WO2000053206A1

WO2000053206A1 - Rosemary having highly efficacious antioxidant extracts

Info

Publication number: WO2000053206A1
Application number: PCT/US2000/006409
Authority: WO
Inventors: John A. Greaves; James E. Haworth; Friedhelm Brinkhaus
Original assignee: Kemin Industries, Inc.
Priority date: 1999-03-11
Filing date: 2000-03-07
Publication date: 2000-09-14
Also published as: AU3876800A

Abstract

Plants of rosemary Rosemarinus officinalis having extracts with antioxidant activity higher than that found in naturally occurring plants and to extracts of rosemary plants extracted using a solvent blend and having an antioxidant activity of at least 50 % that of mixed tocopherols.

Description

ROSEMARY HAVING HIGHLY EFFICACIOUS ANTIOXIDANT EXTRACTS

Background Of The Invention The invention relates generally to rosemary (Rosmarinus officinalis) plants having extracts with antioxidant activity and, more particularly, to rosemary plants having surprisingly efficacious antioxidant activity when extracted using a solvent blend.

Worldwide demand for natural antioxidants has been rising due to safety concerns about synthetic food and feed additives and the public perception that natural food and feed supplements provide certain health benefits. The most important natural antioxidants being exploited commercially today are tocopherols. Tocopherols have a potent ability to inhibit lipid peroxidation in vivo by trapping peroxy-radicals (Burton, G. W., and K. U. Ingold (1989), in Vitamin E: Biochemistry and Health Implications, edited by A. T. Diplock, L. J. Machlin, L. Packer and W. A. Pryor, The New York Academy of Sciences, New York, pp. 7-22). Various herbal extracts for use as natural antioxidants are being explored. Possibilities include the extraction of rosemary or other botanical sources. Such new antioxidants may play a role in combating carcinogenesis as well as the aging process, and may be applicable in the nutraceutical industry.

Among the various natural extracts available in the market are rosemary extracts, which are reported to be highly effective in retarding lipid oxidation and protecting living cells from the damaging oxidative stress (Chen, Q., H. Shi and C-T

Ho (1992), "Effects of rosemary extracts and major constituents on lipid oxidation and soybean lipoxygenase activity", J Am Oil Chem Soc 69: 999-1002; Wong, J. W., K. Hashimoto and T. Shibamoto (1995), "Antioxidant activities of rosemary and sage extracts and vitamin E in a model meat system", J Agric Food Chem 43: 2707-2712). These extracts are described as being superior to vitamin E, a well-known natural antioxidant and food supplement, in many food model systems (Lolinge, J. (1983), Natural antioxidants in Allen, J. C. and R. J. Hamilton eds, Rancidity in Foods, Elsevier Applied Science, London, Chapter 6). However, opposite findings are also documented. Wong et al. (1995) revealed that vitamin E is more effective than rosemary extract in a cooked beef homogenate. Additionally, rosemary extract is shown to be a synergist of vitamin E in stabilizing or retarding oxidation in sardine oil and fish muscle (Fang, X. and S. Wanda (1993), "Enhancing the antioxidant effect of

α-tocopherol with rosemary extract in inhibiting catalyzed oxidation caused by Fe²⁺

and hemoprotein", Food Res Int 26: 405-411 ; Wanda, S. and X. Fang (1992), "The

synergistic antioxidant effect of rosemary extract and α-tocopherol in sardine oil

model system and frozen-crushed fish meat", J Food Process Preserv 16: 263-274). As to the extraction of rosemary, many authors report that polar solvents yield extracts with higher antioxidant activities (Chang, S. S., B. Ostric-Matijasevic, C-L Huang and OA-L Hsieh (1977), "Natural antioxidants from rosemary and sage", J Food Sci 42: 1102-1106). Chen et al. (1992) found that hexane extracts of rosemary contained a higher content of carnosic acid and carnosol than methanol extracts do. Carnosic acid and carnosol are among the most effective antioxidant molecules in rosemary. Carnosic acid and carnosol have been suggested to account for over 90% of the antioxidant activity of rosemary extracts (Aruoma, O. I, B. Halliwell, R. Aeschbach and J. Loligers (1992) "Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid", Xenobiotica 22: 257-268). Antioxidant molecules in general, and rosemary antioxidants specifically, are by nature labile molecules especially when exposed to heat and/or air. During the harvest, the drying, and the regular solvent extraction of rosemary, some oxidation is likely to occur. Through a process of chemical reactions, carnosic acid, the naturally- occurring antioxidant molecule in rosemary, is believed to be the precursor to carnosol and many other antioxidants found therein (Wenkert, E., A. Fuchs, J. D. McChesney (1965), "Chemical artifacts from the family labiate", J. Org. Chem. 30: 2931-2934). It can be demonstrated that the freshly cut leaves of rosemary do not contain carnosol (Aeschbach, R. and L. Philippossian (1993), "Carnosic acid obtention and uses", U.S. Patent No. 5,256,700). Carnosic acid is about 10 times more effective as an antioxidant than carnosol (Aruoma et al, 1992), and it, therefore, is important for the high activity of a rosemary extract to minimize the damage to carnosic acid.

