be induced into producing the compound under appropriate conditions. For example, 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.

[0045] 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.

[0046] Thus, in some embodiments, 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. In some embodiments, the culturing medium is sterile filtered and / or autoclaved.

[0047] 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.

[0048] Thus, in some embodiments, 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. For instance, the plant cell biomass comprises plant cells produce, or are capable of producing a compound of Formula la, wherein R1is methyl, and / or a compound of Formula lb, wherein R1is methyl.

[0049] The present document is also directed to an isolated explant / s or fragment / s thereof obtained or originating from a method as disclosed herein.

[0050] Thus, in some embodiments, 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. For instance, 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 R1is methyl.

[0051] 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. In some embodiments, 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.

[0052] By "recovered from", we include the meaning of "isolated from", which enriches or purifies the concentration of the compound away from other material.

[0053] 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.

[0054] 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.

[0055] In some embodiments, 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.

[0056] 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 R1preferably is methyl and R2is a straight, branched or cyclic optionally substituted alkyl group with 1 to 6 carbon atoms; or a compound of Formula VI.

[0057] The present document is also directed to the use wherein Formula V, R1preferably is methyl and R2is isopropyl.

[0058] 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.

[0059] Brief description of the drawings:

[0060] 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.

[0061] 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.

[0062] 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).

[0063] 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.

[0064] Figure 6 shows a LC-MS graph of post-methanolysis extracts on cell culture medium from Entandrophragma caudatum cell suspension cultures comprising medium III. 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 *.

[0065] Figure 7 shows photographic documentation of the cultures for three plates with 10 explants, which were prepared for each medium of Example 5.

[0066] Figure 8 shows photographic documentation of the cultures for three plates with 8 explants, which were prepared for each medium of Example 5.

[0067] 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. In both A and B, 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. For A, 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; SEA 4a shows RT = 3.07 minutes, and phragmalin shows RT = 3.51 minutes; TOF MS ES+ BPI 1.53e7. For B, peaks are seen at 1.59, 2.40, 3.04, 3.96 minutes; TOF MS ES+ BPI 2.43e7.

[0068] 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. For all spectra, 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.

[0069] 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.

[0070] 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.

[0071] Figure 13 shows callus formation on E. caudatum explants (A), and isolated primary callus material on solid medium (B).

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Detailed description :

[0077] 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. In one embodiment of the method 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.

[0078] The wording callus as used herein describes a growing mass of unorganized plant parenchyma cells.

[0079] The wording callus culture used herein describes a plant tissue culture comprising said unorganized growing and dividing mass of callus cells.

[0080] The wording friable callus used herein is meant to describe a specific class of callus which can be used to generate for instance cell suspension cultures.

[0081] The wording explant used herein is describing a part and / or fragment of a plant taken to be cultured and grown in nutrient medium.

[0082] A method for obtaining the compounds of formula Ila and / or lib:

[0083] 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). Today, 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. However, current processes are lengthy, complex and give a low yield, making production inefficient. Thus, there is a need to find better methods which make the production and recovery of these compounds faster, simpler, and more efficient. In particular, there is a need for 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.

[0084] In one aspect 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:

[0085] Formula Ila Formula lib wherein R1is 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 R1is a methyl, wherein R2, R3and R4independently are selected from hydrogen, nicotinoyl or -C(O)R5; where R5is 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, R3and R4independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso- pentanoyl or acyl, said method comprising :

[0086] I. culturing plant cells in a nutrient medium in a cell culture, wherein the cells produce the compound of Formula Ila and / or lib and / or one or more esters or derivatives thereof;

[0087] II. recovering the compound of Formula Ila and / or lib and / or the one or more esters or derivatives thereof produced in step I.

[0088] Formula Ila

[0089] The compound of Formula Ila can have any possible molecular structure by combination of the varying groups R1, R2, R3, R4and R5as described in this document. R1can 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 R1is a methyl. R2, R3and R4independently are selected from hydrogen, nicotinoyl or -C(O)R5; where R5is 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, R3and R4independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso-pentanoyl or acyl.

[0090] Formula lib

[0091] The compound of Formula lib can have any possible molecular structure by combination of the varying groups R1, R2, R3and R5as described in this document. R1can 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 R1is a methyl. R2, R3are independently selected from hydrogen, nicotinoyl or -C(O)R5; where R5is 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.

[0092] Formula la and lb

[0093] In another aspect the present invention seeks to protect the method wherein the method obtains at least one of the compounds of Formula la or lb:

[0094] Formula la Formula lb wherein R1is 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.

[0095] In another embodiment of the invention R1is a methyl or a trifluoromethyl group.

[0096] 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. By altering the culturing conditions used in the method 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.

[0097] Culturing plant cells

[0098] 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.

[0099] In another embodiment of the invention 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. With 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. With 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.

[0100] 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. For example, the cells of the cell culture can be a finite cell line. Alternatively, 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.

[0101] The cells of the invention cultured in step I of the method can be adherent cells, suspension cells and / or a mix thereof.

[0102] In one embodiment of the invention the cells of the cell culture in step I of the method are adherent cells.

[0103] In another embodiment of the invention the cells of the cell culture in step I of the method are suspension cells.

[0104] In another embodiment of the invention the cells of the cell culture in step I of the method are suspension cells cultured as a suspension cell culture.

[0105] In another embodiment of the invention said suspension cell culture is a continuous culture for growing cells in suspension.

[0106] In another embodiment of the invention the cells of the cell culture in step I of the method are a mix between adherent and suspension cells.

[0107] 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). After the culture is stopped, the culture, comprising the cultured cells and / or culturing medium and / or explant / s, is recovered / harvested as a batch. 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. These 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.

