Method for the separation of the isoprenic constituents of guayule

Abstract

Method for the separation of at least one isoprenic constituent from the resin of a plant of guayule and/or of the guayule type comprising the steps of: a) providing a defatted resin of guayule and/or of the guayule type; b) subjecting the defatted resin to partitioning of the liquid-liquid type with solvents that are immiscible in each other thus obtaining an apolar extract containing the isoprenic constituents guayulin A, guayulin B and argentatin B; and a polar extract containing the isoprene constituents argentatin A, argentatin C and argentatin D; and c) separating at least one isoprenic constituent from said polar extract and/or from the apolar extract thus obtained, wherein step c) comprises a step in which the polar extract is subjected to partitioning of the liquid-liquid type with solvents immiscible in each other and/or a step in which the apolar extract is subjected to partitioning of the solid-liquid type.

Claims

1. A method for separating at least one isoprenic constituent from a resin of a guayule (Parthenium argentatum) and/or of a guayule type plant selected from the group consisting of Euphorbia lathyris, Parthenturr incanum, Chrysothamnus nauseosus, Pedilanthus macrocarpus, Cryptostegia grandiflora, Asclepias syriaca, Asclepius speciosa, Asclepias subulata, Solidago altissima, Solidago gramnifblia, Salidago rigida, Cacalia atriplicifolia, Taraxacum koksaghyz, Pvcnanthemum incanum, Teucreum canadence and Campanula Americana, the method comprising: a) forming a defatted resin of the guayule and/or the guayule type plant; b) liquid-liquid partitioning the defatted resin with solvents that are immiscible in each other to obtain: an apolar extract comprising the isoprenic constituents guayulin A, guayulin B and argentatin B; and a polar extract comprising the isoprenic constituents argentatin A, argentatin C and argentatin D; and c) separating at least one isoprenic constituent from the polar extract, the apolar extract, or both, wherein the separating c) comprises: liquid-liquid partitioning the polar extract with solvents that are immiscible in each other, solid-liquid partitioning the apolar extract, or a combination thereof.

2. The method according to claim 1, wherein the at least one isoprenic constituent is selected from the group consisting of guayulin A, guayulin B, argentatin A, argentatin B, argentatin C, argentatin D, and mixtures thereof.

3. The method according to claim 1, wherein the defatted resin is obtained by defatting a crude extract of the guayule, the guayule type plant, or both.

4. The method according to claim 1, wherein, in the liquid-liquid partitioning b), the solvents that are immiscible in each other comprise a polar solvent and an apolar solvent.

5. The method according to claim 1, wherein: the separating c) comprising the solid-liquid partitioning of the apolar extract; and the solid-liquid partitioning comprises: subjecting the apolar extract, dissolved in a combination of two solvents miscible in each other, to adsorption on a solid matrix, and treating the solid matrix thus obtained with a combination of two solvents miscible in each other, to obtain a solution comprising a mixture of guayulin A and guayulin B, and a solid matrix comprising argentatin B.

6. The method according to claim 5, wherein the solid matrix comprising argentatin B is further treated to obtain a solution of argentatin B.

7. The method of claim 1, wherein: the separating c) comprises the liquid-liquid partitioning of the polar extract; and the liquid-liquid partitioning comprises separating the polar extract in a combination of a polar solvent and an apolar solvent to obtain an organic phase comprising argentatin A and an aqueous phase comprising a mixture of argentatin C and argentatin D.

8. The method according to claim 7, wherein in the liquid-liquid partitioning c): the apolar solvent is selected from the group consisting of cyclohexane, n-hexane, petroleum ether, and combinations thereof; and the polar solvent is selected from the group consisting of water, methanol, ethanol, isopropanol, tert-butanol, and combinations thereof.

9. The method according to claim 1, further comprising: d) purifying at least one isoprenic constituent obtained in the separating c).

10. The method according to claim 9, wherein the purifying d) comprises crystallizing at least one isoprenic constituent, to obtain the at least one isoprenic constituent in substantially pure crystalline form.

11. The method according to claim 9, wherein the purifying d) comprises d1) subjecting a mixture of guayulin A and guayulin B obtained in the separating c) to precipitation of the guayulin A by treatment with an apolar solvent with cooling, to obtain a precipitate of purified guayulin A and a supernatant comprising a mixture of guayulin A and guayulin B.

