CARNOSIC ACID, CARNOSOL AND ROSMARINIC ACID ISOLATION METHOD

Abstract

The invention relates to a method of separation by fractionalization of the antioxidant active ingredients of Rosemary plant, which are the diterpenes Carnosic acid and Camosol and the phenolic acid Rosmarinic Acid, through column chromatography method. The production process involves the passage of rosemary extract, which is obtained in/with a proper solvent, through a column packed with a suitable packing material, the separation of the part rich in carnosic acid and carnosol, and the obtainment of a deodorized product in powder form after drying. During the process, another extract containing rosmarinic acid with high antioxidant activity is also obtained. The invention thus allows the use of an extract rich in carnosic acid and carnosol and another extract containing rosmarinic acid—which are obtained at once with no need for additional processes like acidification, precipitation, membrane filtration or deodorization—in food, pharmaceutical and cosmetic sector applications.

Claims

1. A method for carnosic acid, carnosol and rosmarinic acid isolation, comprising the sequential steps of: 1) Extraction of deoiled rosemary (A) using ultrasonic water bath at 60° C. for 20 min with a 50% ethanol:water (v:v) solution, 2) Filtration (B) of the extract of step (A) by vacuum filtration and the filtered liquid extract is filtered for a second time using a fine filter with 1-5 micrometer pore diameter, 3) Passing of a deionized water as a mobile phase through a macro-porous adsorbent-containing column (C) before feeding the filtered liquid extract into the column then eluent from the column goes to waste, 4) Feeding of the filtered liquid extract into the column in the amount of 0.01 to 5 times of the column volume, then wasting of eluent observed at 284 nm wavelength corresponding to waste, 5) Passing a 50% ethanol-water solution through the column as mobile phase, 6) Collection of eluent corresponding to liquid extract containing rosmarinic acid, 7) Evaporation-1 (D) of ethanol in the eluate of step 6, containing rosmarinic acid, either under vacuum in rotary evaporator, lyophilizer, vacuum dryer, vacuum oven or spray dryer, 8) Freeze drying (lyophilizing) (E) of the remaining aqueous part and obtainment of deodorized rosmarinic acid, 9) Passing a 99% ethanol through the column as mobile, 10) Collection of the eluent of step 9, liquid extract containing carnosic acid and carnosol, 11) Evaporation-2 (F) of ethanol in the eluate of step 10, containing carnosic acid and carnosol either under vacuum in rotary evaporator at 40° C., until it dries completely and obtainment of deodorized carnosic acid and carnosol.

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9. An isolation method according to claim 1, characterized in that, during the feeding of the extract into the column, the ratio of extract:column volume is preferably 1.5.

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21. An isolation method according to claim 1, characterized in that, the column packing material, is selected from the materials; styrene divinylbenzene, macro-porous polyamide, polyacrylate, polymethacrylate and polyvinylpyrolidone adsorbents.

22. An isolation method according to claim 1, characterized in that, during the feeding of the extract into the column, the ratio of extract:column volume is preferably 1.5.

Description

DESCRIPTIONS OF THE FIGURES

[0019] The flow diagram of the purification process designed in line with the purpose of this invention is shown in the attached FIG. 1.

[0020] FIG. 1. Carnosic acid, carnosol and rosmarinic acid isolation process

DESCRIPTIONS OF THE REFERENCES IN THE FIGURES

[0021] (A) De-oiled rosemary extraction step [0022] 1. De-oiled rosemary [0023] 2. 50% ethanol:water solution [0024] 3. Heating [0025] 4. Solid-liquid extract [0026] (B) Filtration step [0027] 5. Vacuum [0028] 6. Insoluble solid matter [0029] 7. Liquid extract [0030] (C) Step of passing through a macro-porous adsorbent-containing column [0031] 8. Gradient mobile phase consisting of water and/or ethanol [0032] 9. Waste [0033] 10. Eluent containing rosmarinic acid (Liquid extract containing rosmarinic acid) [0034] 11. Eluent containing carnosic acid and carnosol (Liquid extract containing camosic acid and carnosol) [0035] (D) Step of Evaporation-1 [0036] 12. Temperature [0037] 13. Vacuum [0038] 14. Liquid extract rich in rosmarinic acid [0039] (E) Freeze drying step [0040] 15. Vacuum [0041] 16. Cooling [0042] 17. Rosmarinic acid extract [0043] (F) Step of Evaporation-2 [0044] 18. Temperature [0045] 19. Vacuum [0046] 20. Camosic acid+carnosol extract