[0108] 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. By culturing the suspension cells of the invention in a continuous fashion 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.

[0109] 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. Alternatively, 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.

[0110] Optimal cell culturing conditions:

[0111] 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.

[0112] 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).

[0113] Hormones:

[0114] In one embodiment of the method of the invention 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. Examples of 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 peptide hormones, nitric oxide (NO), strigolactone biosynthesis inhibitor (SBI), melatonin, 6- benzylaminopurine, kinetin (KIN) or thidiazuron (TDZ).

[0115] In another embodiment of the method of the invention 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).

[0116] In another embodiment of the method of the invention two or more of said plant hormones and / or growth regulators are used in the method in any combination. In another embodiment of the method of the invention 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.

[0117] In yet another embodiment of the method of the invention at least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).

[0118] For the cells of the cell culture in the method of the invention to proliferate and produce as high yield as possible of 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, 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. 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.

[0119] In one embodiment of the method of the invention 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.

[0120] Illumination-cells:

[0121] For plant cell cultures the access to light can affect the proliferation of the cultured cells and / or the yield of compounds produced by said plant cells. However, depending on what type of plant cell that is cultured, 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.

[0122] In one embodiment of the method of the invention the plant cells of the cell culture are cultured under controlled illumination.

[0123] With illumination herein 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.

[0124] As with optimal concentration of plant hormone / s and / or growth regulator / s, as discussed above, the cells exposure to light / photoperiod is optimally tested before the start of culturing the cells for production of compound / s.

[0125] With photoperiod is meant the total time a plant is exposed to light / illumination per 24h.

[0126] The plant cells of the method of the invention can be cultured with a photoperiod from 0- 24h.

[0127] In one embodiment of the method of the invention 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.

[0128] In another embodiment of the method of the invention 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.

[0129] In yet another embodiment of the method of the invention 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.

[0130] In yet another embodiment of the method of the invention 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.

[0131] 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.

[0132] In one embodiment of the invention any photoperiod, from l-23h, can be spread out over the total 24h of a day. Thus, 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.

[0133] 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.

[0134] Temperature:

[0135] 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.

[0136] In one embodiment of the method of the invention 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.

[0137] In another embodiment of the method of the invention the plant cells of the cell culture are cultured at 25°C + / - 2°C, most preferably at 25°C + / - 2°C.

[0138] Culturing time:

[0139] The duration of a plant cell culture varies depending on tissue or cell sample. For new cell cultures 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.

[0140] Culturing medium:

[0141] 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. For instance, cells of the invention can be cultured in MS basal media complemented with plant hormones and / or growth regulator / s of the invention.

[0142] By optimizing these factors 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.

[0143] Recovering the compound of Formula Ila and / or lib, and / or compounds of Formula la and / or lb.

[0144] 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. With "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.

[0145] 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. Alternatively, 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.

[0146] 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.

[0147] In one embodiment of the invention said suitable extraction method comprises methanol extraction, followed by methanolysis.

[0148] For further details please see the experimental section below.

[0149] In one embodiment of the invention 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.

[0150] In another embodiment of the invention 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.

[0151] In another embodiment of the invention 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.

[0152] Origin of the plant cells, cell culture or explants:

[0153] 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.

[0154] Alternatively, 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.

[0155] In one embodiment of the method of the invention 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.

[0156] In another embodiment of the method of the invention 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.

[0157] In another embodiment of the invention the explant / s of said plant is obtained from fresh seeds that first has been germinated.

[0158] Culturing explants on nutrient medium :

[0159] 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. Depending on from what part of the plant the explant is obtained or originate from, 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.

[0160] In one embodiment of the invention 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.

[0161] In another embodiment of the invention 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.

[0162] In one embodiment of the method of the invention 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.

[0163] In another embodiment of the method of the invention 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.

[0164] In another embodiment of the method of the invention the explant / s from said plant are cultured under controlled illumination.

[0165] Culturing medium-explants:

[0166] 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.

[0167] 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.

[0168] For instance, 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. When explants are cultured on 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.

[0169] Growth regulators / plant hormones-explants:

[0170] In another embodiment of the method of the invention the nutrient media for culturing the explant / s are supplemented with at least one or more plant hormone / s or growth regulator / s. Examples of 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, nitric oxide (NO), strigolactone biosynthesis inhibitor (SBI), melatonin, 6- benzylaminopurine, kinetin (KIN) or thidiazuron (TDZ).

[0171] In another embodiment of the method of the invention 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). In another embodiment of the method of the invention two or more of said plant hormones and / or growth regulators are used in the method in any combination.

[0172] In another embodiment of the method of the invention 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.

[0173] In yet another embodiment of the method of the invention at least one of the plant hormones or growth regulators preferably is 2,4-dichlorophenoxyacetic acid (2,4-D) or 1- naphthaleneacetic acid (NAA).

[0174] Plant hormone and growth regulator concentrations:

[0175] For the explants of the method of the invention to generate cells that grow and / or proliferate and / or to stimulate the formation of a friable callus comprising cell / s which can be used / are suitable for inducing a cell culture, 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. 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.

[0176] In one embodiment of the method of the invention 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.

[0177] Illumination - explants:

[0178] For explants 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. However, depending on what type of explant that is cultured, the effect of light stimulation varies. For some explants 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.

[0179] In one embodiment of the method of the invention the explant / s from said plant are cultured under controlled illumination.

[0180] With illumination herein 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.

[0181] As with optimal concentration of plant hormone / s and / or growth regulator / s, as discussed above, the explant / s exposure to light / photoperiod is optimally tested before the start of culturing the cells for production of compound / s.

[0182] 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.

[0183] In one embodiment of the method of the invention 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.