Principal among the antioxidants of rosemary are carnosic acid, carnosol, isorosmanol, rosmanol, rosmaridiphenol, rosmarinic-acid, and rosmariquinone. Duke, J., (http://www.ars-grin.gov/duke). The presence and quantities of antioxidants varies substantially, however, from variety to variety. Moreover, the efficacy of the antioxidants extracted from rosemary plants varies depending on the variety and on the method of extraction. Of nineteen different varieties tested, the antioxidant activity of an extract made in a single extraction at room temperature with 6 ml/g of methanol varied between 9.8 and 34 percent relative activity compared against the reference activity of Eisai tocopherols (Eisai Co., Ltd., Tokyo, Japan) in an oxygen bomb using chicken fat as the test matrix. Summary Of The Invention An antioxidant extract derived from Rosmarinus officinalis is provided which exhibits at least 50 percent by weight of the relative antioxidant efficacy of Eisai tocopherols, which extract was obtained from plant material possessing genetic means for the expression of the antioxidants at the stated relative efficacy.

Plant material of rosemary is provided which yields an extract wherein the antioxidant efficacy of the extract is surprisingly high via genetic control.

A process is provided for the formation of rosemary plants that yield an extract wherein the efficacy of the antioxidants in the extract are atypically highly efficacious that comprises: (a) self-pollinating flowers of a rosemary plant that has a lineage which includes the variety Primley Blue, (b) screening Si progeny plants of step (a) for elevated antioxidant activity, (c) selecting from the progeny of step (b) a plant that exhibits in a solvent blend extract a level of antioxidant activity that is elevated, and (d) forming plants that include the selection of step (c) in their lineage that continue to exhibit an elevated antioxidant activity wherein the level of antioxidant activity continues to be under genetic control.

It is an object of the present invention to provide novel rosemary plants which contain levels of antioxidants that are provided in an atypical combination via genetic control. It is an object of the present invention to provide a novel rosemary extract which exhibits an atypical combination of concentrations of antioxidants that is under genetic control.

It is an object of the present invention to provide novel rosemary plants which contain an atypical combination of levels of antioxidants formed under conventional rosemary field growing conditions while under genetic control.

It is an object of the present invention to provide a novel rosemary extract that is suitable for substitution of tocopherols in personal care products, food products and

animal feeds. These and other objects and advantages of the invention will be apparent to those skilled in the art from the following description and appended claims.

Brief Description of the Drawings Figure 1 is a graphical representation of the antioxidant efficacy of methanol extracts of rosemary measured using an oxygen bomb.

Figure 2 is a graphical representation of induction times of poultry fat treated with three various "potent rosemary varieties in comparison to mixed tocopherols.

Figure 3 is a graphical representation of induction times of rosemary extracts using tetrafluoroethane or methanol for extraction. Figure 4 is a schematic diagram of an extraction method of the present invention.

Description Of The Preferred Embodiments It has been found that rosemary plants of the present invention can be created through the combination of rosemary genetic determinants that have not previously been recognized to make possible the extraction of antioxidants wherein the efficacy of the extract was at least 50% relative to Eisai tocopherols.

A number of varieties of rosemary are grown and samples of plant material are harvested from each variety. The samples are processed to extract the antioxidants present in the plant material, and the antioxidant efficacy of the extract is measured.

Plants of the varieties that exhibit high levels of antioxidant efficacy upon extraction are evaluated for phenotypic and agronomic characteristics that will affect the economy of commercial growing and harvesting of plant material from the variety, such as growth habit, hardiness, disease resistance, and so on. At least one variety is selected for advancement.

