METHOD FOR PRODUCING A COMPOSITION COMPRISING A 3-O-p-COUMAROYL ESTER OF TORMENTIC ACID FROM A PLANT CELL CULTURE, APPLICATIONS THEREOF AS ANTIPARASITIC AGENT FOR THE TREATMENT OF TRYPANOSOMIASIS

Abstract

The present invention relates to a method for the production of a (poly)hydroxylated pentacyclic triterpene composition including a 3-O-p-coumaroyl ester of tormentic acid from a plant suspension cell culture, to a pharmaceutical composition comprising at least 3-O-p-coumaroyl ester of tormentic acid for a use in the prevention and/or the treatment of trypanosomiasis, optionally in admixture with other (poly)hydroxylated pentacyclic triterpenes, and to 3-O-p-coumaroyl ester of tormentic acid for its use as an antiparasitic agent for the prevention and/or the treatment of trypanosomiasis, optionally in admixture with other (poly)hydroxylated pentacyclic triterpenes.

Claims

1. A method for producing, from a plant cell suspension culture, a composition comprising a mixture of (poly)hydroxylated pentacyclic triterpenes including at least a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof, wherein said method comprises at least the following steps: 1) providing a suspension-cultured cell line capable of producing a mixture of (poly)hydroxylated pentacyclic triterpenes including at least a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof, said suspension-cultured cell line being cultured in a liquid culture medium from a callus of a plant selected in the group of Rosaceae and Sapotaceae families; 2) adding in said liquid culture medium at least one elicitor and culturing the suspension-cultured cell line of step 1) in said liquid culture medium during a period of time sufficient to produce said mixture of (poly)hydroxylated pentacyclic triterpenes including at least a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof; 3) extracting said mixture of (poly)hydroxylated pentacyclic triterpenes including at least a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof from the liquid culture medium with a solvent, to obtain a first composition comprising said mixture of (poly)hydroxylated pentacyclic triterpenes including a first concentration C1 of a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof in said solvent, 4) submitting the first composition of step 3) to a silica gel chromatography to obtain a second composition comprising a mixture of (poly)hydroxylated pentacyclic triterpenes including a second concentration C2 of said 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof, with C2 being higher than C1.

2. The method according to claim 1, wherein the plant of the group of the Rosaceae family is selected in the group comprising the species Malus x domestica and the plant of the group of Sapotaceae is selected in the group comprising the species Vitellaria paradoxa.

3. The method according to claim 1, wherein the callus used to prepare the suspension-cultured cell line of step 1) is obtained from the fruits when the plant belongs to the Rosaceae family or from the leaves when the plant belongs to the Sapotaceae family.

4. The method according to claim 1, wherein the callus used to prepare the suspension-cultured cell line of step 1) is a callus obtained from a fruit of the Rosaceae family, more preferably from fruits of apples.

5. The method according to claim 1, wherein said derivative of 3-O-p-coumaroyl ester of tormentic acid is represented by formula (II) below: ##STR00005## wherein R.sup.1 to R.sup.15 are each individually selected in the group consisting of H, OH, O-alkyl, alkyl, ═O, CH.sub.2OH, COOH and COO-alkyl; R.sup.16 to R.sup.20, are each individually selected in the group consisting of H, OH, O-alkyl, alkyl, ═O, CH.sub.2OH, COOH and X, wherein X=F, Cl or Br; the bond represented by a continuous line doubled with a dotted line corresponds either to a single bond or a double bond.

6. The method according to claim 1, wherein said elicitor is selected in the group comprising abscisic acid, auxins, brassinosteroids, cytokinins, ethylene, gibberellins, salicylic acid, strigolactones and jasmonates.

7. The method according to claim 1, wherein said elicitor is selected in the group comprising jasmonates.

8. The method according to claim 1, wherein the liquid culture medium used during step 2) comprises sugar as a carbon source and at least one additional plant hormone in the auxin family.

9. The method according to claim1, wherein the liquid culture medium during step 2) is a Linsmaier and Skoog medium further comprising sucrose as carbon source, and 1-naphtaleneacetic acid and 2,4-dichlorophenoxyacetic acid as additional plant hormone in the auxin family.

10. The method according to c;aim1, wherein step 2) is carried out at a temperature ranging from 20 to 25° C., during a period of time ranging from 1 week to 4 weeks.

11. The method according to claim 1, wherein the solvent used during step 3) is chosen among ethyl acetate, hexane, n-butanol, dichloromethane, ethanol, methanol, acetone, and mixtures thereof.

12. The method according to claim 1, wherein the mixture of (poly)hydroxylated pentacyclic triterpenes present in the first composition obtained at the end step 3) comprises 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid and at least one pentacyclic triterpene compound selected in the group comprising tormentic acid, maslinic acid, annurcoic acid and corosolic acid.

13. The method according to claim 1, wherein at the end of step 4), the second composition comprises a concentration C2 of 3-O-trans-p-coumaroyltormentic acid and/or of 3-O-cis-p-coumaroyltormentic acid of at least 10 weight % with regards to the total weight of the mixture of (poly)hydroxylated pentacyclic triterpenes present in said second composition.

14. The method according to claim 1, wherein when the callus used in step 1) is obtained from a plant of Rosaceae family, in particular Malus x domestica, then the mixture of (poly)hydroxylated pentacyclic triterpenes of the second composition obtained at the end of step 4) comprises from 10 to 33 weight % of 3 -O-trans-p-coumaroyltormentic acid, from 1 to 6 weight % of 3-O-cis-p-coumaroyltormentic acid, from 6 to 20 weight % of tormentic acid, from 9 to 21 weight % of maslinic acid, from 7 to 32 weight % of annurcoic acid and from 5 to 12 weight % of corosolic acid with regards to the total weight of the mixture of (poly)hydroxylated pentacyclic triterpenes present in said composition.

15. The method according to claim 14, wherein the second composition obtained at the end of step 4) comprises: i) 26 weight % of 3-O-trans-p-coumaroyltormentic acid, 4 weight % of 3 -O-cis-p-coumaroyltormentic acid, 20 weight % of tormentic acid, 16% weight % of maslinic acid, 12 weight % of annurcoic acid and 9 weight % of corosolic acid, or ii) 16 weight % of 3-O-trans-p-coumaroyltormentic acid, 5 weight % of 3-O-cis-p-coumaroyltormentic acid, 11 weight % of tormentic acid, 21% weight % of maslinic acid, 31 weight % of annurcoic acid and 12 weight % of corosolic acid, or iii) 33 weight % of 3-O-trans-p-coumaroyltormentic acid, 1.5 weight % of 3-O-cis-p-coumaroyltormentic acid, 8 weight % of tormentic acid, 29 weight % of annurcoic acid, 13.5 weight % of maslinic acid, and 7.5 weight % of corosolic acid.