12. The method according to claim 9, Wherein the purifying d) comprises d2) precipitating the argentatin B obtained in the separating c) with an apolar solvent with cooling, to obtain a precipitate of purified argentatin B.

13. The method according to claim 9, wherein the purifying d) comprises d3) precipitating the argentatin A obtained in the separating c) an apolar solvent with cooling, to obtain a precipitate of purified argentatin A.

14. The method according to claim 9, wherein the purifying d) comprises d4) precipitating a mixture of argentatin C and argentatin D obtained in the separating c) with an apolar solvent with cooling, to obtain a precipitate of purified argentatin D and a supernatant comprising argentatin C.

15. The method according to claim 14, wherein the precipitating d4) is followed by evaporating d5) solvent of the supernatant and dissolving the residue in an apolar solvent with cooling, to obtain a precipitate of purified argentatin C C.

16. The method of claim 11, wherein the separating c) is followed by e1) performing a base hydrolysis reaction of the guayulin A obtained in the precipitation d1) and/or of the mixture of guayulin A and guayulin B obtained in the separating c) to obtain a solution comprising partheniol.

17. The method according to claim 16, wherein the base hydrolysis reaction e1) is followed by e2) esterification of the thus obtained partheniol.

18. The method according to claim 17, further comprising evaporating a solution comprising guayulin B obtained in the esterification e2), and dissolving a resulting residue in an apolar solvent to obtain a precipitate of purified guayulin B.

19. The method according to claim 12, wherein the separating c) is followed by e3) performing a reduction reaction of the argentatin B obtained in the separating c) and/or in the precipitating d2), to obtain a solution comprising argentatin D.

20. The method according to claim 19, wherein, following the reduction reaction, the solution comprising argentatin D is evaporated and a resulting residue is dissolved in an apolar solvent, to obtain a precipitate of purified argentatin D.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents a complete block-diagram of an embodiment of the method of the present invention, starting from a crude extract obtained from the bagasse of Parthenium argentatum. The numbers in brackets represent the amount of product expressed in grams.

(2) FIG. 2 represents a block-diagram of an embodiment of the method of the present invention, starting from a crude extract obtained from branches and stem of Parthenium argentatum, which shows the obtaining of guayulin A, and argentatin A and B. The numbers in brackets represent the amount of product expressed in grams.

(3) FIG. 3 represents a block-diagram of an embodiment of the method of the present invention, starting from a crude extract obtained from leaves of Parthenium argentatum, which shows the obtainment of guayulin A, and argentatin A and B. The numbers in brackets represent the amount of product expressed in grams.

(4) FIG. 4 represents a partial block-diagram of an embodiment of the method of the present invention, which shows the semi-synthesis of lower natural guayulins and argentatins starting from the non-polymer higher isoprenic constituents (guayulin A and argentatin B) separated and purified by the method of the present invention. The numbers in brackets represent the yield by weight of the depicted products obtained by semisynthesis.

DETAILED DESCRIPTION OF THE INVENTION

(5) The invention will be now further described with reference to some embodiments by way of example only, and are not limitative of the present invention.

(6) All the indicated percentages are expressed as weight percentages and all the proportions of the mixtures of solvents are expressed as proportions by volume, unless otherwise indicated.

EXAMPLE 1

(7) Winterization of the crude extract of Parthenium argentatum (FIG. 1)

(8) 108 g of crude extract obtained from the bagasse of Parthenium argentatum were slowly dissolved under continuous stirring at 40 C. in methanol (800 mL) by using a volume of solvent of 1.0 L/100 g of crude extract.

(9) After the complete dissolution the mixture was cooled at room temperature for 1 hour and then at 0 C. for 24 hours. A compact precipitate was formed and then recovered by filtration under 1.5 mbar.

(10) The residue having a gummy consistency was then washed with methanol, cooled to 0 C. and air-dried at room temperature obtaining 44 g of fats and rubbers with a low molecular weight.

(11) The polar liquid fraction (mother liquors) containing the defatted resin was recovered for the subsequent steps.