EXPLANATION OF THE INVENTION

[0047] The invention aims to produce rosemary extract rich in camosic acid and carnosol which are used as natural antioxidants in food and cosmetic sectors. To this end, the liquid extract obtained from rosemary plant with ethanol-water mixture is fractionated by column chromatography method using a suitable packing material and mobile phase flow program; and a deodorized extract is obtained containing 42-48% carnosic acid and carnosol. In addition to the carnosic acid and carnosol extract, an extract containing rosmarinic acid is also obtained in the column chromatography system. In this method, by making use of the suitable solution (ethanol-water) and solvent ratio (50%) for the most efficient extraction of the active ingredients from rosemary and taking advantage of the active ingredients' differences of solubility in water and ethanol and their adsorption affinities against the packing material, it is made possible to produce an extract containing 42-48% carnosic acid and carnosol and another product containing 7-8% rosmarinic acid with high antioxidant activity, at once with a more practical method compared to other methods. The liquid extracts obtained from column chromatography are dried and powdered, in rotary evaporator or lyophilizer, depending on their ethanol and water content.

[0048] Process flow diagram of the developed method is shown in FIG. 1. [0049] 1—De-oiled rosemary (1) is extracted with 50% ethanol:water (v:v) solution (2). Extraction method is ultrasonic water bath at 60° C. for 20 min (3). [0050] 2—The extract (4) is separated from solid matter (6) by vacuum filtration (5). The filtered liquid extract is filtered for a second time using a fine filter with 1-5 micrometer pore diameter (B). [0051] 3—The liquid extract obtained (7) is now ready for feeding into the column (C) containing a macro-porous adsorbent. Before feeding the liquid extract (7) into the column packed with styrene divinylbenzene adsorbent and water, deionized water (8) as mobile phase is passed through the column and the eluent from the column goes to waste (9). [0052] 4—The liquid extract (7) is fed into the conditioned column. Within this time period, the eluent observed at 284 nm wavelength corresponding to peak-1 zone in chromatogram goes to waste (9). [0053] 5—When the feeding of the liquid extract (7) is completed, deionized water (8) as mobile phase is passed through the column and this part goes to waste (9). [0054] 6—Then, 50% ethanol-water solution (8) is passed through the column as mobile phase; meanwhile the eluent corresponding to the peak-2 zone (liquid extract containing rosmarinic acid) (10) is collected for evaporation. [0055] 7—In the next step, 99% ethanol (8) is passed through the column as mobile phase, and in the meantime, the eluent corresponding to the peak-3 zone (liquid extract containing carnosic acid and carnosol) (11) is collected for evaporation. [0056] 8—The column is washed away and cleaned by passing ethanol (8) as mobile phase so that it becomes ready for the next run. Then, deionized water (8) is passed as mobile phase and the column becomes ready for the next run (C). [0057] 9—The eluent (10) corresponding to the peak-2 zone in the column chromatography contains 50% ethanol-water, while the eluent (11) corresponding to the peak-3 zone contains only ethanol. The eluent corresponding to the peak-2 zone is subjected to vacuum (13) in rotary evaporator at 40° C. (12) until the ethanol evaporates completely (D). The remaining aqueous part (14) is freeze dried at lyophilizer (15), dried (E) under vacuum (16), and rosmarinic acid extract (17) is obtained. Since the eluent (11) corresponding to the peak-3 zone is composed of ethanol only, it is subjected to vacuum (19) in rotary evaporator at 40° C. (18) until it dries completely. As a result of this process (F), an extract of camosic acid and carnosol (20) is obtained in powder form.

Example

[0058] 200 grams of deoiled rosemary is extracted with 2 liters of (50% ethanol:water, v:v) solution. Extraction method is ultrasonic water bath at 60° C. for 20 min. The extract is separated from solid matter by vacuum filtration. The filtered liquid extract is filtered for a second time using a fine filter with 1-5 micrometer pore diameter.