16. The method according to claim 1, wherein said method further comprises an additional step 5) of isolating said 3-O-p-coumaroyl ester of tormentic acid from the second composition obtained at the end of step 4), in order to obtain a third composition containing only 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid.

17. A composition comprising a mixture of (poly)hydroxylated pentacyclic triterpenes including at least a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof, wherein said composition may be obtained by the method defined in claim 1, and wherein the mixture of (poly)hydroxylated pentacyclic triterpene comprises at least 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid and at least one triterpenic compound selected in the group comprising tormentic acid, maslinic acid, annurcoic acid and corosolic acid, and wherein the concentration C2 of 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid is of at least 10 weight % with regards to the total weight of the mixture of (poly)hydroxylated pentacyclic triterpenes present in said composition.

18. The composition according to claim 17, in which the callus used in step 1) is obtained from a plant of Rosaceae family, in particular Malus x domestica, then said composition comprises from 10 to 33 weight % of 3-O-trans-p-coumaroyltormentic acid, from 1 to 6 weight % of 3-O-cis-p-coumaroyltormentic acid, from 6 to 20 weight % of tormentic acid, from 9 to 21 weight % of maslinic acid, from 7 to 32 weight % of annurcoic acid and from 5 to 12 weight % of corosolic acid with regards to the total weight of the mixture of (poly)hydroxylated pentacyclic triterpenes present in said composition.

19. A 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof for a use as a drug for the prevention and/or the treatment of trypanosomiasis.

20. The 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof for a use as a drug according to claim 19, wherein the 3-O-p-coumaroylester of tormentic acid is 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid.

21. A pharmaceutical composition comprising, as an active principle, a 3-O-p-coumaroyl ester of tormentic acid and/or a derivative thereof and at least one pharmaceutically acceptable excipient for a use in the prevention and/or the treatment of trypanosomiasis.

22. The pharmaceutical composition for a use in the prevention and/or the treatment of trypanosomiasis according to claim 21, wherein said pharmaceutical composition comprises 3-O-trans-p-coumaroyltormentic acid and/or 3-O-cis-p-coumaroyltormentic acid.

23. The pharmaceutical composition for a use in the prevention and/or the treatment of trypanosomiasis according to claim 21, wherein said pharmaceutical composition further comprises at least one additional pentacyclic triterpenic compound selected in the group comprising tormentic acid, maslinic acid, annurcoic acid and corosolic acid.

24. The pharmaceutical composition for a use in the prevention and/or the treatment of trypanosomiasis according to claim 21, wherein said pharmaceutical composition comprises a mixture of (poly)hydroxylated pentacyclic triterpenes comprising from 10 to 33 weight % of 3-O-trans-p-coumaroyltormentic acid, from 1 to 6 weight % of 3-O-cis-p-coumaroyltormentic acid, from 6 to 20 weight % of tormentic acid, from 9 to 21 weight % of maslinic acid, from 7 to 32 weight % of annurcoic acid and from 5 to 12 weight % of corosolic acid with regards to the total weight of the mixture of (poly)hydroxylated pentacyclic triterpenes present in said composition.

Description

[0092] Besides the arrangements above, the invention also comprises other arrangements which will emerge from the following description, which refers to examples of preparation of a mixture of (poly)hydroxylated pentacyclic triterpenes comprising 3-O-cis/trans-p-coumaroyltormentic acid according to the invention, to in vitro demonstration of the anti-trypanosomial activity of 3-O-cis/trans-p-coumaroyltormentic acid and to in vivo demonstration of the anti-trypanosomial activity of 3-O-cis/trans-p-coumaroyltormentic acid and also to the annexed FIGS. 1 to 3 in which: [0093] FIG. 1 gives the chromatographic profiles recorded at 200 nm of three pentacyclic triterpene compositions respectively obtained from callus of Malus x domestica “Cox's Orange Pippin” cultivar, Malus x domestica “Spartan” cultivar and Malus x domestica “Golden Delicious” cultivar according to the method of the invention; [0094] FIG. 2 reports the evolution of parasitemia (Log Trypanosoma/mL) as a function of time (in days) in mice infested intraperitoneally with 10.sup.4 Trypanosoma brucei brucei and treated intraperitoneally by 3-O-cis/trans-p-coumaroyltormentic acid or ursolic acid (UA) at 50 mg/kg at day 3 after infection with the parasite and then every day until day 7 post-infection. Positive control: suramine (0.5 mg/kg); [0095] FIG. 3 reports the results of mice survival. On this figure, the percentage of survivals in given as a function of time (in days). The values with full squares correspond to the negative control, the values with crosses correspond to mice administered with 3-O-cis/trans-p-coumaroyltormentic acid, the values with full triangles correspond to mice administered with ursolic acid and the values with full dots correspond to the positive control, i.e. to mice administered with suramine.

EXAMPLES

Example 1: Preparation of a (poly)hydroxylated pentacyclic triterpenes Composition According to the Method of the Invention

[0096] In this example, a (poly)hydroxylated pentacyclic triterpenes compositions including 3-O-trans-p-coumaroyl ester of tormentic acid and 3-O-cis-p-coumaroyl ester of tormentic acid and other triterpene compounds has been produced from three different calli of Malus x domestica cultivars: “Cox's Orange Pippin”, “Spartan” and “Golden Delicious”. These compositions have further been fractionated to isolate the different pentacyclic triterpenes comprised therein.

1) Materials and Method

1.1. Preparation of the Suspension-Culture Cell Line

[0097] The callus of the apple cultivar (Malus x domestica “Cox's Orange Pippin”, “Spartan” and “Golden Delicious”) were obtained from the Leibniz Institute DSMW (Germany). The calli were cultured on solid fresh Linsmaier and Skoog medium in the dark and subcultured monthly. Cell suspensions were established by resuspending 2-cm callus pieces in liquid Linsmaier and Skoog medium, and subculturing weekly by transferring 30-90% (v/v) of the culture into 50 mL fresh liquid Linsmaier and Skoog medium and incubating at 23° C., with an orbital shaking speed of 140 rpm. Once the cell suspension culture was established, the cells were further subcultured at 15-d intervals by transferring 50% (v/v) into fresh liquid Linsmaier and Skoog medium.