[0184] In another embodiment of the method of the invention 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.

[0185] In yet another embodiment of the method of the invention 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.

[0186] In yet another embodiment of the method of the invention 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.

[0187] 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.

[0188] In one embodiment of the invention any photoperiod, from l-23h, can be spread out over the total 24h of a day. Thus, 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.

[0189] 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.

[0190] Plant origin:

[0191] 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.

[0192] In one embodiment of the method of the invention said plant cells and / or said explant / s and / or fragments thereof originate from a plant selected from the Meliaceae family.

[0193] In another embodiment of the method of the invention 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. In another embodiment of the method of the invention said plant selected from the Meliaceae family is from Swietenia macrophylla, Khaya senegalis, Neobeuga mahafalensis, Chukrasia tabularis or Entandrophragma caudatum; most preferably Entandrophragma caudatum.

[0194] In yet another embodiment of the method of the invention said plant selected from the Meliaceae family is from Swietenia macrophylla or Entandrophragma caudatum.

[0195] In yet another embodiment of the method of the invention said plant selected from the Meliaceae family is Entandrophragma caudatum.

[0196] Notice that the plant Xylocarpus moluccensis mentioned herein also have the name Xylocarpus mekongensis.

[0197] Isolated products:

[0198] 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.

[0199] In another embodiment of the invention 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.

[0200] These compounds, and / or esters and / or derivatives thereof are then used in synthesis of pharmaceutically active limonoid compounds. By executing the method of the invention compounds of Formula Ila and / or lib, and / or one or more esters and / or derivatives thereof, and / or one of the compounds of Formula la and / or lb, are obtained. Other products obtained from the method are for example isolated plant cells, plant cell lines, plant cell batches, suspension cultures, plant cell biomass, plant cell culturing medium comprising non-cell biomass and explants. All these products comprise and / or can be stimulated, by using the method herein, to produce 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 and / or lb.

[0201] 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.

[0202] Thus, in one embodiment the invention also seeks to protect an isolated plant cell, obtained from the method. In another embodiment the invention seeks to protect an isolated plant cell line obtained or originating from the method.

[0203] In another embodiment the invention seeks to protect an isolated plant cell batch obtained or originating from the method.

[0204] In another embodiment 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.

[0205] With non-cell biomass herein is meant any biomass, other than the cells, obtained or originating from the cell culture of the method. Examples of non-cell biomass herein 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.

[0206] Other examples of a non-cell biomass are, but not limited to, cellular fragments, intracellular structures, organelles, nuclei, vesicles, microsomes, exosomes, lipid structures, proteins, peptides, polysaccharides, compounds and / or molecules.

[0207] 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.

[0208] In another embodiment 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.

[0209] In another embodiment the invention seeks to protect an isolated explant / s or fragment / s thereof obtained or originating from the method.

[0210] In another aspect 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.

[0211] 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. Alternatively, said plant cell / s can also be obtained from an explant / s of the method herein.

[0212] In another aspect 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.

[0213] Use:

[0214] 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).

[0215] In another aspect the invention seeks to protect the use of the cell culturing medium, the non-cell biomass, or the plant cell biomass as a medicament.

[0216] In another aspect 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 R1preferably is methyl and R2is a straight, branched or cyclic optionally substituted alkyl group with 1 to 6 carbon atoms; or a compound of Formula VI:

[0217] Formula V Formula VI.

[0218] In an example, there is provided a compound of Formula V, wherein R1is methyl and R2is isopropyl. In another aspect the invention seeks to protect the use wherein Formula V, R1preferably is methyl and R2is isopropyl.

[0219] It will be appreciated that the compound of Formula VI as described herein is a combination of:

[0220] (i) a compound of Formula V, wherein R1is methyl and R2' is isopropyl, and

[0221] (ii)water wherein (i) and (ii) are taken in a ratio of 1: 1.

[0222] In other words, the compound of Formula VI is a monohydrate of the compound of Formula V, wherein R1is methyl and R2is isopropyl.

[0223] In another aspect 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.

[0224] It will be appreciated that the compounds described herein may be depicted without the stereochemistry indicated. In this case, all chemical bonds are drafted as straight lines. For example, phragmalin (i.e. the compound of Formula la, wherein R1is methyl) may be depicted without the stereochemistry as shown in Table 9 or Table 13 herein.

[0225] Alternatively, the compounds of described herein may be depicted as a stereoisomer. For instance, phragmalin may be depicted as

[0226]

[0227] Conclusion :

[0228] 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. In one embodiment of the novel method said cells are retrieved from friable calli material produced by first culturing explant / s taken from plants of the Meliaceae family. The 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.

[0229] 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.

[0230] Examples

[0231] Note that the compound of Formula la, wherein R1is methyl, is referred to as SAE4, SAE 4, SAE-4 or phragmalin, and the compound of Formula lb, wherein R1is methyl, is referred to as SAE4a, SAE 4a, or SAE-4a, in the following experimental section. Additionally, SAE may be referred to as a compound of Formula III, and SAE4a may be referred to as a compound of Formula IV.

[0232] Example 1. Seed germination

[0233] 1.1. Plant material Seeds from the plant Entandrophragma caudatum and Chukrasia tabularis were used for germination. The seeds used had the following specifications.

[0234] Producer Parceval Pharmaceuticals, South Africa

[0235] Species Entandrophragma caudatum

[0236] Origin Zimbabwe

[0237] Batch D0377 / 22

[0238] Species Chukrasia tabularis

[0239] Origin Ordered from rarepalmseeds.com

[0240] 1.2. Sowing in sterile conditions for in vitro culture

[0241] 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.