EXAMPLE 2

(12) Partitioning of the liquid-liquid type of the resin (FIG. 1)

(13) 800 mL of methanol solution obtained from the process of winterization, for separating the lipophilic component of the crude resin, were diluted to a final volume of 1 L with 200 mL of water obtaining a mixture of methanol/water 4:1.

(14) The obtained mixture was subjected to a partitioning process with petroleum ether (200 mL). After having reached the equilibrium between the phases, the organic phase was separated from the aqueous phase.

(15) The latter was extracted again with 200 mL of petroleum ether for 3 times. The resultant organic phase was then washed with 200 mL of a mixture of methanol/water (4:1), recovered, anhydrified and concentrated under low pressures up to the complete drying thus obtaining 7.7 g of product.

(16) The partitioning process was monitored by TLC (Thin layer chromatography).

EXAMPLE 3

(17) Solid-liquid partitioning of the apolar extract (FIG. 1)

(18) 7.7 g of apolar extract obtained by the liquid-liquid partitioning process of 108 g of crude resin were dissolved in 60 mL (7 mL/g) of a mixture of petroleum ether and ethyl acetate 9:1 and adsorbed on a bed of 200 mL of silica gel (70-230 mesh, 25 mL/g).

(19) The solvent was removed from the suspension by evaporation under reduced pressure or vacuum filtration, and the resultant solid matrix was washed three times with 100 mL of the solvent mixture used for the absorption.

(20) This operation was carried out into a reaction flask according to a method known to the person skilled in the art. The de-absorption solution was evaporated, thus obtaining a mixture of guayulins (5.0 g). The solid matrix was then washed with 200 mL of ethyl acetate, thus recovering argentatin B (2.3 g).

EXAMPLE 4

(21) Liquid-liquid partitioning of the polar fraction (FIG. 1)

(22) 1.2 L of methanol/water 4:1 solution obtained from the first process of partitioning of the liquid-liquid type described in the example 2, were subjected to a further partitioning process with cyclohexane (200 mL).

(23) After having reached the equilibrium between the phases, the organic phase was separated from the aqueous phase. The latter was extracted again with 200 mL of cyclohexane for 3 times.

(24) The resultant organic phase was then washed with 200 mL of a mixture of methanol/water (4:1), recovered, anhydrified and concentrated at low pressures up to the complete drying thus obtaining 5.25 g of extract.

(25) The methanol phase of the partitioning, comprising argentatins C and D, was extracted with 200 mL of ethyl acetate for 3 times, anhydrified and concentrated under low pressures up to the complete drying thus obtaining 1.77 g of extract.

(26) The partitioning process was monitored by TLC (Thin layer chromatography).

EXAMPLE 5

(27) Direct crystallization of guayulins and argentatins (FIG. 1)

(28) a. Guayulin A: 5.0 g of guayulin mixture obtained by partitioning of the solid-liquid type described in the example 3, were dissolved in 10 mL of n-hexane. A copious precipitate was obtained by cooling the solution at 23 C. (acetone and dry ice-bath), which was then recovered by filtration.

(29) The residue was washed with n-hexane at 23 C. up to the obtaining of a white crystalline product identified as guayulin A (3.2 g). IR max (KBr): 3081, 2977, 2925, 2860, 1709, 1640, 1496, 1310, 1180, 927, 766, 708, 662, 563, 486 cm.sup.1. .sup.1H NMR (250 MHz, CDCl.sub.3) ppm: 1.08 (s, 1H), 1.13 (s, 1H), 1.55 (d, J=1.2 Hz, 3H), 1.67 (d, J=1.4 Hz, 3H), 2.79 (dd, J=12.3, 5.3 Hz, 2H), 4.52 (d, J=11.6 Hz, 1H), 4.92 (td, J=11.1, 5.3 Hz, 2H), 5.12 (m), 6.43 (d, J=16 Hz, 1H), 7.40 (m, 3H), 7.50 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3) ppm: 166.2; 144.3; 135.8; 134.5; 130.1; 130.0; 128.8; 128.1; 128.0; 125.0; 118.7; 75.4; 42.9; 40.3; 32.9; 28.8; 28.5; 25.2; 21.4; 20.4; 16.5; 15.4.

(30) The supernatant was recovered and the solvent was evaporated under reduced pressures thus obtaining 1.3 g of a mixture of guayulins A and B.