1.2. Elicitation

[0098] Methyl jasmonate (Sigma Aldrich 392707-25ML) at 50 μM was added 7-days after sub-subculturing. Cells were harvested after 8-days of incubation at 23° C.

1.3. Extraction of (poly)hydroxylated pentacyclic triterpenes

[0099] 50 mL of the suspension cells obtained hereabove at the end of step 1.2. were mixed with 200 mL of ethanol, homogenized, sonicated (37 kHz, 1200 W) for 10 min and shaken for 4 h at 4° C. Samples were then vacuum filtered and stored at 4° C. until fractionation.

1.4. Fractionation

[0100] A 5 g C18 Isolute® Solid-Phase Extraction (SPE) cartridge (Biotage, Sweden) was conditioned with 10 mL of ethanol (EtOH), then 10 mL of 1:1 EtOH/H.sub.2O (v:v), and then 10 mL of 25:75 EtOH/H.sub.2O (v:v). Each of the three extracts obtained hereabove at the end of step 1.3. (250 mL) (“Cox's Orange Pippin”, “Spartan” and “Golden Delicious” respectively) was coated onto 5 g C18 (silica gel) by rotary evaporation at 40° C. and applied to the preconditioned SPE cartridge. This was eluted with 2×10 mL each of 25:75 EtOH/H.sub.2O (v:v) (Fractions (F) 1 and 2), 50:50 EtOH/H.sub.2O (v:v) (F3-F4), 65:35 EtOH/H.sub.2O (v:v) (F5-F6), 75:25 EtOH/H.sub.2O (v:v) (F7-F8), and 85:15 EtOH/H.sub.2O (v:v) (F9-F10), and 100:0 EtOH/H.sub.2O (v:v) (F11-F12).

1.5. Triterpene Identification and Quantification

[0101] For each extract, F7, F8, and F9 contained all triterpenes.

[0102] The different fractions F7-F9 obtained hereabove at step 1.4 for each of the three extracts were compared at 200 nm with a Waters Acquity UPLC (Ultra-Performance Liquid Chromatography) system (Milford, MA, USA) hyphenated to a Diode Array Detector (UPLC-DAD). The separation of the 5 ∥L aliquot was performed on a reverse-phase Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm particle size, Waters, Milford, Mass., USA). The eluents were 0.05% o-phosphoric acid in water (A) and 0.05% o-phosphoric acid in methanol (B). The gradient was as follows: 0 min, 75% B; 2 min, 75% B; 16 min, 82% B; 25 min, 100% B; 26.5 min, 100% B; 27 min, 75% B; 30 min, 75% B. The flow rate was of 0.3 mL min.sup.−1 and the column temperature was 40° C. For identification, a high-resolution time of flight mass spectrometer (HR-MS) (TripleTOF 5600+, AB Sciex, Concord, Ontario, Canada) was used.

2) Results

[0103] For each extract, F7, F8, and F9 contained all triterpenes.

[0104] The chromatographic profiles of the pentacyclic triterpene compositions thus obtained for each of the three fractionated extracts are reported on FIG. 1 annexed. On this figure, intensity of the peaks (in arbitrary units: AU) is expressed as a function of times (in minutes). On this figure, the dotted line corresponds to the profile of the triterpene composition obtained starting from the Malus x domestica “Cox's Orange Pippin” callus, the full line corresponds to the profile of the triterpene composition obtained starting from the Malus x domestica “Spartan” callus, and the dashed line corresponds to the profile of the triterpene composition obtained starting from the Malus x domestica “Golden Delicious” callus. All three triterpene compositions included tormentic acid (peak 1), annurcoic acid (peak 2), 3-O-cis-p-coumaroyltormentic acid (peak 3 (cis)), 3-O-trans-p-coumaroyltormentic acid (peak 4 (trans)), maslinic acid (peak 5), corosolic acid (peak 6), oleanolic acid (7), and ursolic acid (8).

[0105] It emerges from these profiles that 3-O-trans-p-coumaroyltormentic acid is the major constituent of the three (poly)hydroxylated pentacyclic triterpene compositions thus obtained. It can also be noted, that the fractions obtained from the callus of Malus x domestica “Cox's Orange Pippin” cultivar have the highest quantity of (poly)hydroxylated pentacyclic triterpenes and coumaroyl derivatives.

[0106] The quantitative composition of the (poly)hydroxylated pentacyclic triterpene mixture present in the fractions obtained from the callus of Malus x domestica “Cox's Orange Pippin” cultivar is given in Table 1 below:

TABLE-US-00001 TABLE 1 TRITERPENES Amount (weight %) 3-O-trans-p-coumaroyltormentic acid 26 3-O-cis-p-coumaroyltormentic acid 4 Tormentic acid 20 Maslinic acid 16 Annurcoic acid 12 Corosolic acid 9 Other triterpenes 13

Example 2: Up-Scaled Preparation of a (poly)hydroxylated Pentacyclic Triterpenes Composition According to the Method of the Invention

[0107] In this example, the culture of the “Cox's Orange Pippin” cell line described in example 1 paragraph 1.1. was further up-scaled in a lab scale bioreactor instrument.

1) Materials and Method

1.1. Environmental Parameters Set for the Cell Suspension Culture in Bioreactor

[0108] A 4 L bioreactor instrument with flat bottom vessel (Infors HT—minifors 2) and equipped with two 5 cm-diameter impellers adjusted at 0 and 16 cm from the bottom end of the stirring bar was used for the present example. The cell line was inoculated at 20% (v/v) into the reactor filled with a Linsmaier and Skoog medium supplemented with 30 g/L sucrose, 0.2 mg/L of 1-naphtaleneacetic acid and 0.2 mg/L 2,4-dichlorophenoxyacetic acid. The stirring speed was at 150 rpm (revolutions per minute) to prevent (i) any deposition of the cell aggregates at the bottom of the vessel and (ii) an excessive shearing stress. The oxygenation of the medium was supported by an air sparging system set at 0.125 v.v.m. (Vessel Volume per minute). Using these environmental conditions, the kLa (liquid phase mass transfer coefficient) measured in the medium without cells at 23° C. was equal to 0.0735 min.sup.−1. The batch was run for three weeks to reach to stationary phase.