[0242] Example 2. Entandrophragma caudatum and Chukrasia tabularis callus cultures

[0243] 2.1. Callus initiation

[0244] Entandrophragma caudatum and Chukrasia tabularis seedlings germinated in sterile conditions were used to initiate the culture. The fragments of hypocotyl and / or 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 (Table 1) and solidified with 8 g / L agarose. The cultures were maintained in 16h / 8h day / night photoperiod, 25°C ± 2°C.

[0245] Three plates with 10 explants each (30 explants overall) per plant were prepared for each medium. Explants produced different callus tissue on most of the media. For Entandrophragma caudatum the best outcome in terms of the callus size and percentage of explants producing callus was noted on media containing auxin 2,4-D combined with KIN and IAA, NAA combined with TDZ after three weeks of culture (Table 2). Root-like structures were forming on the cotyledons cultured on some of the media (Table 2). For Chukrasia tabularis the best outcome in terms of the callus size and percentage of explants producing callus was noted on media containing 2,4-D combined with KIN for both types of explants (hypocotyl and cotyledons) after three weeks of culture (Table 3 and 4). Rootlike structures were forming on the hypocotyl cultured on several of the media (Table 3).

[0246] Table 1. The combinations of plant growth regulators used in Entandrophragma caudatum and Chukrasia tabularis in vitro callus cultures trials.

[0247] Table 2. Entandrophragma caudatum cotyledon callus cultures overview. Three plates with 10 explants each (30 explants overall) were prepared for each medium.

[0248] Table 3. Chukrasia tabularis hypocotyl callus cultures overview. Three plates with 10 explants each (30 explants overall) were prepared for each medium. Table 4. Chukrasia tabularis cotyledon callus cultures overview. Three plates with 10 explants each (30 explants overall) were prepared for each medium.

[0249] 2.2. Extraction, methanolvsis, and LC-MS analysis

[0250] Processing of the callus samples

[0251] 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.

[0252] Each replicate / sample was grounded to a powder in a mortar and weighted. Subsequently, the samples were extracted for 24h with methanol on a shaking table at 40°C (0,25 ml per mg of initial dry weight of callus) then the hot extract was centrifuged, and the supernatant was collected. Pre-methanolysis raw methanol extracts were collected for LC-MS analysis. Subsequently, appropriate amounts of ethyl acetate and water were added to the extract to obtain phase separation and after phase extraction, the organic phase was collected. The organic phase was evaporated to dryness and the resulting oil extract was weighted (oil sample).

[0253] For the methanolysis reaction, 24 ml of methanol and sodium methoxide (2 mmol per mg of oil sample dry weight) was added to each oil sample. The methanolysis reaction was carried out under reflux at 64°C for 24h. 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 30 min 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 5 ml of ethyl acetate. The organic phase was separated, evaporated to dryness (post-methanolysis oil), and weighed. Overall, for Entandrophragma caudatum 72 methanolysis reactions were carried over. The extraction and methanolysis conditions have been outlined in Table 5. Overall, for Chukrasia tabularis 30 methanolysis reactions were carried over. The sample id, organ origin (cotyledon or hypocotyl) and the media organs were grown in are outlined in Table 6. The extraction and methanolysis conditions are outlined in Table 7.

[0254] Table 5. Outline of Entandrophragma caudatum callus extraction and methanolysis conditions.

[0255] ♦Seed samples

[0256] Table 6. Outline of Chukrasia tabularis callus extraction (in triplicates) and methanolysis conditions. Note that in column for organ Cot. is short for cotyledon and Hip for hypocotyl. 17.2

[0257] Table 7. Outline of Chukrasia tabularis callus extraction and methanolysis conditions.

[0258] 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).

[0259] Processing of seed samples

[0260] Seeds of Chukrasia tabularis, Entandrophragma caudatum and five additional species (Swietenia mahagoni, Swietenia macrophylla, Cedrela odorata, Cedrela montana, Khaya senegalis') were also analyzed for limonoid compounds such as SAE-4 (compound of Formula la, wherein R1is methyl) and / or SAE-4a (compound of Formula lb, wherein R1is methyl). Each repetition / seed 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 in 40°C (40 ml per g of initial dry weight of grounded seeds) then hot extract was filtered. Pre-methanolysis methanol extracts were collected for HPLC and LC-MS analysis (1 pl diluted with 50pl of methanol). Subsequently, ethyl acetate (4ml) and water (1,7ml) were added to the extract and the organic phase was separated. The organic phase was evaporated to dryness and the resulted oil extract was weighted (oil sample). The methanolysis conditions were similar to callus samples. The methanolysis reaction was carried out under reflux at 64°C for 24h. 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.

[0261] Table 8. Outline of extraction and methanolysis conditions for seeds of Chukrasia tabularis, Entandrophragma caudatum, Swietenia mahagoni, Swietenia macrophylla, Cedrela odorata, Cedrela montana and Khaya senegalis. LC-MS analysis

[0262] 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. For Chukrasia tabularis post-methanolysis samples of different series were analyzed (Table 6). Reference samples (Entandrophragma caudatum post-methanolysis seed extract and purified SAE 4a compound) were analysed with the same LC-MS system and conditions as other samples (Fig. 1). All analyses were performed using MassLynx 4.2 software (Waters).

[0263] As can be seen in figure 1 both limonoid compounds SAE4a (compound of Formula lb, wherein R1is methyl) and SAE4 (compound of Formula la, wherein R1is methyl) were possible to isolate and identify in seeds from Entandrophragma caudatum.

[0264] 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.

[0265] Mass chromatograms for each of the compounds were generated using the compound characteristics (Table 9) and mass filtering tool.

