Composition comprising a lupeol or beta-sitosterol derivative for use in the treatment of breast or colon cancer

Abstract

The present invention relates to a bioactive composition, with an antitumor effect, obtained from the transformation by alkylation and esterification reaction of an extract of the «Stipa tenacissima L» esparto plant. Said composition comprises at least one lupeol and / or β-sitosterol derivative selected from formulae (III) to (VI) obtained by alkylation or esterification reaction with a very high cytotoxicity efficiency.

Description

[0001] COMPOSITION COMPRISING A LUPEOL DERIVATIVE OR BETA-SITOSTEROL FOR USE IN THE TREATMENT OF BREAST OR COLON CANCER

[0002] Field of invention

[0003] The present invention relates to a composition having an improved antitumor effect. Said composition is obtained from an extract of the Alfa plant “Stipa tenacissima L” of the Poaceae family.

[0004] Prior art

[0005] Lupeol, a natural compound, is known for its antioxidant, anti-inflammatory, and anticancer activities (Liu K, Zhang X, Xie L, Deng M, Chen H, Song J, Long J, Li X, Luo J, Lupeol and its derivatives as anticancer and antiinflammatory agents: molecular mechanisms and therapeutic efficacy, Pharmacological Research (2020), doi: https: / / doi.org / 10. 1016 / j.phrs.2020. 105373). Lupeol is a natural triterpenoid that widely exists in edible fruits and vegetables and medicinal plants. Recent studies have demonstrated that lupeol possesses a wide range of pharmacological activities such as anticancer, antioxidant, anti-inflammatory, and antimicrobial activities. Pharmacokinetic studies indicated that the absorption of lupeol by animals was rapid despite its non-polar characteristics, and lupeol belongs to BCS (Biopharmaceutical Classification System) Class II compounds.

[0006] In addition, the bioactivities of some isolated or synthesized lupeol derivatives have been studied, and these results have shown that, with modification at C-3 or C-19, some derivatives exhibit stronger activities, for example, antiprotozoal or anticancer activity.

[0007] One of the sources of lupeol is the alfa plant "Stipa tenacissima L", which is a very abundant grass in North Africa, and typically Mediterranean. It is found in Algeria, Morocco, Tunisia, Libya, southern Spain and also southern Portugal. It occupies nearly 8.5 million hectares around the Mediterranean, especially on the large Algerian-Moroccan plateaus. The territorial distribution known to date is estimated at 4,000,000 ha in Algeria, 3,155,700 ha in Morocco; 600,000 ha in Tunisia, 350,000 ha in Libya and 300,000 ha in Spain.

[0008] Alfa has special properties that make it ideal for various applications, both industrial and artisanal. Alfa is often used for the extraction of cellulose fibers and as a pulp for paper production due to its excellent impermeability, bulkiness, strength, stability, flexibility, porosity, softness, opacity, and satisfactory elasticity.

[0009] Therefore, only works on the extraction of nano- and microcrystalline cellulose from Alfa have been described in the literature. However, on the therapeutic properties of the plant, no work has been described, especially regarding the extraction of active molecules from the Alfa plant.

[0010] For example, patent applications FR-643384_A and TN2012000241 _A1 relate to processes for extracting cellulose from Alfa fibers (stipa tenacissima).

[0011] Hence the interest of the present invention which aims at a new form of valorization of the Alfa plant through the extraction of bioactive elements mainly lupeol and |3-S itosterol and the use of their derivatives as anticancer agents.

[0012] Brief description of the invention

[0013] The present invention relates to an extract from the Alfa plant rich in lupeol and |3-S itosterol and its extraction process.

[0014] Lupeol (formula I) belongs to the triterpene family and in particular to that of triterpene alcohols.

[0015] Lupeol is of interest because of its many biological activities. In particular its anti-inflammatory (Singh S. et al., Filoterapia, 1997, 68, No. 1, 9) and analgesic (De Miranda G et al., Planta Med 2000, 66 (3) 284) properties, its nephroprotective action against heavy metals (Nagaraj M. et al., J. Appl. Toxicol, 2000, 20 (5), 413), its antihistamine action (De MedranoNillar M. j. et al.,MethodsFindExp. Clin. Pharmacol, 1997, 19, 0 8, 515), its antimitotic activities (Zachariah R. et al., Indian J. Pharm. Sci. 1994, 56, No. 4, 129. Prasad S, Kalra N, Singh M, Shukla Y, Asian J Androl, vol. 10, no. 2, 2008, p. 313-8) and antiviral (Kahlos K., Filoterapia, 1996, 61, No. 4, 344).

[0016] Lupeol can also be used as a synthetic intermediate, particularly for the preparation of phytohormones and steroid analogues.