1.2. Elicitation

[0109] Methyl jasmonate (Sigma Aldrich 392707-25ML) was added at the beginning of the stationary phase to reach a final concentration of 50 μM. Cells were harvested after 8-days of incubation at 23° C. Cells were separated from the medium using vacuum filtration, flash frozen in liquid nitrogen and freeze-dried.

1.3. Extraction of (poly)hydroxylated Pentacyclic Triterpenes

[0110] The total dried cells material (65 g) obtained at the end of step 1.2 was added to 10 L ethanol using a custom-made pilot scale Pignat Solid-liquid extraction system. The mixture was sonicated (37 kHz, 1200 W) for 10 min followed by a mixing step of 2 h at room temperature. The extract was collected and evaporated using a Büchi R-300 rotavapor and re-suspended in 100% EtOH solution.

1.4. Triterpene Purification

[0111] The sample extract obtained after step 1.3 was pre-conditioned using 5 g C18 (Aldrich octadecyl-functionalized silica gel). The triterpene extract was purified using a Reveleris flash chromatography system and a 12 g Reveleris C18 column (Büchi) using a solid type injection, a 30 mL/min flow rate and 5 min cartridge equilibration. Pentacyclic triterpenes were detected using UV wavelength set at 220 nm and 240 nm. A gradient table was set as followed: step 1: time 0 min-65% EtOH, step 2: time 8 min-75% EtOH. Fractions were collected from step2 and were further analyzed as described in example 1 paragraph 1.5.

2) Results

[0112] The quantitative composition of the (poly)hydroxylated pentacyclic triterpene composition of the fractions obtained from the callus of Malus x domestica “Cox's Orange Pippin” cultivar is given in Table 2 below:

TABLE-US-00002 TABLE 2 TRITERPENES Amount (weight %) 3-O-trans-p-coumaroyltormentic acid 16 3-O-cis-p-coumaroyltormentic acid 5 Tormentic acid 11 Maslinic acid 21 Annurcoic acid 31 Corosolic acid 12 Other triterpenes 4

Example 3: In Vitro Anti-Trypanosomal Activity of 3-O-cis/trans-p-coumaroyltormentic Acid in Comparison to Different Pentacyclic Triterpene Compounds

[0113] In this example, the in vitro antiparasitic activity of 3-O-trans-p-coumaroyltormentic acid isolated from the fractions obtained at the end of step 1.4 of example 1 with the callus of Malus x domestica “Cox's Orange Pippin” cultivar, was compared to that of suramine, a commercial anti-trypanosomal drug and to different pentacyclic triterpene compounds.

1) Materials and Method

1.1. Isolation and Identification

[0114] For isolation, the different fractions F7-F9 obtained hereabove at step 1.4 of example 1 with the callus of Malus x domestica “Cox's Orange Pippin” cultivar, were submitted to preparative High Pressure Liquid Chromatography (HPLC) consisting of a Shimadzu ® LC-20AP pump hyphenated with a Spd-20AV UV detector. The column used was a Phenomenex Luna® C18, 250×30 mm.sup.2 packed with 5 μm particles. The flow rate was 42 mL/min of acetonitrile/methanol/water 45:35:20 (v:v:v). Ten peaks were collected using a detection at 210 nm and 310 nm. A Liquid Chromatography (LC) system consisting in a Thermo Accela pump, autosampler, coupled with a photodiode array UV detector (PAD) and a Thermo Scientific LTQ orbitrap XL mass spectrometer (MS) LC-PAD-MS was used to verify the purity of isolated peaks. The column used was a Phenomenex Luna ® C18, 250×4.6 mm.sup.2 packed with 5 μm particles. The flow rate was 1 mL/min using an isocratic binary solvent system: solvent A (20%), H.sub.2O pH=6 (CH.sub.3COONH.sub.4 0.02M); solvent B (80%), ACN/MeOH 40:35. Peaks were detected at 210 nm. High-resolution MS was measured with APCI source in the negative mode. The following inlet conditions were applied: capillary temperature 250° C., APCI vaporizer temperature 400° C., sheath gas flow 20.00 u.a., auxiliary gas flow 5.00 u.a., sweep gas flow 5.00 u.a. Data acquisition and processing were performed with Xcalibur software.

1.2. Parasites, Cells and Media

[0115] Antiparasitic activities were evaluated in vitro on Trypanosoma brucei brucei bloodstream forms (strain 427) (Tbb BSF). Tbb BSF were cultured in vitro at 37° C. with 5% CO.sub.2 in HMI9 medium containing 10% heat-inactivated fetal bovine serum, 3-mercaptoethanol (20 mM) and L-cysteine (150 mM).

[0116] The cytotoxicity of tested compounds was evaluated in parallel on a Human normal fibroblast cell line (WI-38) cultivated in a humidified atmosphere with 5% CO.sub.2 at 37° C. Human normal fibroblast cell line (WI-38) was cultivated in DMEM medium (Life Technologies) containing 4 mM L-glutamine, 1 mM sodium pyruvate supplemented with 10% fetal bovine serum (Sigma) and penicillin-streptomycin (100 UI/mL).

1.3. In Vitro Activity

[0117] In vitro tests were performed as previously described by Hoet S. et al. (Planta Med., 2004, 70, 407-413, doi:10.1055/s-2004-818967). Suramine (a commercial anti-trypanosomal drug) and camptothecin were used as positive controls. Stock solutions of compounds to be tested were prepared at a concentration of 10 mg/mL in DMSO. The solutions were further diluted in medium (described in 1.1) to give 0.1 mg/mL stock solutions. Extracts and compounds were tested in eight serial three-fold dilutions (final concentration range: 50-0.02 mg/L) in 96-well microliter plates. All tests were performed at least in duplicate.