Plants of the variety are self-pollinated either flower by flower or by cross- pollinating flowers on the individual plant. Mature seed is harvested and planted. The plants grown form the seed are genetically distinct. Specifically, seed collected from a single parent plant of the variety Primley Blue that, because of isolation of the growing plant are understood to have been primarily self-pollinated seed of the parent plant, were planted in a greenhouse and produced 210 Primley Blue plants. These plants are being grown in a greenhouse under identical growing conditions ie. light, fertilization, watering, soil, etc. These plants exhibit considerable heterozygosity in their phenotypic characteristics. Vegetative samples of each growing plant are collected and extracted using a blend of solvents. Antioxidant efficacy of the extract is tested. Individual plants exhibiting the highest antioxidant efficacy are selected for advancement.

The selected plants are again self-pollinated and mature seed is collected. The seed is planted and samples of plant material from the genetically diverse growing plants is again extracted and tested for antioxidant activity. The individual plants representing the highest antioxidant activity are evaluated for desired agronomic and phenotypic traits and at least one of them is used as the parent plant for asexually propagating plants to be used in the commercial production of rosemary for production of the highly efficacious antioxidant extract.

Rosemary is a perennial and is cultivated primarily by asexual reproduction using propagules as it does not reproduce true from seed. Accordingly, all varieties that are publicly available are vegetative clones that are genetically identical. Within a collection of plants of a particular variety, there is no genetic variation to allow for selection of novel individuals or improved traits. In the present invention, a plants of a number of publicly available varieties of rosemary were screened for the presence and efficacy of antioxidants.

Rosemary Extraction A number of various rosemary accessions were obtained from the Chart Co.,

Papa Geno's Herb Garden, and the North Carolina Botanical Garden. The poultry fat, used as a test matrix, was supplied through Tyson. All solvents were purchased from

Fisher Scientific Co.

Method 1 : Rosemary leaves were dried in a Virtis freeze dryer (Model 125-ml, Gardiner, NY), and were ground in a coffee grinder. At room temperature, 100 g of rosemary was placed in a 1000 ml flask with 600 ml of methanol for 48 hours. The extract was then filtered through a Whatman no. 4 filter paper. After filtration the

filtrate was evaporated to dryness at 40° C using a rotary evaporator.

Method 2: 2.0 g of dried, ground rosemary were introduced into a glass vial extractor. The sample was then extracted with 20 ml tetrafluoroethane or tetrafluoroethane with

10% acetone or 10% hexane. The extraction was performed under pressure. The sample then was further extracted according to the method described in U.S. Pat. No.

5,512,285.

Method 3: One g of dried, ground rosemary leaves were introduced into a closed glass vial extractor. The sample was then extracted with 6 g of a solvent blend comprised of 85% TFE, 10% acetone and 5% methanol solvent blend for two hours. At this time the filtrate was quantitatively transferred into a glass collection vial. The rosemary was then washed with 10.0 g of the extraction solution for five minutes. This liquid portion was added to the first filtrate collected. The rosemary was washed a second time with 10.0 g of the extracting solution and this was also added to the collection vial. After all of the filtrate solutions had been combined, the pressure in the vial was slowly released. After all of the TFE had evaporated, the other organic solution was removed under a stream of nitrogen gas under moderate heating. The extraction process is illustrated diagrammatically in Fig. 4. Oxygen Bomb Method

Experiments were conducted in an oxygen bomb apparatus, purchased from Koehler Instruments (Bohemia, NY), according to established procedures with modifications (12-14). Five grams poultry fat (control or treated) was weighed into five glass dishes, 1.0 g fat each. The dishes were placed in a holder and then into a bomb (stainless steel sample container). The bomb was sealed, pressurized with O₂

gas to 50 psi, and immersed in a tank with silicon oil circulating at 100° C. The

pressure inside the bomb reached its maximum when the bomb's temperature had

equilibrated to 1.0^'0° C. A decline was seen as the sample started oxidizing and

absorbing O₂. The pressure was converted to a simulated digital signal and monitored continuously by a computer. At any time (t;) after the peaking of the pressure (P_maχ), the O₂ absorption (Aj) and the rate of absorption (Rj) of the sample were calculated as:

(2) R, = ₊₁ ^A, t,+ι - 1,

where 23.6 is a conversion factor in mg/psi; and t, P, A, and R are in min, psi, mg, and mg/min, respectively. The endpoint was taken as the induction time (t_max) at which the maximum rate of absorption (R_max) occurred (Figure 1). t_max was most consistent and easier to be decided than the time at the starting induction point. The effective induction time was obtained by subtracting the induction time of the control from that of the treated sample:

y-¹) t_s._c ^«- t_max, s_ample " m_aχ, control

The relative activity was expressed as the t_s._c ratio of the sample over the reference treatment:

(4) Relative activity, % = t_s,_{Cj samp}i_e x 100

ts-c, reference

Rancimat Method

The antioxidant activity of an extract was also evaluated using a Rancimat

operated at 100° C, using standard procedures. The samples tested included a control

or unprotected fat and the fat treated with an antioxidant for comparison. The protection against oxidation provided by the antioxidant was measured by the change

in the induction point over the control, unprotected fat, reported as a ΔT.

HPLC Tests The high performance liquid chromatography (HPLC) results were generated using a Hewlet Packard Series 1050 HPLC instrument. The samples were prepared by adding 5 mg or the extract to 1 ml of a mixture of 80% methanol and 20% acetone.

The column was a Phenomenex Luna 5 μ CI S, 250 X 4.60 mm. Each shot consisted

of 10 μl injections and the flow was adjusted to 1 ml per minute. Detection was made

at 230 nm.

Antioxidant Activity of Different Rosemary Varieties

Nineteen different accessions of rosemary were obtained for extraction with methanol. The antioxidant efficacy of these methanol extracts were measured using the oxygen bomb. Table 1, column 3, displays the results of the analysis in terms of percent of the antioxidant activity of mixed tocopherols (mixed tocopherols = 100%>). Figure 2 shows the induction times of untreated chicken fat, chicken fat treated with methanol extracts of three potent accessions of rosemary (500 ppm), and fat treated with Eisai tocopherols (125 ppm) for comparison. The antioxidant efficacy of the various rosemary varieties ranges from 9.8%> to 34.0% of the activity of mixed tocopherols. The variety "Santa Barbara" displayed the highest antioxidant activity with 34%> of the activity of mixed tocopherols.

Thirty-one rosemary varieties were extracted using extraction Method 3, including "Arp", the highest efficacy found in a preceding publication (Huang, Z. and F. Brinkhaus (1996), "Antioxidant efficacy of rosemary and curcuma extracts", Kemin Industries, Inc. publication) for a methanol extract of that variety was 10%> of the tocopherol activity. Results found in Table 1 , column 2, for "Arp" and the other varieties show a significant improvement over the previous results wherein Method 1 was used in the extraction. Also included in Table 1 are measurements taken from HPLC tests performed on the extracts made by using Method 3 of the weight percent carnosic acid in the extract (column 4) and the weight percent carnosic acid content calculated from the data of column 4 on the basis of the dried rosemary used as starting material (column 5).

TABLE 1 : ANTIOXIDANT CONTENT AND EFFICACY OF ROSEMARY

VARIETIES IN TERMS OF PERCENT OF THE ANTIOXIDANT ACTIVITY OF

MIXED TOCOPHEROLS USING TWO EXTRACTION METHODS

Wild varieties available in commercial quantities; obtained from Turkey

Extraction of Rosemary Using Tetrafluoroethane An extraction technique based on tetrafluoroethane (Method 2) was also investigated. Using a tetrafluoroethane/acetone 9:1 blend as a solvent we observed an increase in antioxidant efficacy of 74% over the methanol extracted material when the variety "Arp" was extracted (Figure 3). Expressed as percent of the activity of mixed tocopherols the tetrafluoroethane extract had 44% antioxidant activity. Testing in a Rancimat was conducted to compare the antioxidant efficacy of the Method 2 and Method 3 extracts obtained from vegetation of the Arp variety. In addition to the control fat, the fat was treated with 500 ppm of the Arp extract of Method 2, with 500 ppm of the Arp extract of Method 3, with 100 ppm of the Arp extract of Method 3, and with 250 ppm of mixed tocopherols (from the Naturox® product of Kemin Industries, Inc.). The Arp extract of Method 2 at 500 ppm extended the induction time over the control by 73.4 hours, the Arp extract of Method 3 at 500 ppm extended the induction time over the control by 102.6 hours, the Arp extract of Method 3 at 100 ppm extended the induction time over the control by 30 hours, and the mixed tocopherols at 250 ppm extended the induction time over the control by 31.5 hours.