(31) b. Argentatin B: 2.3 g of argentatin fraction obtained by the partitioning of the solid-liquid type described in the example 3, were dissolved in 10 mL of petroleum ether. A copious precipitate was obtained by cooling the solution at 0 C. (ice bath), which was then recovered by filtration.

(32) The residue was washed with petroleum ether at 0 C. up to the obtaining of a white crystalline product identified as argentatin B (2.2 g). IR max (KBr): 3523, 3048, 2967, 2871, 2723, 1704, 1448, 1372, 1336, 1244, 1171, 1112, 1059, 910, 578 cm-1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 4.55 (m, 1H,), 3.56 (dd, 5, 5, 1H), 1.11 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.87 (d, 7, 3H), 0.85 (s, 3H), 0.83 (s, 3H), 0.51 (d, 5, 1H) and 0.31 (d, 5, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3); ppm: 18.83, 19.51, 20.8, 21.03, 21.12, 21.65, 22.24, 23.68, 24.06, 25.15, 26.25, 26.32, 26.42, 29.27, 29.78, 32.93, 33.62, 35.79, 37.66, 45.07, 46.06, 47.73, 48.67, 50.35, 57.61, 75.14, 80.14, 92.93, 217.00.

(33) c. Argentatin A: 5.25 g of extract derived from the organic phase obtained by the process of partitioning of the liquid-liquid type described in the example 4, were dissolved in 30 mL of ethyl ether. The solution was cooled at 23 C. (acetone and dry ice-bath) and n-hexane was slowly added as long as a turbidity of the solution with the consequent precipitation of argentatin A was observed.

(34) The precipitate was recovered by filtration and then it was subjected to washes with ethyl ether and n-hexane up to obtain a white crystalline product.

(35) 0.3 g of product identified as argentatin A were obtained. IR max (KBr): 3386, 2966, 2870, 1705, 1462, 1380, 1249, 1175, 1051, 954, 890, 837 cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 3.83 (dd, 11, 1H), 3.58 (m, 1H), 1.67 (s, 3H), 1.43 (s, 3H), 1.27 (s, 3H), 1.23 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H), 0.92 (s, 3H), 0.77 (d, 7, 1H), 0.43 (d, 7, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3); ppm: 20.4, 20.9, 21.1, 21.5, 23.9, 25.6, 26.0, 26.2, 26.4, 26.7, 27.4, 30.2, 33.4, 37.4, 37.6, 46.6, 46.7, 47.8, 48.7, 50.3, 56.1, 70.9, 73.4, 87.2, 84.7, 216.1.

(36) d. Argentatins C and D: 1.77 g of extract derived from the aqueous phase obtained from the process of partitioning of the liquid-liquid type described in the example 4, were dissolved in 10 mL of petroleum ether.

(37) A white precipitate was obtained by cooling the solution at 23 C. (acetone and dry ice-bath), which was then recovered by filtration. The residue was washed with petroleum ether at 23 C. up to the obtaining of a white crystalline product identified as argentatin D (0.54 g). IR max (KBr): 3499, 3036, 2972, 2922, 2862, 1710, 1440, 1337, 1162, 1111, 1055, 913, 779, 564 cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 4.55 (m, 1H), 3.56 (dd, 5, 5, 1H), 3.44 (br t, 1H), 1.11 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.87 (d, 7, 3H), 0.85 (s, 3H), 0.83 (s, 3H), 0.51 (d, 5, 1H), 0.31 (d, 5, 1H). .sup.13C-NMR (75 MHz; CDCl.sub.3): ppm: 18.83, 19.51, 20.85, 21.03, 21.12, 21.65, 22.24, 23.68, 24.06, 25.15, 26.25, 26.32, 26.42, 29.27, 29.78, 32.93, 33.62, 35.79, 37.66, 45.07, 46.06, 47.73, 48.67, 50.35, 57.61, 75.14, 78.9, 80.14, 92.93.

(38) The supernatant derived from the purification process of the argentatin D was recovered and the solvent was completely evaporated under reduced pressures. The residue was dissolved in 10 mL of ethyl ether.

(39) The solution was cooled at 23 C. (acetone and dry ice-bath) and n-hexane was slowly added as long as a turbidity of the solution and the consequent precipitation of argentatin C were observed.