2) Results

[0118] The anti-trypanosomal activity and toxicity of tested compounds and extracts are reported in Table 3 below:

TABLE-US-00003 TABLE 3 WI38 Tbb (IC.sub.50 WI38 (IC.sub.50 Tbb μg/mL) (IC.sub.50 μM) μg/mL) (IC.sub.50 μM) SI Whole fractions 80.40 ± 5.56 — 0.87 ± 0.32 — 92.4 F7-F9 of example 1 Tormentic acid 26.48 ± 0.33 54.22 ± 0.67 7.49 ± 0.33 15.33 ± 0.67  3.5 3-O-cis/trans-p- 28.43 ± 0.79 44.81 ± 1.25 0.48 ± 0.36 0.75 ± 0.57 59.2 coumaroyl- tormentic acid Maslinic acid 20.85 ± 3.97 44.11 ± 8.4  4.45 ± 0.64 9.41 ± 1.35 4.6 Corosolic Acid  9.34 ± 2.47 19.82 ± 5.22 3.45 ± 0.23 7.30 ± 0.59 2.7 Annurcoic acid 70.17 ± 6.82 144.10 ± 14.01 27.39 ± 2.24  56.29 ± 4.60  2.6 Ursolic acid  5.08 ± 0.10 11.14 ± 0.22 1.08 ± 0.11 2.38 ± 0.24 4.8 Oleanolic acid 28.89 ± 0.34 63.26 ± 0.74 2.66 ± 0.32 5.83 ± 0.70 10.9 Camptothecin  0.04 ± 0.01  0.12 ± 0.03 — — — Suramine — — 0.05 ± 0.01 0.038 ± 0.008 —

[0119] In 2005, Pink et al. (Nat Rev Drug Discov, 2005, 4, 727-740, doi:10.1038/nrd1824) published in “Nature Reviews”, criteria to select a pure compound as a hit for the treatment of parasitic diseases: this compound has to be active in vitro against whole protozoa with a IC50≤1 mg/L as well as to be selective (being at least tenfold more active against parasite than against a mammalian cell line). Our results show that the whole fraction F7-F9 of example 1 has a significant antitrypanosomal activity (IC50≤1 mg/L) with a high selectivity index, which could be due to its high content in 3-O-cis/trans-p-coumaroyltormentic acid, the only pure compound which could be considered as a hit antitrypanosomal compound (IC.sub.501; SI>10).

Example 4: In Vivo anti-trypanosomal Activity of 3-O-cis/trans-p-coumaroyltormentic Acid in Mice

[0120] In this example, the anti-trypanosomal activity of 3-O-cis/trans-p-coumaroyltormentic acid was tested in vivo in mice in comparison with ursolic acid.

1) Materials and Method

1.1. Animals

[0121] NMRI mice (6-8 weeks of age) obtained from Envigo Laboratories (The Netherlands) were used. All in vivo experiments performed were approved by the Ethical Committee for animals use at the Health Sciences Sector of the Catholic University of Louvain (2017/UCL/MD/017).

1.2. In Vivo Acute Toxicity Test

[0122] The assessment of the highest tolerated dose was based on a DNDi protocol by Loset J.-R. et al. (V. Drug Screening for Kinetoplastids Diseases. A Training Manual for Screening in Neglected Diseases-DNDi, 2009) and adapted by Beaufay C. et al. (Malar J., 2017, 16, doi:10.1186/s12936-017-2054-y). Briefly, 3-O-cis/trans-p-coumaroyltormentic acid isolated from fractions F-F9 obtained from a callus of Malus domestica Cox cultivar of example 1 above was given intraperitoneally every 2 hours to 2 mice using increasing doses: 10-15-25-50 mg/kg from stock solutions of 10 mg/mL. Mice were controlled for any health problem symptoms or behavioral changes and monitored for weight and hematocrit after each injection and every day during 48 h after administration. Main organs (heart, liver, spleen, lung and kidney) of treated mice were observed and weighed wet during autopsy. Control group received the vehicle, distilled water with 10% of tween 80-ethanol (7:3). The total injected dose was finally recorded and will ensure the non-toxicity of in vivo anti-trypanosomal test.

1.3. In Vivo anti-trypanosomal Activity

[0123] Mice were randomly divided into 6 mice per group for 3-O-cis/trans-p-coumaroyltormentic acid (mixture of cis and trans) and ursolic acid, 4 for positive control (Suramine) and 7 for the negative control, and were infested intraperitoneally with 10.sup.4 Trypanosoma brucei brucei. All compounds were solubilized in the vehicle (water-tween 80-ethanol) and administered intraperitoneally. 3-O-cis/trans-p-coumaroyltormentic acid or ursolic acid (UA) were administered at 50 mg/kg at day 3 after infection with the parasite and then every day until day 7 post-infection. Suramine (0.5 mg/kg) was administered while vehicle was used as a negative control. From day 3 post infection, a drop of blood collected each day from mouse-tail was used to count parasitemia.

2) Results

2.1. In Vivo Acute Toxicity

[0124] The results show that no acute toxic symptom was observed in each group (UA and 3-O-cis/trans-p-coumaroyltormentic acid) after the repeated injections of the treatments which did not impact neither weights nor haematocrits. As autopsy of treated mice did not reveal any macroscopic signs of toxicity and organs weights were normal, the total cumulative highest tolerated doses were evaluated as 100 mg/kg for both compounds.

2.2. In Vivo anti-trypanosomal Activity

[0125] The results are reported on FIGS. 2 and 3 annexed.

[0126] FIG. 2 reports the results of the antitrypanosomal activity in vivo. On this figure, parasitemia (Log Trypanosoma/mL) is given as a function of time (in days). The curve with full squares corresponds to the negative control, the curve with crosses corresponds to 3-O-cis/trans-p-coumaroyltormentic acid, the curve with full triangles corresponds to ursolic acid and the curve with full dots corresponds to the positive control (Suramine).

[0127] FIG. 3 reports the results of survival. On this figure, the percentage of survivals in given as a function of time (in days). The values with full squares correspond to the negative control, the values with crosses correspond to 3-O-cis/trans-p-coumaroyltormentic acid, the values with full triangles correspond to ursolic acid and the values with full dots correspond to the positive control (Suramine).

[0128] The results presented on FIG. 2 show that the mixture of 3-O-cis/trans-p-coumaroyltormentic acids exhibit a significant decrease of the parasitemia on day 4 post-infection when administered intraperitoneally at 50 mg/kg/day. This compound was more active than UA which did not show any effect on the parasitemia count.

[0129] Concerning survival analyses (FIG. 3), the esters treatment significantly improved the survival of infected mice in comparison to the untreated group, contrarily to UA for which no significant difference was observed at day 19 post-infection. Positive control mice survive during all the experimental period while all mice died after 12 days in the negative control group and in both treated groups only one mouse survived till the end of the experiment. Remaining mice were euthanized on day 19 post-infection. Of note, on day 12 post-infection, survival increases of 16.7% and 33.3% were observed for UA and esters treated mice respectively.

Example 5: Synthesis of Derivatives of Formulae (II-1) to (II-1)

[0130] Derivatives of formulae (II-1) to (II-10) were synthetized starting from oleanolic acid and ursolic acid present in the fractions obtained according to example 1 above.