[0266] Table 9. Characteristics of the compounds of interest used m

[0267] The resulting peaks matched the molecular weights and retention times of the compounds of interest.

[0268] LC-MS Entandrophragma caudatum calli samples

[0269] For Entandrophragma caudatum both SAE 4a (compound of Formula lb, wherein R1is methyl) and SAE 4 (compound of Formula la, wherein R1is methyl) were found in all post- methanolysis calli samples as well as in the Entandrophragma caudatum seeds (Fig. 2). The chromatograms were different depending on the plant hormones that were used to grow the callus (Fig. 2). This shows that callus can be established from plants of Entandrophragma caudatum and that these calli comprises the limonoid compounds SAE4 and SAE4a. Fig. 2 shows LC-MS traces of the Entandrophragma caudatum post-methanolysis seed extract (A) and two different callus samples: no 17 (B) and no 3 (C). The peaks for SAE 4a (compound of Formula lb, wherein R1is methyl) and SAE 4 / Phragmalin (compound of Formula la, wherein R1is methyl) are marked. Note that seed extract (A) is the same as "raw A" seed sample in Table 5 and callus sample no 17 and no 3 are representatives of sample 17 A, B and C and 3 A, B and C respectively also presented in Table 5.

[0270] LC-MS of Chukrasia tabularis calli and seeds of Chukrasia tabularis, Entandofragma cantatum and five additional species

[0271] The resulting peaks, matching the characteristics and retention times of the compounds of interest, were integrated and the area under the curve (AUC) conveyed the relative quantity value (Table 10).

[0272] Phragmalin / SAE4 (compound of Formula la, wherein R1is 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 R1is methyl) (Table 10).

[0273] Phragmalin / SAE4 (compound of Formula la, wherein R1is 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.

[0274] Table 10. LC-MS analysis and relative quantitation of SEA 4 / phragmalin (compound of Formula la, wherein R1is methyl) and SEA 4a (compound of Formula lb, wherein R1is methyl) in post-methanolysis extracts from seeds of different mahogany species and Chukrasia tabularis calli (Table 7).

[0275] 2.4. Quantitation of the compounds of interest from Entandrophragma caudatum calli using HPLC-UV

[0276] 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). Several dilutions of 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. Using the 215 nm channel, peaks in the standard samples and peaks in the callus and seed samples (identified by the same retention time as the standard compounds) 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.

[0277] Fig. 3 shows the average amounts (pg / mg of post-methanolysis oil) of SAE 4a (compound of Formula lb, wherein R1is methyl) and SAE 4 (compound of Formula la, wherein R1is methyl) in the post-methanolysis oil across all the Entandrophragma caudatum calluses and seed samples (Table 5). Bars indicate standard deviation.

[0278] All the calli samples of Entandrophragma caudatum contained both SAE 4a (compound of Formula lb, wherein R1is methyl) and SAE 4 (compound of Formula la, wherein R1is methyl). The quantities of the compounds in the post-methanolysis samples were in several cases similar to or surpassing the amounts in the seeds. The highest content of SAE 4a was obtained in callus grown on media III (sample number 3 in the figure).

[0279] Example 3. Entandrophragma caudatum cell suspension cultures

[0280] 3.1. Initiation of the cultures

[0281] Three media 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). After two weeks of culture, 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.

[0282] Fig. 4 shows the example morphology of suspension cells grown in media III, with visible organelles and nuclei (n).

[0283] 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.

[0284] 3.2. Extraction, methanolysis, and LC-MS analysis

[0285] Detection of SAE-4a and SAE-4 in the cell biomass

[0286] The samples were treated in the same manner as the callus samples and all procedures are described in Section 2.2. The extraction and methanolysis conditions are outlined in Table 11.

[0287] Table 11. Outline of Entandrophragma caudatum suspension culture cell biomass extraction and methanolysis conditions.

[0288] 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 R1is methyl) and SAE 4a (compound of Formula lb, wherein R1is 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.

[0289] Fig. 5 shows the example LC-MS traces for the post-methanolysis extract from the cell biomass grown in media III. A, B, the SAE 4a and SAE 4 peaks separated using mass filtering tool. C, crude LC-MS trace of the extract.

[0290] Detection of SAE4a and SAE4 in cell suspension medium

[0291] The three different media compositions in which the cell suspension cultures were grown were tested for the presence of SAE4 (compound of Formula la, wherein R1 is methyl) and SAE4a (compound of Formula lb, wherein R1is methyl) (Table 12).

[0292] 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. However, note that a column with different characteristics (longer, operated in lower flow rate) was used in the LC-MS analysis and therefore the exact retention times (RT) for SAE-4 (compound of Formula la, wherein R1is methyl) and SAE-4a (compound of Formula lb, wherein R1is methyl) are different than for the other analyses presented herein. The compounds were identified by molecular weights and retention time (Fig. 6). Mass chromatograms for each compound were generated using the compound characteristics (Table 13) and mass filtering tool.

[0293] As shown in figure 6, 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.

[0294] 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 *.

[0295] Example 4. Summary of Entandrophragma caudatum cell fast growing suspension cultures

[0296] 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.

[0297] Seed germination

[0298] Fresh seeds were provided by Dicot that were viable and germinated.

[0299] 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.

[0300] • Different types of callus tissue proliferated on most of the media.

[0301] • The best results in terms of callus proliferation were obtained on media supplemented with 2,4-D alone (1-2 mg / L) or in combination with KIN (1-2 mg / L) or NAA / IAA combined with TDZ.

[0302] Extraction, methanolysis, LC-MS, and HPLC-UV quantitative analysis

[0303] ® Small-scale extraction methanolysis and LC-MS analysis were trialled.