(40) The precipitate was recovered by filtration and then it was subjected to washes with ethyl ether and n-hexane up to obtain a white crystalline product. The supernatant was recovered and re-crystalized in order to recover further product.

(41) 0.2 g of product identified as argentatin C were obtained. IR max (KBr): 3346, 2936, 2870, 1714, 1455, 1373, 1288, 1167, 1098, 1064, 917, 732, 669, cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 4.55 (m, 1H), 3.58 (s, 2H), 3.56 (dd, 5, 5, 1H), 1.11 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.87 (d, 7, 3H), 0.85 (s, 3H), 0.83 (s, 3H), 0.51 (d, 5, 1H) 0.31 (d, 5, 1H).

EXAMPLE 6

(42) Synthesis of the minor isoprenic constituents starting from the extracts (FIG. 4)

(43) Starting from the guayulin mixture obtained as described in the example 3 or from the pure guayulin A obtained as described in the example 5 or from its mother liquors, it was possible to obtain natural products contained in the resin by semi-synthesis, wherein they are hardly obtainable by non-chromatographic methods, because of their lower concentrations.

(44) a. Partheniol: Pure partheniol was obtained by basic hydrolysis reaction starting from the guayulin mixture obtained from the extract in petroleum ether/ethyl acetate 9:1 of the process of partitioning of the solid/liquid type or from pure guayulin A. 23.8 mL of a basic solution of potassium hydroxide and methanol (5% w/v; 425.4 mmol; 60 eqv) and 510 L of water were added to 5.00 g of guayulin mixture. The reaction was maintained under stirring at 40 C. for 24 hours, monitoring its progress by TLC (Thin layer chromatography). The reaction was stopped by dilution with a saturated solution of water and sodium chloride and extracted with petroleum ether. The organic phase is anhydrified, filtered and the solvent was evaporated under reduced pressures. The residue (5.0 g) was then purified by crystallization in petroleum ether at 0 C. (into an ice-bath). A white crystalline product was obtained (2.6 g) identified as partheniol or deacylguayulin. IR max (KBr): 3294, 3014, 2976, 2924, 2854, 2730, 1654, 1454, 1204, 1004, 851, 655, 534 cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 0.73 (dd), 1.15 (s), 1.16 (s), 1.19 (s), 1.42 (dd), 1.44 (s), 1.63 (s), 3.6 (ddd), 4.36 (d), 4.91 (dd). .sup.13C-NMR (75 MHz; CDCl.sub.3): 15.5, 16.5, 20.5, 20.8, 25.3, 28.8, 29.2, 36.0, 40.5, 46.3, 72.6, 125.3, 126.9, 129.2, 136.9.

(45) b. Guayulin B: to a solution of 100 mg of anisic acid (molecular mass 152.15; 0.657 mmol) dissolved in 2 mL of dichloromethane, 380 L of a solution of 98% oxalyl chloride in dichloromethane (molecular mass 126.93; density 1.335 g/mL; 2.63 mmol; 4 equivalents) and 66 L of DMF (100 L/mmol of acid) were added at 0 C. and under stirring for 1 hour. The reaction was maintained under stirring at room temperature for further 2.5 hours thus monitoring the development by TLC (Thin layer chromatography). The reaction was stopped by solvent evaporation under reduced pressures thus obtaining 100 mg of anisoyl chloride. Later, 77 mg of anisoyl chloride (molecular mass 170.15; 0.454 mmol; 1 equivalent) were added to a solution of 100 mg of partheniol (molecular mass 220.35; 0.454 mmol) dissolved in 6 mL of pyridine. The reaction was maintained under stirring at room temperature for 4 hours thus monitoring the development by TLC (Thin layer chromatography). The reaction was stopped by dilution with a saturated solution of water and sodium chloride and extracted with petroleum ether. The organic phase was anhydrified, filtered and the solvent was evaporated under reduced pressure. The residue was then purified by crystallization in petroleum ether at 0 C. (into an ice bath). A white crystalline product (0.15 g), identified as guayulin B, was obtained. IR max (KBr): 3504, 2932, 2854, 2790, 2657, 2123, 1707, 1606, 1449, 1359, 1166, 1045, 890, 770, 645, 539 cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 1.08 (s), 1.13 (s), 1.55 (d, J=1.2 Hz, 3H), 1.67 (d, J=1.41 Hz, 3H), 2.79 (dd, J=12.3, 5.3 Hz, 2H), 3.85 (s), 4.52 (d, J=11.6 Hz, 1H), 4.92 (td, J=11.1, 5.3 Hz, 2H), 5.12 (m), 6.82 (d, J=9 Hz, 1H), 7.95 (d, J=9 Hz, 2H).