[0131] Hydroxyl function at position 3 from the oleanolic and ursolic acids template was targeted to synthetize cinnamic esters following the Steglich esterification. The triterpenic acid (1.3 equivalents) was treated with dicyclohexylcarbodiimide (DCC: 2.2 equivalents) and 4-dimethylaminopyridine as a catalyst (DMAP: 0.2 equivalent) in toluene at 80° C. under agitation and argon or nitrogen gas as described by Lee et al. (Planta Med., 2008, 74 (12), 1481-1487). Aromatic acids reagents: cinnamic and hydrocinnamic acids or some fluorophenylpropionic acid isomers (ortho/meta/para) were firstly incubated during two hours with DMAP to ensure carboxylic function activation. After filtration, a purification was performed on a silica gel column (Merck, silica gel 60, 0.065-2 mm) with a toluene-ethyl acetate gradient. When necessary, an additional purification step by semi-preparative HPLC was performed with a Phenomenex Luna C18 (2) column (250×10 mm.sup.2 with 5 μm as particle size) on a Shimadzu Prominence system (LC20-AP pumps and SPD-20AV UV/VIS detector) with 100% methanol at 3 mL/min. The purity was checked at 210 nm with the analytical column (250×4.6 mm.sup.2), a flow rate of 1 mL/min and a binary solvent system composed with acetonitrile and Milli-Q water as followed: 50% acetonitrile 0-2 min, 100% acetonitrile 27-42 min, 50% acetonitrile 43-50 min.

[0132] The derivative of formula (II-1), named 3-O-hydrocinnamic ursolic acid (C.sub.39H.sub.56O.sub.4) was obtained with a yield of 51.8% and a purity >95%.

[0133] HRMS (APCI): m/z=587.41 (M-H.sup.+) (587.40949 calculated for C.sub.39H.sub.55O.sub.4), 437.34 (C.sub.30H.sub.45O.sub.2=M-H.sup.+ —C.sub.9H.sub.10O.sub.2); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.28-7.25 (m, 2H, H-2′/-4′), 7.20 (d, J=7.6 Hz, 3H, H-1′/-3′/-5′), 5.23 (d, J=3.6 Hz, 1H, H-12), 4.50 (dd, J=9.7, 6.2 Hz, 1H, H-3), 2.95 (t, J=7.8 Hz, 2H, H-7′), 2.63 (ddd, J=9.0, 6.8, 1.5 Hz, 2H, H-8′), 2.17 (d, J=11.3 Hz, 1H, H-18), 2.1-0 (m, 43H).

[0134] The derivative of formula (II-2), named 3-O-cinnamic ursolic acid (C.sub.39H.sub.54O.sub.4) was obtained with a yield of 17.6%) and a purity >95%.

[0135] HRMS (APCI): m/z=585.39 (M-H.sup.+), 437.34 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.8O.sub.2); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.67 (d, J=16.0 Hz, 1H, H-7′), 7.53-7.38 (m, 5H, H1′-5′ aromatic), 6.45 (d, J=16.0 Hz, 1H, H-8′), 5.26 (d, J=3.5 Hz, 1H, H-12), 4.65 (t, J=8.1 Hz, 1H, H-3), 2.19 (d, J=11.2 Hz, 1H, H-18), 2.08-0.75 (m, 43H).

[0136] The derivative of formula (II-3), named 3-O-parafluorophenylpropionic ursolic acid (C39H5504F) was obtained with a yield of 5.8% and a purity >95%.

[0137] HRMS (APCI): m/z=605.40027 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.34 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.16 (m, 2H, H-17-5′), 6.96 (m, 2H, H-2′/-4′), 5.23 (t, J=3.4 Hz, 1H, H-12), 4.50 (dd, J=8.0, 6.9 Hz, 1H, H-3), 2.93 (t, J=7.6 Hz, 2H, H-7′), 2.69-2.53 (m, 2H, H-8′), 2.18 (d, J=11.1 Hz, 1H, H-18), 2.10-0.60 (m, 43H).

[0138] The derivative of formula (II-4), named 3-O-metafluorophenylpropionic ursolic acid (C.sub.39H.sub.55O.sub.4F) was obtained with a yield of 7.9% and a purity >95%.

[0139] HRMS (APCI): m/z=605.40 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.34 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.22 (dd, J=8.0, 6.1 Hz, 1H, H-2′), 6.98-6.85 (m, 3H, H-1′/-3′/-5′), 5.25 (t, J=3.6 Hz, 1H, H-12), 4.53-4.45 (m, 1H, H-3), 2.95 (t, J=7.7 Hz, 2H, H-7′), 2.63 (dd, J=8.4, 6.9 Hz, 2H, H-8′), 2.18 (d, J=11.2 Hz, 1H, H-18), 2.08-0.70 (m, 43H).

[0140] Derivative of formula (II-5), named 3-O-orthofluorophenylpropionic ursolic acid (C.sub.39H.sub.55O.sub.4F) was obtained with a yield of 51.7% and a purity >95%.

[0141] HRMS (APCI): m/z=605.40 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.34 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.24-7.13 (m, 2H, H-17-5′), 7.07-6.97 (m, 2H, H-27-4′), 5.23 (t, J=3.5 Hz, 1H, H-12), 4.55-4.42 (m, 1H, H-3), 2.98 (t, J=7.8 Hz, 2H, H-7′), 2.64 (dd, J=8.5, 7.1 Hz, 2H, H-8′), 2.18 (d, J=11.2 Hz, 1H, H-18), 1.99 -0.70 (m, 43H).

[0142] Derivative of formula (II-6), named 3-O-hydrocinnamic oleanolic acid (C.sub.39H.sub.56O.sub.4) was obtained in the form of an amorphous white powder with a yield of 5.2% and a purity >95%.

[0143] HRMS (APCI): m/z=587.53970 (M-H.sup.+) (587.40949 calculated for C.sub.39H.sub.55O.sub.4), 437.45272 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.10O.sub.2) and m/z=589.42331 (M+H.sup.+) (587.40949 calculated for C.sub.39H.sub.57O.sub.4), 439.35543 (C.sub.30H.sub.47O.sub.2=M+H.sup.+—C.sub.9H.sub.10O.sub.2, major one), 393.35082 (C.sub.29H.sub.45=M+H.sup.+—C.sub.9H.sub.10O.sub.2—CH.sub.2O.sub.2); .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.28-7.01 (m, 5H, H1′-5′ aromatic), 5.20 (d, J=3.5 Hz, 1H, H-12), 4.42 (dd, J=10.2, 5.7 Hz, 1H, H-3), 2.88 (t, J=7.8 Hz, 2H, H-7′), 2.75 (dd, J=13.9, 4.5 Hz, 1H, H-18), 2.56 (dd, J=8.9, 6.7 Hz, 2H, H-8′), 2.00-0.51 (m, 43H).