[0304] • LC-MS traces of the reference compounds and extracts were obtained: phragmalin (SAE4), SAE4a, Entandrophragma caudatum seed extract pre- and post- methanolysis.

[0305] • SAE4 and SAE4a were found in the E. caudatum seeds but not in the calluses. This indicates that the methanolysis process on a small scale poses difficulty and requires further development.

[0306] • The LC-MS analysis of raw methanol extracts showed multiple different limonoids and that the composition can be manipulated with plant growth regulators used in in vitro culture.

[0307] • Fragments specific to SAE4 and SAE4a were found in LC-MS / MS spectra of extracts and pure compounds hinting at the possibility of developing a method of detection and quantitation of the compounds in pre-methanolysis samples.

[0308] • All LC-MS data and extracts have been stored for future re-evaluation.

[0309] Suspension culture initiation

[0310] ® Fast-growing suspension cultures were successfully initiated from callus grown on media III and X, prepared using Gamborg B5 basal media.

[0311] ® The material from in vitro culture produced during the project was used to initiate fast-growing suspensions ready to be tried in a bioreactor setup.

[0312] 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.

[0313] 5.1 Seed germination

[0314] 5.1.1 Plant material

[0315] The seed germination was performed October-November 2023 (Exp. 1) and April-May 2024 (Exp. 2). Dicot supplied the following seeds in October 2023:

[0316] Producer Parceval Pharmaceuticals, South Africa

[0317] Species Entandrophragma caudatum

[0318] Origin Zimbabwe

[0319] Batch D0377 / 22

[0320] 5.1.2 Sowing in sterile conditions for in vitro culture

[0321] 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.

[0322] The age of the seeds appeared to be important for the germination rates. The same batch of seeds, collected in the year 2022, was used in the Dicot I Project, where the germination was performed in late 2022-early 2023. There, the germination rates were >90%, whereas, in Exp. 2 of the current project (mid-2024), the rates fell below 50%.

[0323] 5.2 Entandrophraama caudatum callus cultures

[0324] 5.2.1 Callus initiation

[0325] 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.

[0326] Three plates with 10 (Exp. 1; Figure 7) or 8 (Exp. 2; Figure 8) explants each (30 or 24 explants overall) were prepared for each medium. Explants produced different callus tissue on most of the media. The photographic documentation of the cultures is presented in Figures 7 and 8. The best outcome in terms of the callus size and percentage of explants producing callus was noted on media containing auxin 2,4-D combined with KIN and IAA, NAA combined with TDZ after three weeks of culture (Table 14, 15, 16, 17 and Figures 7 and 8). Root-like structures formed on the cotyledons cultured on some of the media (Table 14, 15, 16, 17 and Figures 7 and 8).

[0327] Table 14. The combinations of plant growth regulators used in E. caudatum in vitro callus cultures trials Exp. 1

[0328] Table 15. The combinations of plant growth regulators used in E. caudatum in vitro callus cultures trials Exp. 2

[0329] Table 16. Exp. l E. caudatum cotyledon callus cultures overview. Three plates with 10

[0330] Table 17. Exp. 2 E. caudatum cotyledon callus cultures overview. Three plates with 10 explants each (30 explants overall) were prepared for each medium.

[0331] 5.2.2 Extraction, methanolysis, and LC-MS analysis

[0332] The extraction and methanolysis procedures were standardized across all the samples to adjust the methanolysis process to a small scale and simplify it. We assumed that the quantities of the compounds would not differ greatly between samples and used fixed amounts of reagents and solvents for extraction and methanolysis. This allowed us to avoid weighting sub-mg amounts of oil, likely introducing a lot of variation. Enhancing and speeding up the phase separation process by performing it in Falcon test tubes and using centrifugation, was also possible.

[0333] Extraction of the callus samples

[0334] 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).

[0335] 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.

[0336] Methanolysis

[0337] All samples from Exp. 1 and selected samples from Exp. 2 underwent methanolysis (98 reactions overall). To obtain the pre-methanolysis oil, 25 ml of water and 10 ml of ethyl acetate were added to raw methanol extracts in 50 ml Falcon tubes. The mixture was shaken and then centrifuged at 3000 rpm for phase separation. Subsequently, the upper ethyl acetate layer was collected and evaporated to dryness. For methanolysis, the pre- methanolysis oil was dissolved in 10 ml of methanol, transferred to a 50 ml round bottom flask, and 1 mg of sodium methoxide in 1 ml of methanol was added. 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.

[0338] LC-MS analysis

[0339] 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. 1 were analyzed with LC-MS, and the raw data can be sent upon request. The reference samples (Entandofragma cantatum post-methanolysis seed extract and purified SAE 4 / 4a compounds) were supplied by Dicot and analyzed with the same LC-MS system and conditions as other samples (Figs. 9 and 10). All analyses were performed using MassLynx 4.2 software (Waters). Mass chromatograms for each of the compounds were generated using the compound characteristics (Table 18) and mass filtering tool.

[0340] Table 18. Characteristics of the compounds of interest used in LC-MS data analysis

[0341] The resulting peaks, matching the characteristics and retention times of the compounds of interest, were considered the compounds of interest.

[0342] SAE 4a and SAE 4 were not found in any of the post-methanolysis calli samples. Problems with small-scale methanolysis probably caused this result, e.g., high instability of sodium methoxide necessary for the process.

[0343] Possibility of detection and semi-quantitation of SAE 4 / 4a in pre-methanolysis extracts The LC-MS analysis revealed a host of other limonoids present in the samples identified based on the fragmentation spectra. The composition differed depending on the plant hormones used to grow the callus. All the raw LC-MS data is stored on the server at Pharmacognosy, Uppsala University, and can be supplied upon request.