(46) c. Semisynthetic argentatin D: To a solution of 100 mg of argentatin B (molecular mass 456.70; 0.218 mmol) obtained by the partitioning of the solid-liquid type described in the example 4 or from the mother liquors derived from the process of crystallization described in the example 5, dissolved in 4 mL of methanol, 25 mg of sodium borohydride NaBH.sub.4 (molecular mass 37.83; 0.661 mmol; 3.0 equivalents) were slowly added, into an ice-bath at 0 C. The reaction was maintained under stirring at room temperature for 5 minutes, monitoring the development by TLC (Thin layer chromatography). The reaction was stopped by dilution with a saturated solution of water and sodium chloride, addition of a solution of water and 5% sulfuric acid up to neutral pH and extraction of the aqueous phase with ethyl acetate. The organic phase was anhydrified, filtered and the solvent was evaporated under reduced pressures. The residue was then purified by crystallization in petroleum ether at 0 C. (into an ice-bath). A white crystalline product (0.070 g) identified as argentatin D, was obtained. IR max (KBr): 3499, 3036, 2972, 2922, 2862, 1710, 1440, 1337, 1162, 1111, 1055, 913, 779, 564 cm.sup.1. .sup.1H-NMR (250 MHz, CDCl.sub.3); ppm: 4.55 (m, 1H), 3.56 (dd, 5, 5, 1H), 3.44 (br t, 1H), 1.11 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.87 (d, 7, 3H), 0.85 (s, 3H), 0.83 (s, 3H), 0.51 (d, 5, 1H), 0.31 (d, 5, 1H). .sup.13C-NMR (75 MHz; CDCl.sub.3): ppm: 18.83, 19.51, 20.85, 21.03, 21.12, 21.65, 22.24, 23.68, 24.06, 25.15, 26.25, 26.32, 26.42, 29.27, 29.78, 32.93, 33.62, 35.79, 37.66, 45.07, 46.06, 47.73, 48.67, 50.35, 57.61, 75.14, 78.9, 80.14, 92.93.

EXAMPLE 7

(47) Plant material: crude extract of branches and stems of parthenium argentatum (FIG. 2)

(48) 3.89 g of crude extract, obtained from branches and stem of Parthenium argentatum were processed in accordance with this invention for separating guayulin A and argentatins A and B. Following the separation process of the lipophilic component comprising fats and low molecular weight rubbers, by the winterization method, the processes of separation and direct crystallization with the same procedures described in the Examples 2-6 and in relation to the amount of the crude extract used, were carried out. 1.7 g of lipophilic mixture of fats and low molecular weight and high molecular weight (HMW) rubbers, 0.15 g of crystalline guayulin A, 0.08 g of argentatin A, 0.18 g of argentatin B, were obtained.

EXAMPLE 8

(49) Plant material: crude extract of Parthenium argentatum leaves (FIG. 3)

(50) 4.6 g of crude extract, obtained from leaves of Italian Parthenium argentatum from Basilicata, were processed in accordance with this invention, for separating the guayulins component and argentatins A and B. Following the separation process of the lipophilic component comprising fats and low molecular weight rubbers, by the winterization method, the processes of partitioning and direct crystallization with the same procedures described in the Examples 2-6 and in relation to the amount of the crude extract used, were carried out. 0.30 g of guayulin mixture were obtained, from which by direct crystallization with n-hexane at 23 C. (acetone and dry ice-bath) 0.06 g of crystalline guayulin A, 0.06 g of argentatin A and 0.17 g of argentatin B, were obtained.