[0144] Derivative of formula (II-7), named 3-O-cinnamic oleanolic acid (C.sub.39H.sub.54O.sub.4) was obtained in the form of a yellow powder with a yield of 21.8% and a purity >95%.

[0145] HRMS (APCI): m/z=585.39478 (M-H.sup.+) (585.39384 calculated for C.sub.39H.sub.53O.sub.4), 437.34171 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.8O.sub.2) and m/z=587.39413 (M+H.sup.+) (587.40949 calculated for C.sub.39H.sub.55O.sub.4), 439.35565 (C.sub.30H.sub.47O.sub.2=M+H.sup.+—C.sub.9H.sub.8O.sub.2, major one), 391.28323 (C.sub.29H.sub.43=M+H.sup.+—C.sub.9H.sub.8O.sub.2—CH402); .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.68 (d, J=16.0 Hz, 1H, H-7′), 7.50-7.41 (m, 5H, H-1′-5′ aromatic), 6.45 (d, J=16.0 Hz, 1H, H-8′), 5.27 (d, J=3.5 Hz, 1H, H-12), 4.49 (m, 1H, H-3), 2.8 (dd, J=13.8, 4.5 Hz, 1H, H-18), 2.03-0.65 (m, 43H).

[0146] Derivative of formula (II-8), named 3-O-parafluorophenylpropionic oleanolic acid (C.sub.39H.sub.55O.sub.4F) was obtained in the form of white crystals with a yield of 13.0% and a purity >95%.

[0147] HRMS (APCI): m/z=605.40161 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.34286 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F) and m/z=607.41408 (M+H.sup.+) (607.41571 calculated for C.sub.39H.sub.56O.sub.4F), 439.35565 (C.sub.30H.sub.47O.sub.2=M+H.sup.+—C.sub.9H.sub.9O.sub.2F, major one), 393.35065 (C.sub.29H.sub.45=M+H.sup.+—C.sub.9H.sub.9O.sub.2F—CH.sub.2O.sub.2); .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.19-7.11 (m, 2H, H-1′-5′), 7.00-6.90 (m, 2H, H-2′/-4′), 5.26 (t, J=3.6 Hz, 1H, H-12), 4.55-4.41 (m, 1H, H-3), 2.92 (t, J=7.3 Hz, 2H, H-7′), 2.82 (dd, J=13.8, 4.5 Hz, 1H, H-18), 2.60 (dd, J=8.5, 6.8 Hz, 2H, H-8′), 2.01-0.67 (m, 43H).

[0148] Derivative of formula (II-9), named 3-O-metafluorophenylpropionic oleanolic acid (C.sub.39H.sub.55O.sub.4F) was obtained in the form of a yellow solid with a yield of 36.8% and a purity >95%.

[0149] HRMS (APCI): m/z=605.51654 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.51715 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F) and m/z=607.41416 (M+H.sup.+) (607.41571 calculated for C.sub.39H.sub.56O.sub.4F), 439.35569 (C.sub.30H.sub.47O.sub.2=M+H.sup.+—C.sub.9H.sub.9O.sub.2F, major one), 393.35102 (C.sub.29H.sub.45=M+H.sup.+—C.sub.9H.sub.9O.sub.2F—CH.sub.2O.sub.2); .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.40-7.08 (m, 1H, H-2′), 7.06-6.80 (m, 3H, H-17-37-51 5.27 (d, J=3.5 Hz, 1H, H-12), 4.50 (dd, J=10.0, 6.1 Hz, 1H, H-3), 2.95 (t, J=7.7 Hz, 2H, H-7′), 2.82 (dd, J=13.9, 4.5 Hz, 1H, H-18), 2.63 (t, J=7.7 Hz, 2H, H-8′), 2.12-0.53 (m, 43H).

[0150] The derivative of formula (II-10), named 3-O-orthofluorophenylpropionic oleanolic acid (C39H5504F) was obtained in the form of an amorphous yellow powder with a yield of 1.8% and a purity >95%.

[0151] HRMS (APCI): m/z=605.54545 (M-H.sup.+) (605.40006 calculated for C.sub.39H.sub.54O.sub.4F), 437.56130 (C.sub.30H.sub.45O.sub.2=M-H.sup.+—C.sub.9H.sub.9O.sub.2F) and m/z=607.41409 (M+H.sup.+) (607.41571 calculated for C.sub.39H.sub.56O.sub.4F), 439.35565 (C.sub.30H.sub.47O.sub.2=M+H.sup.+—C.sub.9H.sub.9O.sub.2F, major one), 393.35097 (C.sub.29H.sub.45=M+H.sup.+—C.sub.9H.sub.9O.sub.2F—CH.sub.2O.sub.2); .sup.1H-NMR (400 MHz, CDCl.sub.3): 7.12 (m, 2H, H-1′/-5′), 7.03-6.86 (m, 2H, H-2′/-4′), 5.27-5.13 (m, 1H, H-12), 4.49-4.35 (m, 1H, H-3), 2.91 (t, J=7.9 Hz, 2H, H-7′), 2.75 (dd, J=13.8, 4.5 Hz, 1H, H-18), 2.57 (t, J=7.8 Hz, 2H, H-8′), 2.00-0.58 (m, 43H).

