JPH0129558B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to a method for producing rosmarinic acid by tissue culture of plants of the Labiatae family. BACKGROUND OF THE INVENTION Certain spice plants, such as Rosmarinus officinalis and S. officinalis, are known to contain useful antioxidant compounds. See, e.g., U.S. Pat. , large amounts of spicy plant material are required to obtain even relatively small fractions with antioxidant properties.Also, it is necessary to remove the strong spicy odor normally characteristic of each plant, which often requires large amounts of expensive plant material. A solvent is required. It is also possible to use synthetic antioxidants (e.g. BHA and BHT), but the major drawback of synthetic antioxidants is that they are not soluble in water. BHA is a water-dispersible rubber. However, foods without synthetic additives are more popular with consumers.Therefore, it would be beneficial to develop water-soluble antioxidants from natural sources that do not have the problems associated with spicy plant extracts. SUMMARY OF THE INVENTION It is an object of the invention to provide a process for the production of non-synthetic, water-soluble antioxidants. Another object of the invention is the production of rosmarinic acid by suspension culture of Lamiaceae plant cells. Another object of the present invention is to provide an antioxidant obtained from plants of the Lamiaceae family, without the strong odor characteristic of spice plants. Still another object of the invention The object of the present invention is to provide a method for propagating tissue cells of plants of the family Lamiaceae in cell suspension culture. These and other objects are achieved by the following embodiments. Cells of the production plant are suspended and grown in a specific non-morphogenesis-related nutrient liquid growth medium, (b) at least a portion of the cells are collected from the medium, (c) from the collected cells,
comprising collecting a rosmarinic acid-containing fraction;
A method for producing rosmarinic acid by cell suspension culture is provided. DESCRIPTION OF THE PREFERRED EMBODIMENTS Part of the present invention is the discovery that rosmarinic acid is a naturally occurring antioxidant and that at least a portion of the antioxidant activity of extracts of plants of the Lamiaceae family is attributable to rosmarinic acid. . The present invention also provides a method for producing a rosmarinic acid composition by growing tissue cells of a Lamiaceae plant in a liquid growth medium and collecting a rosmarinic acid-containing fraction from the cells grown in a cell suspension culture. give you a way to do it. The invention has several advantages. First, compared to extraction from commercially available dried spices, the present invention produces 3 to 4 times more rosmarinic acid. Second, rosmarinic acid is water-soluble, making it extremely useful in the production of foods (eg, meat, fruit drinks, etc.). Thirdly, rosmarinic acid is a natural product derived from plants belonging to the Labiatae family (for example, Stonewort, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Amaranthica japonica, Amarinthus japonica, etc.), and therefore does not have the negative impression associated with synthetic food additives. The present invention includes the use of cells of any species of Lamiaceae that produce rosmarinic acid. The present invention
Rosmarinus, Salvia, Hyssopus, Ibozia, Lavandula, Lycopus, Mentha, Monarda, Origanum, Perowskia, Includes, but is not limited to, rosmarinic acid producing species of Plectranthus, Satureia and Thymus. Among these, Rosmarinus and Salvia are preferred. Rosmarinus oficinalis (R.
officinalis) or Salvia oficinalis (S.
Particular preference is given to selecting cell tissues of the present invention (C. officinalis) or of their variants. Representative plants of the present invention are commonly known by the names Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, Salvia japonica, and Salvia japonica. Those skilled in the art can easily find other rosmarinic acid producing plants of the Lamiaceae family. for example,
Ellis and Towers, Biochem.J, Vol. 118, 291−
p. 297 (1970); Reschke, “Z. Lebensm.
176, pp. 116-119 (1983
21b, pp. 604-605 (1966). After finding the species and strain, select the tissue cells that produce the highest amount of rosmarinic acid. Such tissue cells can be found by observing with a fluorescence microscope. 331 or 325nm
Cells that emit fluorescence usually produce the highest amount of rosmarinic acid. One way to initiate a cell suspension culture is to inoculate a liquid medium with a callus of plant tissue grown on a solid, non-morphogenetic medium. The tissue used for inoculating the culture medium is preferably from leaves of plants. Several fixed media are known in the art (e.g. Gamborg's B
-5 times, White's times and Schenk-Hildebrandt times). For example, Gamborg et al., “In vitro,” Vol. 12,
See pages 473-478 (1976). The best media for many species are Murashige and Skoog.
and Skoog) (MS) medium. "Physiol. Plant." Volume 15, pp. 473-479 (1962
year) reference. A friable callus is obtained after a few weeks, suitable for starting cell suspension. It is usually preferred to include growth regulators in solid media. For example, a suitable growth regulator for R. officinalis is 2,4-
They are dichlorophenoxylacetic acid (2,4-D) and kinetin. Typical concentration ranges for these growth regulators are about 1 to 20 μM for 2,4-D and 0 to about 40 μM for kinetin. However, one skilled in the art may also use concentrations outside this range. A particularly preferred medium for R. officinalis is MS medium containing approximately 1 μM each of 2,4-D and kinetin. For Salvia species, it is preferable to include indole-3-acetic acid (IAA) and 6-benzyladenine (6-BA) in the medium. Typical concentration ranges are about 1 to about 40 μM for IAA and about 10 to about 40 μM for 6-BA.