EXAMPLE 9 (COMPARATIVE)

(51) Liquid-liquid partitioning of a Non-winterized crude extract of Parthenium argentatum

(52) 5.0 g of crude extract obtained from the bagasse of Parthenium argentatum were slowly dissolved under continuous stirring at 40 C. in methanol (400 mL) using a solvent volume of 1.0 L/100 g of crude extract. After complete dissolution, the mixture was cooled at room temperature and diluted to a final volume of 500 mL with 100 mL of water, obtaining a mixture methanol/water (4:1).

(53) The obtained mixture was subjected to a partitioning process with petroleum ether (100 mL). 250 mL of methanol/water (4:1) mixture were further added to the mixture, and finally the establishment of an equilibrium between the phases was obtained; thereafter the organic phase was separated from the aqueous phase.

(54) The latter was subjected again to extraction with 100 mL of petroleum ether for six times. The resulting organic phase was subsequently washed with 200 mL of a methanol/water (4:1) mixture, recovered, anhydrified and concentrated under reduced pressure till complete drying, obtaining 2.5 g of product. The partitioning process was monitored through TLC (Thin layer chromatography).

EXAMPLE 10 (COMPARATIVE)

(55) Solid-Liquid Partitioning of the Apolar Extract Deriving from a Non-Winterized Crude Extract of Parthenium Argentatum

(56) 2.5 g of apolar extract obtained from the liquid-liquid partitioning process of 5.0 g of crude extract obtained as described in comparative Example 9 were dissolved in 20 mL of a mixture of petroleum ether and ethyl acetate (9:1) and adsorbed on a bed of 70 mL of silica gel (70-230 mesh, 25 mL/g). The solvent was removed from the suspension by evaporation under reduced pressure or by filtration under vacuum, and the resulting solid matrix was washed three times with 100 mL of the solvent mixture used for adsorption.

(57) The process resulted difficult and ineffective, as any efficient separation of the components of the starting apolar crude extract could not be observed, due to the presence of a component consisting of fats and low molecular weight rubbers, which negatively affected the solid-liquid partitioning process.

EXAMPLE 11 (COMPARATIVE)

(58) Liquid-liquid partitioning of the polar fraction deriving from a non-winterized crude extract of Parthenium argentatum

(59) 950 mL of a methanol/water (4:1) solution obtained from the first liquid-liquid partitioning process described in comparative Example 9 were subjected to a further partitioning process with 100 mL of cyclohexane. After the establishment of an equilibrium between the phases, the organic phase was separated from the aqueous phase. The latter was subjected again to extraction with 100 mL of cyclohexane, for three times. The resulting organic phase was subsequently washed with 100 mL of a methanol/water (4:1) mixture, recovered, anhydrified and concentrated under low pressure till complete drying, obtaining 300 mg of extract. The methanol phase from the partitioning, comprising argentatins C and D, was extracted with 100 mL of ethyl acetate for three times, anhydrified and concentrated under low pressure till complete drying, obtaining 100 mg of extract.

EXAMPLE 12 (COMPARATIVE)

(60) Crystallization of guayulins and argentatins from extracts deriving from a non-winterized crude extract of Parthenium argentatum

(61) a. Guayulin A and argentatin B: 2.5 g of apolar extract obtained from the liquid-liquid partitioning described in comparative Example 9, were dissolved in 10 mL of n-hexane. By chilling the solution to 23 C. (into a dry ice/acetone bath) the expected formation of any precipitate of guayulin A and/or argentatin B could not be observed.

(62) b. Argentatin A: 300 mg of extract deriving from the organic phase obtained from the liquid-liquid partitioning process described in comparative Example 11, were dissolved in 5 mL of ethyl ether. The solution was chilled to 23 C. (into a dry ice/acetone bath) and n-hexane was slowly added until a faint turbidity of the solution, resulting in an inefficient precipitation of argentatin A, was observed.

(63) c. Argentatins C and D: 100 mg of extract deriving from the aqueous phase obtained from the liquid-liquid partition process described in comparative Example 11, were dissolved in 2 mL of petroleum ether. After chilling the solution to 23 C. (into a dry ice/acetone bath) no precipitate formation was observed.

(64) These comparative examples clearly demonstrate that omitting the crucial winterization step from the separation method of the present invention results in a drastic reduction of its effectiveness in obtaining the isoprenic constituents of guayule or guayule-like resin in purified form.