Example 6: Antitrypanosomal Activities of Derivatives of Formula (II-1) to (II-10)

[0152] Semi-synthetized derivatives of 3-O-p-coumaroyl tormentic acid of formulae (II-1) to (II-10) were tested for their antitrypanosomal activities and selectivity towards mammalian cells according to the same methods as described above in example 3. The results of the activity and selectivity are given in Table 4 below:

TABLE-US-00004 TABLE 4 Biological activities expressed in IC.sub.50 (μM, Mean ± Sd) SI Antitrypanosomal Cytotoxicity (IC.sub.50WI38/ DERIVATIVES (Tbb) (WI38) IC.sub.50Tbb) (II-1) 2.22 ± 0.66 142.01 ± 3.45  64.0 (II-2) 68.19 ± 2.15  nd nd (II-3) 2.45 ± 0.30 >164.90 >67.3 (II-4) 2.67 ± 0.83 >164.90 >61.5 (II-5) 1.66 ± 0.38 >131.92 >79.5 (II-6) 2.59 ± 0.87 23.54 ± 6.56 9.1 (II-7) 6.31 ± 0.96 22.31 ± 1.88 3.5 (II-8) 2.88 ± 1.11 16.33 ± 3.26 5.7 (II-9) 1.72 ± 0.07 20.74 ± 2.25 12.1 (II-10) 4.63 ± 0.28 18.61 ± 4.47 4.0

[0153] Activity of all tested 3-O-ursane esters, except the cinnamic one (derivative of formula (II-2)), was similar to ursolic acid with an enhanced selectivity, especially for aromatic esters (Derivatives of formulae (II-1) and (II-3) to (II-5)). For 3-O-oleanane derivatives, activity also remained similar than oleanolic acid except the hydrocinnamic derivative (derivative of formula (II-6)) and para/meta-fluorophenylpropionic derivatives (derivatives of formulae (II-8) and (II-9)) showing a significantly increased activity but also cytotoxicity leading to similar selectivity.

Example 7: Evaluation of the Triterpene Composition in Malus x domestica (Rosaceae) and Vitellaria paradixa (Sapotaceae)

[0154] In this example, the triterpene composition obtained using the Malus x domestica “Cox's Orange Pippin” cell line described in examples 1 and 2 was compared to those obtained using “Cox's Orange Pippin” apple fruit skin (raw material—example not forming part of the present invention), Vitellaria paradoxa leaf (raw material—example not forming part of the present invention) and Vitellaria paradoxa calli (cell) samples (obtained according to the process of the present invention).

1) Materials and Method

1.1. Sample Preparation

[0155] The “Cox Orange Pippin” cell sample was obtained after a batch culture using a 6 L custom-made stirred-bioreactor instrument with round bottom vessel (Biostream) and equipped with two 8.5 cm-diameter marine impellers adjusted at 0 and 24 cm from the bottom end of the stirring bar. The cell line was inoculated at 13% (v/v) into the reactor filled with a Linsmaier and Skoog medium supplemented with 30 g/L sucrose, 6 mg/L of 1-naphtaleneacetic acid. The stirring speed was at 105 rpm (revolutions per minute). The oxygenation of the medium was supported by an air sparging system set at 0.125 v.v.m. (Vessel Volume per minute) as already described in the example 2. Methyl jasmonate (Sigma Aldrich 392707-25ML) was added at the beginning of the stationary phase to reach a final concentration of 50 μM. Cells were harvested after 8-days of incubation at 23° C. Cells were separated from the medium using vacuum filtration.

[0156] Vitellaria paradoxa calli culture was initiated and multiplied from leaf explant placed on a Murashige and skoog medium supplied with 30 g agar, 0.2 mg/l 1-naphtaleneacetic acid and 0.2 mg/l 2,4-dichlorophenoxyacetic acid. Calli were obtained after 2 months and were further spitted each month.

[0157] Vitellaria paradoxa leaf and Malus x domestica ‘Cox Orange pippin’ skin samples have to be considered as comparative examples not forming part of the present invention. Vitellaria paradoxa leaf samples were collected from a two years-old plant grown in a pot filed with a soil/sand (70/30, v/v) mixture at 30° C. and 60% relative humidity. Cox Orange Pippin skin samples were obtained from fruits collected in October 2016 and peeled with scalpels.

[0158] Vitellaria paradoxa leaf and cell (calli) samples as well as Malus x domestica “Cox Orange pippin” fruit skin and cell samples were collected, directly flash-frozen in liquid nitrogen, freeze-dried and ground.

1.2. Laboratory Scale Wide Spectrum Extraction Procedure

[0159] In order to evaluate the triterpene content of these heterogeneous samples, a wide spectrum extraction procedure was used. 10 ml of an ethyl acetate/hexane mixture (50/50, v/v) was added to 500 mg dried samples in a 15 ml Tube. Samples were homogenized using vortex and sonicated for 10 min, and finally shaken at 20 Hz for 20 min in a (mill grinder Retsch). After centrifugation at 4700 g/2 0min/20° C., the supernatant was collected and evaporated. The remaining pellet was re-extracted using 10 mL ethanol/water mixture (80/20, v/v), homogenized using vortex and sonicated for 10 min, and finally shaken at 20 Hz for 20 min in a (mill grinder Retsch). After centrifugation at 4700 g/20 min/20° C., the supernatant was combined to the lipophilic dried extract and further evaporated during 7 h. The final extract was re-suspended in 1.5 mL and filtrated on PTFE 0.2 μm. Samples were analyzed as described in example 1 paragraph 1.5.

2) Results

[0160] The quantitative composition of the (poly)hydroxylated pentacyclic triterpene composition obtained from Malus x domestica “Cox's Orange Pippin” cultivar and Vitellaria paradoxa samples is given in Table 5 below:

TABLE-US-00005 TABLE 5 Amount (weight %) Cox orange Vitellaria Vitellaria Cox Orange Pippin fruit paradoxa paradoxa TRITERPENES Pippin cell skin .sup.(1) cell leaf .sup.(1) Tormentic acid 8.25 2.49 39.92 0.00 Annurcoic acid 29.28 29.67 0.00 0.00 3-O-trans-p-coumaroyl 32.88 0.00 7.35 1.57 tormentic acid 3-O-cis-p-coumaroyl 1.59 0.00 0.00 0.00 tormentic acid Maslinic acid 13.69 2.76 13.02 0.00 Corosolic acid 7.47 3.03 15.09 0.87 Betulin 0.00 1.65 0.00 0.84 Betulinic acid 0.00 3.15 0.00 0.00 Oleanolic acid 1.55 15.53 4.58 11.89 Ursolic acid 3.50 30.85 10.20 43.70 Betulinaldehyde 0.00 1.32 0.00 0.00 Oxidosqualene 0.00 0.93 14.91 4.24 Lupeol 1.77 8.61 0.00 36.95 .sup.(1) comparative example not forming part of the present invention

[0161] It clearly emerges from these results that the fractions obtained according to the process according to the invention exhibit a very higher amount of 3-O-trans-p-coumaroyl tormentic acid compared to the samples obtained by a simple extraction, for both Cox Orange Pippin and Vitellaria paradoxa.

[0162] The same applies to the amount of 3-O-cis-p-coumaroyl tormentic acid obtained from Cox Orange Pippin when the process according to the invention is used.