[0059] The liquid extract obtained is now ready for feeding into the column containing a macro-porous adsorbent. Before feeding the liquid extract into the column packed with styrene divinylbenzene Diaion HP20 adsorbent and water; deionized water as mobile phase in the amount of 0.2 times of the column volume is passed through the column and the eluent from the column goes to waste. The conditioned column is fed with liquid extract in the amount of 1.5 times of the column volume. Within this time period, the eluent observed at 284 nm wavelength corresponding to peak-1 zone in chromatogram goes to waste. When the feeding of the liquid extract is completed, deionized water as mobile phase in the amount of the column volume is passed through the column and this part goes to waste. Then, 50% ethanol-water solution as mobile phase in the amount of 2 times of the column volume is passed through the column; and the eluent corresponding to the peak-2 zone (liquid extract containing rosmarinic acid) is collected for evaporation. In the next step, 99% ethanol as mobile phase in the amount of 2 times of the column volume is passed through the column, meanwhile, the eluent corresponding to the peak-3 zone (liquid extract containing carnosic acid and carnosol) is separately collected for evaporation. The column is washed away and cleaned by passing ethanol as mobile phase in the amount of the column volume, so that it becomes ready for the next run. Then, deionized water is passed as mobile phase in the amount of the column volume and the column becomes ready for the next run.

[0060] The eluent corresponding to the peak-2 zone in the column chromatography contains 50% ethanol-water, while the eluent corresponding to the peak-3 zone contains only ethanol. The eluent corresponding to the peak-2 zone is subjected to vacuum in rotary evaporator at 40° C. until the ethanol in it evaporates completely. The remaining aqueous part is freeze dried at lyophilizer, dried under vacuum, and rosmarinic acid extract is obtained. Since the eluent corresponding to the peak-3 zone is composed of ethanol only, it is subjected to vacuum in rotary evaporator at 40° C. until it dries completely. As a result of this process, an extract of carnosic acid and carnosol is obtained in powder form.

[0061] Analysis results on total phenolic compounds and antioxidant activity of carnosic acid, carnosol and rosmarinic acid extracts in dried powder form are given in Table 1 below.

TABLE-US-00001 TABLE 1 Active ingredients and antioxidant activities of extracts Amount of Total phenolic DPPH, active compounds, IC.sub.50 value, ingredient, mg mg ABTS, mg mg/100 mg GAE/100 mg extract/ml TE/100 mg Extract 44.09 22.38 0.19 53.23 containing carnosic acid and carnosol Extract 7.42 29.00 0.21 50.65 containing rosmarinic acid Active ingredient amounts: For the extract containing carnosic acid and carnosol, the amount is determined with the sum of carnosic acid and carnosol content, whereas, for the extract containing rosmarinic acid, the amount is determined with the rosmarinic acid content per 100 mg extract. Total phenolic compounds: Results are given in mg gallic acid equivalent/100 mg extract. DPPH: (2,2-diphenyl-1-picrylhydrazyl) radical scavenging capacity Results are given in IC.sub.50 (sample amount required to halve initial DPPH concentration in mg extract/ml solution). ABTS: Trolox-equivalent (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) antioxidant capacity (TEAC—ABTS) Results are given in mg Trolox-equivalent/100 mg extract.

[0062] This method makes it possible to produce deodorized extracts rich in camosic acid and carnosol, by using plant species such as rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa) from the Labiatae (Lamiaceae) family.

[0063] It is possible to use both deoiled rosemary and oil-containing rosemary, however, in case of oil-containing rosemary use, efficiency may decrease.

[0064] For the extraction of active ingredients, the rosemary:solvent (solid:liquid) ratio can be in the range from 1:1 to 1:200.

[0065] The solvent to be used for the extraction of active ingredients can be ethanol as well as other polar and apolar solvents like methanol, acetone and hexane. The ratio of the solvent used for the extraction of active ingredients can range from 5% to 100%.

[0066] During the feeding of the extract into the column, the ratio of extract:column volume can range from 0.01 to 5.

[0067] During fractionating by column chromatography method, it is possible to use a column chromatography system or apparatus.

[0068] As the column packing material, it is possible to use styrene divinylbenzene Diaion HP20, as well as macro-porous polyamide, polyacrylate, polymethacrylate and polyvinylpyrrolidone adsorbents.

[0069] During fractionating by column chromatography, as mobile phase, it is possible to use ethanol:water, as well as polar and apolar solvents like methanol, acetone and hexane.

[0070] In the mobile phase flow program, it is possible to start with 100% water and switch to 50% ethanol:water flow and finally to 100% ethanol flow; or instead of a stepwise transition, it is possible to switch directly from 100% water to 100% ethanol.

[0071] The obtained liquid extracts could be dried in rotary evaporator or lyophilizer, as well as in a vacuum dryer, vacuum oven or spray dryer.

[0072] If desired, the active ingredient amounts of the extracts can be increased by using a second column.

Industrial Application of the Invention

[0073] The extracts containing carnosic acid, carnosol and rosmarinic acid can be used in food, pharmaceutical and cosmetic products both directly and by encapsulation.