Experiments were conducted using varying amounts of acetone combined with the TFE and varying amounts of hexane combined with the TFE, and to compare the two different groups of solvent systems in terms of yields and specific activity. A steady increase in extraction yields was observed as the TFE is replaced by the two solvents hexane or acetone. As to the specific activity, a rapid increase followed by a long plateau is observed. On average the TFE/acetone extracts outperformed the TFE/hexane extracts by about 10%> in terms of specific activity. However, at a concentration of 30% for both solvents, the extracts were approximately equal in efficacy.

Variations in the antioxidant levels of rosemary found in this study may be attributed to growth factors such as soil conditions, lighting, genetic variation, and other environmental factors. However, the wide range of effective antioxidant potency between the different rosemary accessions that have been tested is likely due to genetic variability. This view is supported by the fact that all of the rosemary was grown under the same environmental conditions in the same growing media and that the varieties displayed significant morphological differences. It is therefore reasonable to believe that genetic variation also exists for the secondary metabolite pathway which yield the antioxidant molecules.

Generally, elimination of heat treatment from the extraction process has increased significantly the activity of the extracted material. A further increase in specific activity was observed when the tetrafluoroethane/acetone blend was used for extraction. Specifically, it was possible to double the efficacy of an extract over material extracted with methanol. This increase can be attributed to several effects. During the extraction process oxygen is excluded and tetrafluoroethane may be extracting a higher percentage of the antioxidants over the other cellular components. Further research will also address the separation technologies available to separate the essential oil and the antioxidants. The primary product of a tetrafluoroethane extraction is a liquid which allows a subsequent distillation process. In contrast, the primary product of a methanol extraction is a dry powder which is difficult to process further. We are currently using a rolled film, ultra-low vacuum distillation technology for further purification of the antioxidant product.

It has been found that the rosemary plants of the present invention can be created through the combination of rosemary genetic determinants that heretofore were not recognized to make possible the formation of a novel plant wherein the levels of carnosic acid and other antioxidant molecules are provided in an atypical combination via genetic control.

An initial rosemary plant is a rosemary plant exhibiting elevated levels of carnosic acid, such as the Santa Barbara and Primley Blue varieties. Representative publicly-available rosemary varieties and sources for their acquisition that have elevated carnosic acid levels and accordingly are suitable for utilization as parental rosemary plants in accordance with the present invention include: Albia Thin Leaf, Collingwood Ingram, Russian River, Salem, Primley Blue, and Santa Barbara. Primley Blue and these representative rosemary varieties have the ability to form an elevated level of carnosic acid in the extracted antioxidants wherein the production of carnosic acid is under genetic control. Best results are achieved when a parental plant is selected that exhibits good agronomic characteristics in the area where the resulting rosemary plant of the present invention is to be grown.

Two or more of these parental varieties are cross-pollinated to form a first generation of heterozygous plants. A selection is made from the resulting first- generation plants that exhibits a level of carnosic acid that is elevated above the levels of the parental varieties used in the cross, wherein the levels of carnosic acid is under genetic control. The level of carnosic acid in the extract is preferably at least 25 percent relative antioxidant efficacy of tocopherols, and most preferably is at least 30 percent (e.g., at least 35 percent) based upon the relative antioxidant activity of tocopherols. In another preferred embodiment the combined levels of carnosic acid and other antioxidant molecules is at least 40 (e.g., approximately 40 to 45 or more) percent based upon the relative antioxidant activity of tocopherols. The formation of each of the principal antioxidant molecules in the extract is under genetic control. Plants in accordance with the present invention reliably can be formed under conventional rosemary field growing conditions. The genetic means for the expression of these traits can also be transferred by standard plant breeding to other rosemary plants where the same atypical levels of antioxidant molecules is exhibited.

The formation of a rosemary plant having a high level of carnosic acid and other antioxidant molecules is considered to be surprising and is incapable of simple explanation since this trait was not publicly available in the past and was not expressed by any of the initial parental plants.

In order to make a determination of the levels of antioxidant molecules present in the rosemary plant, vegetative material from the plants is processed according to any of the known antioxidant extraction methods in accordance with procedures known in the art. In particular, the levels of antioxidant molecules can be determined by extraction using TFE as described above and in pending United States Patent Application Serial No. 09/082,109, filed May 20, 1998.