A particularly suitable medium for Salvia species contains approximately 10 μM each
This is an MS medium containing IAA and 6-BA. Another example of a medium composition for Salvia species is MS medium containing about 5 to about 20 μM 2,4-D and about 1 to about 40 μM kinetin. Another composition is
Gamborg's B containing about 0.2 to about 1.0 μM 2,4-D and about 0.47 μM kinetin
-5 medium is available. After obtaining the callus, cell suspension culture is started using it. Liquid MS is the preferred medium for cell suspension culture.
It is a medium. According to the present invention, sucrose is present in such a medium in an amount of about 4% to about 8% (particularly preferably about 6%) (W/V). Other liquid media known in the art or appropriate modifications of the above-mentioned media may also be used. For example, the growth regulators described above for solid media can be added to cell suspension cultures. By adding growth regulators, cell suspension cultures can be maintained for long periods of time. The addition of this growth regulator is particularly preferred if the cell suspension culture is carried out in a continuous process, in which case small amounts of cells are harvested periodically. However, if you perform a batch process and collect all the cells at once,
Addition of growth regulators is not preferred. This is because cell suspension culture without growth regulators produces more rosmarinic acid per total cell weight. In order to increase the yield of rosmarinic acid, a precursor of rosmarinic acid may be added to the cell suspension culture medium. For example, adding tyrosine to the cell suspension culture medium increases the amount of rosmarinic acid produced. The preferred concentration is approx.
It is 3000μM. However, the actual concentration of the precursor will be determined primarily by economic considerations. Plant cells are preferably harvested from suspension culture on about 7 to about 17 days, most preferably on about 10 to about 14 days. When using precursors such as tyrosine, cells are preferably cultured for longer periods (about 28 days). The cells must be aerated to prevent oxygen depletion and agitated to prevent the cells from clumping into a callus in suspension. Many methods in the art can be used for this purpose (shaking, stirring, shaking, or bubbling sterile air through the medium). To isolate rosmarinic acid, all or part of the plant tissue cells are recovered from the cell suspension. It is convenient to further culture the remaining cells in order to obtain extra cells to collect. Although other cell harvesting methods are known in the art, the preferred method is automated desludging centrifugation used in continuous operation. When culturing cells in suspension using the batch method, filtration may be used. Continuous paper filtration methods, such as drum filtration, are also known in the art. There are various methods for extracting rosmarinic acid from collected cells. One of them is hot water extraction and ethanol extraction. Ethanol extraction provides a suitable product, but this method requires extra steps to evaporate and recover the solvent, special care to avoid fire or explosion, and extra controls due to tax regulations. . A preferred method is therefore hot water extraction. Rosmarinic acid can be extracted by lysing cells with hot water of approximately 140 to 212 degrees. Generally, cells are exposed to hot water for about 10 to about 30 minutes. The exact time depends, inter alia, on the total amount of substance to be extracted, the water temperature, the shape of the extraction vessel and the stirring conditions. For ethanol extraction, cells are separated from approximately 140〓 to approximately
Contact with absolute ethanol at 173°C for about 10 to about 30 minutes. Again, the exact time depends, inter alia, on the total amount of substance to be extracted, the temperature of the ethanol, the shape of the extraction vessel and the stirring conditions. In the case of either hot water or ethanol extraction, the solid material is removed by a suitable method (eg filtration) and the remaining liquid fraction contains the rosmarinic acid. Other extraction methods will be apparent to those skilled in the art. The rosmarinic acid-containing fraction obtained by the above method does not have a strong odor or taste, so it can be used directly in foods such as ground meat (eg, beef, pork, or turkey), margarine, fruit drinks, or tyokolate. The rosmarinic acid fraction can be further purified by standard methods, such as chromatography, if necessary to obtain a substantially tasteless and odorless antioxidant composition. The appropriate amount of rosmarinic acid in a food depends on many factors, but typically about 0.001% to about 0.01% will provide protection against oxidation for a significant period of time. The appropriate amount of rosmarinic acid for a particular food product can be readily determined by one skilled in the art. The following examples are illustrative only and are not intended to limit the invention in any way. Example 1 Rosmarinus oficinalis (R.officinalis)
The production of rosmarinic acid was tested for many varieties of Salvia species. Mature leaves were collected from plants grown in a greenhouse. The leaves were extracted and the percentage of rosmarinic acid per dry weight was determined (shown in Figure 1).