[0344] 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. This emphasizes the need to develop a semi-quantitative, LC-MS-based method for identifying limonoid core structures (SAE 4 and SAE 4a precursors) in raw pre-methanolysis extracts. We have shown that unique fragment signature to SAE 4 core molecule in MSe high-energy MSMS spectra LC-MS analysis of post- and premethanolysis extracts (Fig. 10). Based on this finding, it was possible to identify molecules sharing the same core molecular structure with SAE 4 (Fig. 10). These molecules can be quantified, and the sum of their abundances can be a predictor for quantifying SAE 4 in the sample post-methanolysis.

[0345] All the LC-MS data from the raw methanol extracts will be re-evaluated once the semiquantitative method based on fragments specific for SAE 4 / 4a is established during the separate Dicot Smiquant Project planned for January-March 2025. This will allow us to confirm the presence of the compounds and relatively quantify them without the need for methanolysis.

[0346] 5.3 E. caudatum cell suspension cultures

[0347] 5.3.1 Initiation of the cultures

[0348] 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). After two weeks of culture, the larger pieces of the callus were drained, and 50 ml of fresh liquid media was added to the cultures. The resulting suspensions were placed back in the culture cabinet and shaken for 3 weeks. During that time, the suspensions were sampled for microscopy evaluation. The best results were achieved when Gamborg B5 media was used for both callus production and suspensions. These cultures showed viable, dividing cells, small aggregates (Fig. 11), and visible growth. The cultures are passaged every two weeks by taking 25 ml of cultures and filling up to 100 ml with fresh media.

[0349] The possible explanation for how 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.

[0350] Table 19. Com

[0351] 5.3.2 Suppling material for scale-up trials

[0352] Material from in vitro cultures, including callus and fast-growing suspensions generated on media containing Gamborg B5 basal media, generated in the course of the project, was used for these data. The characteristics of the material used are outlined in Tables 20 and 21.

[0353] Table 20. Characteristics of the first batch of material used in this project, including samples of callus grown on media containing 0.5 mg / L or 1 mg / L TDZ (G 1, G 0.5), callus from media III and samples of biomass filtered from suspensions and suspensions in E- flasks.

[0354] Table 21. Characteristics of the second batch of material used in this project, including samples of suspensions from niedja III and grown

[0355] Example 6. Production of Picot's Entandrophraama caudatum Cell Culture, Part 1

[0356] Nine plates with explants were provided, one plate with cell suspension put on a filter in a plate and one T-flask with cell suspension.

[0357] Upon arrival, 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.

[0358] 6.1 Cultivation Media GUI

[0359] For the preparation of culture medium Gill, a basal salt mixture is used. This mixture derives from PhytoTech Labs (Kansas, USA) and is Gamberg 8-5 Basal Medium (G398). For media preparation, 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.

[0360] 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.

[0361] 6.2 Establishment of cell line ENT006

[0362] 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.

[0363] After an adaptation phase of approximately 4 weeks, a constant increase in biomass was observed. A duplication of cell growth was accomplished in just 7 days. At initiation, 125 ml Erlenmeyer flasks were used. As biomass increased, shake flasks were adjusted in size to accommodate larger media volumes. Presently, the culture is sustained in 1000 ml Erlenmeyer flasks with a culture volume of 200 ml (20 g inoculation density (100 g / L)).

[0364] The quality of cell morphology improved as transfer cycles increased (Figure 15).

[0365] With each transfer, the biomass of the cell culture became lighter and lighter. The suspension is currently light yellow and can be smoothly vacuum-filtrated. Regular tests on foreign growth are performed on bacterial screening agar confirming absence of foreign growth.

[0366] 6.3 Conclusion

[0367] Successful establishment of one suspension culture derived from Entandrophragma caudatum was achieved using premature callus material sent by Dicot. The culture exhibits a good growth performance in liquid culture, with a growth index of approximately 2 - 2.5 (quotient of end fresh weight and inoculum). The culture is currently being maintained on a litre scale and might be ready for a further upscale in Fernbach flasks and possibly for the inoculation of laboratory fermenters. A back-up culture in a 250 ml Erlenmeyer flask is in place, too. Biomass samples of ent006 were taken and freeze-dried. Example 7. Production of Picot's Entandrophraama caudatum Cell Culture, Part 2

[0368] This work is based on the initial work carried out in Example 6.

[0369] 7.1 Establishment of cell line ENT006

[0370] 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.

[0371] 7.2 Cultivation media GUI

[0372] 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). For media preparation, 3.21 g of 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.

[0373] 7.3 Properties of the cell culture ENT0006

[0374] 7.3.1 Cell growth

[0375] 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.

[0376] Cell growth of f. caudatum is represented by the growth index (Gb). This 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 13th.

[0377] 7.3.2 Macroscopic appearance of ent006

[0378] Figure 17 shows three individual pictures of vacuum-filtrated biomass from Erlenmeyer flasks.

[0379] 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.

[0380] 7.3.3 Cell viability of ent006

[0381] The cell viability of the culture was analysed by fluorescence microscopy with an FDA staining (fluorescein diacetate). Only living cells actively convert the nonfluorescent FDA into the green, fluorescent compound fluorescein following enzymatic activity, which is considered a sign of viability. Figure 18 shows some microscopic images of the cell culture ent006 (flask 3 / 3).

[0382] 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%.

[0383] 7.3.4 Tests on foreign growth

[0384] Routine in-house tests using microbiological detection media confirmed that the culture was free of microbial contamination. Since the culture began in May 2024, 43 individual tests for foreign growth have been performed. All of them have been negative. The flasks will be checked regularly.