[0017] p-Sitosterol comes in the form of:

[0018] (II)

[0019] It is a sterol found in almost all plants. It is one of the main components of a group of plant sterols called phytosterols, which are very similar in composition to cholesterol. These plant sterols are the active ingredients in margarine spreads (Take Control, Benecol) used to lower cholesterol (Lees AM et al, Atherosclerosis, 1977;28:325-38).

[0020] [3-Sitosterol] is found in rice bran, wheat germ, peanuts, corn oils, and soybeans. It is also found in high concentrations in plants such as saw palmetto, rye pollen, pygeum, and nettles, which have been shown to be beneficial for BPH. Unlike cholesterol, beta-sitosterol cannot be converted to testosterone. It also inhibits aromatase and 5-alpha-reductase. Beta-sitosterol is likely one of the many reasons why eating vegetables is good for your health. Encourage adequate intake of these plants in your diet.

[0021] Lupeol and 3-Sitosterol are present separately in many plants such as aloe vera and Crataevanurvala bark (in the case of lupeol) or in a mixture in the case of Bergiasuffruticosa. Lupeol has been isolated several times from various plants such as Bresk. However, the two molecules have never been extracted from the alfa plant.

[0022] In the medical applications section of the alfa plant extract, its richness in lupeol and (3-Sitosterol), inexpensive and very available constitutes an option for the treatment of certain tumors as demonstrated in the previous application of the same applicant under number MA53187.

[0023] Certainly, the antitumor effect of the alpha plant extract has been demonstrated in the said application, however the objective of the present application is to provide another option for using alpha plant extracts as an antitumor agent thanks to a compound obtained from the same plant with an improved effect thanks to a transformation of the lupeol molecules and (3-Sitosterol) into derivatives with increased antitumor activity.

[0024] Statement of the invention

[0025] The objective of the invention is achieved by means of a composition containing bioactive molecules obtained from the transformation by alkylation and / or by esterification of lupeol molecules and (3-Sitosterol)

[0026] According to the concept of the invention, the synthesis of the new lupeol-derived compounds is carried out according to the following alkylation or esterification protocol: Alkylation reaction: 5, 7 and 9

[0027] Esterification reaction:

[0028] Or X' can take one of the following formulas from 6 to 8: X' = CH3 (6), Br (7),

[0029] NHCH3 (8) According to the concept of the invention, the synthesis of the new compounds derived from [3-Sitosterol] is carried out according to the following alkylation and esterification protocol:

[0030] - alkylation reaction:

[0031] Or R can take the following forms from 1 to 5 depending on the value of n: or n=1, 3, 5, 7 and 9

[0032] - esterification reaction:

[0033] Or X' can take one of the following formulas from 6 to 8: X' = CH3 (6), Br (7),

[0034] NHCH3 (8)

[0035] The new compounds of formulae (III), (IV), (V) and (VI) obtained by the alkylation and esterification reactions of lupeol and [3-Sitosterol] exhibit improved antitumor activity compared to the natural extract.

[0036] Preferably, the compounds resulting from the alkylation and / or esterification can be used synergistically for the treatment of tumors. Way(s) of carrying out the invention

[0037] To illustrate the object of the invention, we proceeded by experimental reactions to evaluate the antitumor effect of the transformation compounds of lupeol and [3-Sitosterol extracted from the alfa plant.

[0038] - LUPEOL molecule Alkylation reaction: or n=1, 3, 5, 7 and 9 (odd) to avoid cycle formation. The reagents obtained, with a fixed choice of X, are numbered in the sequence from 1 to 5.

[0039] Operating mode:

[0040] For the synthesis of alkyls: at 1.0 mmol of lupeol, 2 mmol of reagent RX from 1 to 5 in 20 ml of Toluene and 10 ml of NaOH (50%) in the presence of a phase transfer agent (TBAB). The reaction mixture was then kept under vigorous stirring, at 60°C, for 48 hours.

[0041] The reaction is monitored by TLC. The product is then extracted with ethyl acetate (2X50mL). The organic phases are combined and dried over anhydrous Na2SO4. The lupeol alkyls obtained from 1 to 5 are subsequently dried under vacuum and packaged for characterization. The reaction yield is greater than 90%.

[0042] Esterification reaction:

[0043]

[0044] With X' = CH3(6), Br (7), NHCH3(8)

[0045] Operating mode:

[0046] For the synthesis of esters: at 1.0 mmol of lupeol, 1.2 mmol of acid chloride of 1 to 3 in 20 ml of dichloromethane. The reaction mixture was then kept under magnetic stirring, at room temperature, for 24 hours. Added 50 ml of DCM and washed the solution with 2X20 ml of a potassium carbonate solution (1M). The lupeol esters are obtained from 1 to 3 after drying on anhydrous Na2SO4 and evaporation of the solvent then conditioned for characterization. The yield of the reaction is greater than 90%.