[Table] Nasuofisinalis〓Prostratus〓
(R.off.〓Prostratus〓)(Very detailed)

【table】

[Table] * Expressed as % of dry weight.
Example 2 The amount of rosmarinic acid produced was tested for four different cell lines of R. officinalis that produce rosmarinic acid. Cell lines of Rosmarinus oficinalis (referred to as A, B, C and D) were obtained from a common type of rosmarinic acid. Cell suspension cultures were started from callus lines grown on solid MS medium containing 1 μM each of 2,4-D and kinetin. on rotary shearer
Five grams (dry weight) of callus cells were transplanted into 75 ml of liquid MS medium containing 1 μM each of 2,4-D and kinetin in a 250 ml Erlenmeyer flask. The suspension culture was further cultured at intervals of 7 days, 7 days, 14 days,
A replica was collected on the 24th. The collected cells were filtered through Watmann No. 1 filter paper in a 7 cm Buchner funnel, washed twice with 10 ml of distilled water, and weighed. 1g of collected wet cells
Rosmarinic acid was extracted by boiling each sample with 5 ml of absolute ethanol for about 2 hours. The hot extract was then filtered through Watmann No. 1 filter paper in a long funnel.
To obtain complete extracts, cells were washed with up to 30 ml of hot ethanol. The filtrate was kept refrigerated until analysis. Filter the sample before analysis, exactly 50 or 100 ml
diluted to The extracted cells were air-dried for 2 days,
Dry weight was measured on the third day. The total dry weight was determined by adding the weight of the dried and extracted cells to the weight of the solid content in the extract. Perkin Elmer 601HPLC
was analyzed using a C-18 reverse phase column. A convex gradient elution from 0.01 M citric acid to ethanol was used. As shown in Figure 2 below, changes in rosmarinic acid synthesis were observed depending on the cell line. Furthermore, a decrease in the amount of rosmarinic acid synthesis was observed between the 17th and 24th days.

[Table] Example 3 Several precursors for rosmarinic acid biosynthesis were added to a cell suspension culture solution, and their effects on rosmarinic acid production were determined. Cell line D of Example 2 was treated with 1 μM each of 2,4-D.
and kinetin in liquid MS medium (PH5.7). Approximately 75 ml of the medium in a 250 ml Erlenmeyer flask was autoclaved at 250 ml for 15 minutes, and then
g (wet weight) of filtered R. officinalis cells. Seven types of media containing different precursors at a concentration of 3000 μM were prepared. Its precursors are L-phenylalanine, cinamic acid, caffeic acid, p-coumarin, tyrosine, DOPA (DL) and DOPA (L). Cells were harvested and extracted at 7 day intervals and analyzed for rosmarinic acid synthesis as described in Example 2.
The suspension culture containing cinamic acid (444.7 mg/) and P-coumarin (492.6 mg/) died in 7 days. The results for the remaining five types are shown in Table 3 below. In the case of tyrosine, the amount of rosmarinic acid produced increased 20 times in 28 days compared to the control.

【table】

[Table] Example 4 The influence of sucrose concentration on rosmarinic acid production was investigated using cell line D of Example 2. Cells were grown in the liquid MS medium described in Example 2, but in addition to the medium containing growth regulators, liquid MS medium without growth regulators was used. 7 as described in Example 2.
On days 1, 14 and 21, rosmarinic acid was extracted and analyzed. Table 4 shows the results of cell suspension culture without growth regulators, and Table 5 shows the results of cell suspension culture containing 1 μM each of 2,4-D
The results are shown for a culture solution containing and kinetin.
The greatest amount of rosmarinic acid accumulation was observed in medium containing only 6% (w/v) sucrose and no growth regulators. The amount of cell proliferation was higher in the cell suspension containing the growth regulator than in the cell suspension without the growth regulator (1.9% with 6% sucrose on day 14).
It was twice as large). Compared to suspensions containing growth regulators, cell suspensions without growth regulators
% sucrose, the production amount of rosmarinic acid was 2 on the 14th day.
It was twice as large (percentage per dry weight).

【table】

【table】

[Table] % number mg mg weight mg
mg weight mg mg weight

Claims (1)

[Scope of Claims] 1. A method for producing a rosmarinic acid composition, comprising: (a) cells of a rosmarinic acid-producing plant of the Labiatae family in a non-morphogenesis-related nutrient liquid growth medium of about 4% to about 8%; (b) collecting at least a portion of the cells from the medium; (c) harvesting. The above method comprises collecting a rosmarinic acid-containing fraction from the cells. 2. The method according to claim 1, wherein the plant is a genus selected from the group consisting of Rosemarinus and Salvia. 3 The plant is Rosmarinus oficinalis (R.
officinalis) and Salvia oficinalis (S.
3. The method according to claim 2, wherein the method is selected from the group consisting of A. offidinalis. 4. The method according to claim 1, which is a continuous process in which the unharvested portion of tissue cells is used to initiate a second cell suspension culture. 5. The method according to claim 2, which is a continuous process in which the unharvested portion of tissue cells is used to initiate a second cell suspension culture. 6. The method according to claim 3, which is a continuous process in which the unharvested portion of tissue cells is used to initiate a second cell suspension culture. 7. The plant cells are from the species R. officinalis, and the non-morphogenic nutrient liquid growth medium contains a growth regulator selected from the group consisting of 2,4-dichlorophenoxylacetic acid and chitinene. The method according to claim 1, wherein: 8 Plant cells are Salvia oficinalis (S.
The method of claim 1, wherein the non-morphogenetic nutrient liquid growth medium contains a growth regulator selected from the group consisting of indole-3-acetic acid and 6-benzyladenine. .