[0385] List of abbreviations

[0386] 2,4-D - 2,4-Dichlorophenoxyacetic acid

[0387] ACN - acetonitrile

[0388] BAP - 6-Benzylaminopurine

[0389] IAA - indole-3-acetic acid

[0390] FA - formic acid

[0391] KIN - kinetin

[0392] NAA - 1-Naphthaleneacetic acid

[0393] MS - Murashige and Skoog Medium

[0394] B5 - Gamborg B5

[0395] TFA - trifluoro acetic acid

[0396] References

[0397] 1. Murashige, T. & Skoog, F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plant. 15, 473-497 (1962).

Claims

Claims:

1. 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:wherein R1is 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 R1is a methyl, wherein R2, R3and R4independently are selected from hydrogen, nicotinoyl or - C(O)R5; where R5is 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, R3and R4independently are selected from hydrogen, acetyl, propyl, isopropyl, butyryl, isobutyryl, pentanoyl, iso- pentanoyl or acyl, said method comprising:I. culturing plant cells in a nutrient medium in a cell culture, wherein the cells produce the compound of Formula Ila and / or lib and / or one or more esters or derivatives thereof;II. recovering the compound of Formula Ila and / or lib and / or the one or more esters or derivatives thereof produced in step I.

2. The method of claim 1, wherein the method obtains at least one of the compounds of Formula la or lb;wherein R1is 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.

3. The method of claim 1 or 2, wherein R1is methyl.

4. The method of any of claims 1-3, 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.

5. The method of claim 4, wherein 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.

6. The method of claim 4 or 5, wherein the explant / s from said plant are cultured for 1- 10 weeks or 2-8 weeks or 3-6 weeks or 2-5 weeks; and / or 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.

7. The method according to any of claims 4-6, wherein the explant / s from said plant are cultured under controlled illumination.

8. The method according to any of claims 1-7, wherein 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; such as gibberellins, auxins, abscisins, ethylene, brassinosteroids, strigolactones and / or cytokinins; optionally wherein the plant hormone / s or growth regulator / s is / are selected from the group consisting of 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-benzylaminopurine, kinetin (KIN), thidiazuron (TDZ), and combinations thereof.

9. The method of claim 8, wherein the auxin plant hormone is at least one of 2,4-D, NAA or IAA; and / or the cytokinin plant hormones is at least one of KIN and / or TDZ.

10. The method of claim 8 or 9, wherein two or more of said plant hormones or growth regulators are used in the method in any combination.

11. The method of claim 10, wherein the combination of plant hormones or growth regulators at least comprises: (i) 2,4-D and KIN; or (ii) NAA and TDZ.

12. The method of claims 8-11, wherein at least one of the plant hormones or growth regulators is 2,4-D or NAA.

13. The method according to any of claims 8-12, wherein the concentration of each plant hormone / s or growth regulator / s is 0.01-10 mg / l in said nutrient media.

14. The method according to any of claims 1-13, wherein the plant cells of the cell culture are cultured under controlled illumination.

15. The method according to any of claims 1-14, wherein 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, preferably at 25°C + / - 2°C.

16. The method according to any of claims 1-15, wherein 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.

17. The method according to any of the preceding claims, wherein the plant cells in step I of the method are cultured as a suspension cell culture.

18. The method according to claim 17, wherein said suspension cell culture is a continuous culture for growing cells in suspension.

19. The method according to any of claims 1-18, wherein at least one of the compounds of Formula Ila or lib and / or one or more esters and / or derivatives thereof, and / 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.

20. The method according to any of claims 1-18, wherein at least one of the compounds of Formula Ila or lib and / or one or more esters and / or derivatives thereof, and / 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.

21. The method according to any of claims 1-18, wherein at least one of the compounds of Formula Ila or lib and / or one or more esters and / or derivatives thereof, and / 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.

22. The method according to any of claims 1-21, wherein said plant cells and / or said explant / s and / or fragments thereof originate from a plant selected from the Meliaceae family.

23. The method according to claim 22, 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.

24. The method according to claim 23, 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.

25. An isolated plant cell obtained from the method of any of claims 1-18 or 22-24.

26. An isolated plant cell line obtained or originating from the method of any of claims 1- 18 or 22-24.

27. An isolated plant cell batch obtained or originating from the method of any of claims 1- 18 or 22-24.

28. Suspension cell culture obtained or originating from the isolated plant cell line of claim 26 and / or the isolated plant cell batch of claim 27.

29. A plant cell culturing medium obtained or originating from the plant cell culture of the method of any of claims 1-24, from which the plant cells have been removed, which comprises a non-cell biomass derived from the plant cells.

30. A plant cell biomass comprising plant cells obtained from the friable callus material according to the method of any of claims 4-7 and / or the cell culture of the method according to any of claims 1-3 or 8-18 or 22-24.

31. An isolated explant / s or fragment / s thereof obtained or originating from the method of any of claims 4-7.

32. Use of the cell culturing medium or the non-cell biomass of claim 29; or the plant cell biomass of claim 30 as a medicament.

33. 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 as presented in any of claims 1-24, respectively; from the plant cell line and / or from the plant cell batch and / or from the suspension cell culture of claims 26-28; from the culturing medium and / or non-cell biomass of claim 29; or from the plant cell biomass of claim 30; for producing a compound of Formula V, wherein R1preferably is methyl and R2is a straight, branched or cyclic optionally substituted alkyl group with 1 to 6 carbon atoms; or a compound of Formula VI:Formula V Formula VI.

34. The use according to claim 33, wherein Formula V, R1 preferably is methyl and R2 is isopropyl.

35. Use of a compound of Formula V or a compound of Formula VI obtained from the use as specified in claim 33 or 34, in a medicament.

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