[0047] - p-Sitosterol molecule

[0048] Alkylation reaction: With: R: or n=1, 3, 5, 7 and 9 (odd) to avoid cycle formation. The reagents obtained, with a fixed choice of X, are numbered in sequence from 1 to 5.

[0049] Operating mode:

[0050] For the synthesis of alkyls: at 1.0 mmol of [3-Sitosterol, 2 mmol of reagent RX from 1 to 5 in 20 ml of Toluene and 10 ml of NaOH (50%) in the presence of a phase transfer agent (TBAB). The reaction mixture was then kept under vigorous stirring, at 60°C, for 48 hours.

[0051] The reaction is monitored by TLC. The product is then extracted with ethyl acetate (2X50mL). The organic phases are combined and dried over anhydrous Na2SO4. The [3-Sitosterol Alkyls] obtained from 1 to 5 are subsequently dried under vacuum and packaged for characterization. The reaction yield is greater than 90%.

[0052] Esterification reaction:

[0053] With X' = CH3(6), Br (7), NHCH3(8)

[0054] Operating mode:

[0055] For the synthesis of esters: at 1.0 mmol of [3-Sitosterol, 1.2 mmol of acid chloride of 1 to 3 in 20 ml of dichloromethane (DCM). The reaction mixture was then kept under magnetic stirring, at room temperature, for 24 hours. Added 50 ml of DCM and washed the solution with 2X20 ml of a potassium carbonate solution (1 M). The esters of [3-Sitosterol are obtained from 1 to 3 after drying on anhydrous Na2SO4 and evaporation of the solvent then conditioned for characterization. The yield of the reaction is greater than 90%. Evaluation of the antitumor effect (cytotoxicity):

[0056] The work described below was carried out with the aim of highlighting the cytotoxic properties of the different derivatives obtained from the previous alkylation and esterification reactions of lupeol and [3-Sitosterol

[0057] Cytotoxicity was performed on two human cancer lines, HT-29 colon (human colorectal adenocarcinoma cell) and MDA-MB231 breast (human breast adenocarcinoma), using a concentration range from 500 pg / ml to 15.62 pg / ml. The comparison also concerns lupeol extracts and [3-Sitosterol] obtained from the alfa plant in order to highlight the interest of the derivatives in antitumor treatment.

[0058] The results are presented in the table below:

[0059] Table I: % Cytotoxicity Yield Against Human Colon Cancer Line HT-29 and Breast Cancer Line MDA-MB231

[0060] With respect to both HT-29 and MDA MB 231 lines, all lupeol and [3-Sitosterol] extracts dose-dependently inhibit cell proliferation. The new compounds derived from lupeol and [3-Sitosterol] obtained by the alkylation and esterification reactions of lupeol (III), (IV), and [3-Sitosterol (V), (VI) exhibit strong antitumor activity compared to the natural extract with respect to both HT-29 and MDA MB 231 lines (Table I).

[0061] The introduction of a long alkyl side chain into the extract of lupeol and / or [3-Sitosterol] resulted in a cytostatic effect on both HT-29 (%R <70 and <65) and MDA MB 231 (%R <55) cell lines. This effect increased with the length of the alkyl chain, which gave the best effect, whereas lupeol and [3-Sitosterol] esters showed a very remarkable cytostatic effect on both cancer lines.

[0062] According to a concept of the invention, the derivatives of formulas (III) to (VI) are used in combinations between at least two formulas to create a synergy between them in the treatment protocol.

Claims

Claims:

1. Bioactive composition obtained by alkylation or esterification process of lupeol and |3-S itosterol extracted from the alfa plant “Stipa tenacissima L” characterized in that it comprises derivatives of lupeol and / or [3-Sitosterol of formulas (III), (IV), (V) and (VI) where: X: is chosen from [Cl, Br, I, OTS, OMes] or any group carrying the radical R with R = and n included in the range 1 to 9. And X' is chosen from the following formulas 6, 7 and 8: CH3 (6), Br (7), NHCH3 (8) 2. Bioactive composition characterized in that the number of chains n is odd to avoid the formation of a cycle.

3. Use of the bioactive compositions (III) to (VI) of claims 1 to 2 characterized in that the bioactive compositions (III) to (VI) are used in the treatment of breast and colon cancers.

4. Use according to claim 3 characterized in that it uses a range of concentrations from 500 to 15.62 pg / ml.

5. Use according to claims 3 and 4 characterized in that compounds (III) to (VI) are used in combinations between at least two formulas to create a synergy between them in the treatment protocol.

Images