The genetic means for the expression of the recited levels of antioxidant molecules in the extracted vegetative material once established reliably can be transferred to other rosemary plants via conventional plant breeding wherein plants are formed and are selected that continue to exhibit such recited levels of antioxidant molecules. The genetic determinants for such fatty acid profile preferably is introduced into varieties that exhibit highly satisfactory agronomic characteristics or into rosemary varieties that are agronomically well adapted for the intended growing site.

The unique levels of antioxidant molecules provided in the extract of the present invention make possible a number of end uses wherein such levels of antioxidant molecules can serve to advantage. For instance, such molecules can be substituted for all or part of the conventional antioxidants, such as tocopherols, used in food, feed and other related products.

Although the invention has been described with preferred embodiments, it is to be understood that variations and modification may be resorted to as will be apparent to those skilled in the art. Such variations and modifications are to be considered within the purview and scope of the claims appended hereto.

Claims

We claim:

1. A method for breeding rosemary plants having enhanced levels of antioxidants, comprising the steps of: pollinating at least one Rosemarinus officinalis plant to produce heterozygous seeds; collecting the segregating (Si) seeds; growing segregating plants from the Si seeds; measuring antioxidant content of vegetation produced from the plants grown from the Si seeds; and selecting plants having desired characteristics including extracts having an antioxidant activity greater than 50 percent of mixed tocopherols.

2. A method according to claim 1, and additionally comprising the step of crossing plants which have been selected according to the method of claim 1 , with a Rosemrinus officinalis plant having desired characteristics including an extract with antioxidant activity greater than 50 percent of mixed tocopherols.

3. A method according to claim 2, wherein the steps of crossing and selecting are repeated at least once.

4. A method according to claim 2, wherein crossing includes sexual crossing.

5. A method according to claim 2, wherein crossing includes asexual crossing.

6. A method according to claim 5, wherein asexual crossing includes somatic cell hybridization.

7. A method according to claim 1, wherein the step of pollinating includes self pollination.

8. A method according to claim 1, wherein the step of pollination includes back crossing with a Rosemarinus officinalis plant.

9. A method according to claim 1, wherein the Rosemarinus officinalis plant is selected from the group of varieties including Primley Blue, Santa Barbara, Russian River, Albia Thin Leaf, Salem, and Collingwood Ingram.

10. A method according to claim 1, and additionally comprising the step of propagating the plants having the desired characteristics.

11. A method according to claim 10, wherein the step of propagating includes the step of vegetative propagation.

12. A method according to claim 10, wherein the step of propagating includes the step of propagation by seed.

13. A rosemary plant produced according to the method of claim 1.

14. Antioxidant-containing vegetation produced by a rosemary plant in accordance with claim 13.

15. An antioxidant-containing extract obtained by collecting vegetation of claim 14 and extracting the antioxidants from the vegetation using a blend of TFE and at least one other organic solvent.

16. The extract of claim 15, wherein the step of extracting the antioxidants comprises: contacting the rosemary vegetation in a vessel with a blend of tetrafluoroethane and at least one organic solvent to dissolve the antioxidant component in the solvent blend; removing the remaining vegetation from the solution of the antioxidant component and the solvent blend; and removing the solvent blend to isolate a liquid, oily product containing the antioxidant component.

17. The process of claim 16, wherein the organic solvent is selected from the group including acetone, butane, ethanol, ethylene chloride, hexane, isopropanol, methanol, methylene chloride, and propylene glycol.

18. The process of claim 16, wherein the solvent blend comprises from between about 60% to about 95%> tetrafluoroethane.

19. The process of claim 18, wherein the solvent blend comprises tetrafluoroethane and at least two organic solvents.

20. The process of claim 19, wherein the organic solvents are selected from the group including acetone, butane, ethanol, hexane, and methanol.

21. The process of claim 20, wherein the solvent blend comprises between about 70%) and about 85%o tetrafluoroethane, between about 1% and about 25% acetone, and between about 1% and about 25% methanol.

22. An extract of vegetation of Rosemarinus officinalis having an antioxidant activity that is at least 50% that of mixed tocopherols.

23. An extract of vegetation of Rosemarinus officinalis having a carnosic acid content that is greater than 30 weight percent of the dried material weight basis of the vegetation.

24. A personal care product protected against oxidation, comprising a personal care product combined with the extract of claim 14.

25. A personal care product protected against oxidation, comprising a personal care product combined with the extract of claim 23.

26. A personal care product protected against oxidation, comprising a personal care product combined with the extract of claim 24.