ES2183745B1 - TAXUS PLANT M0DIFIED AND PROCEDURE FOR OBTAINING. - Google Patents

Abstract

Modified Taxus Plant and procedure for obtaining it. The invention relates to a process for obtaining a modified Taxus plant which comprises subjecting said plant to a water regime of between 50 and 100%, and subjecting said plant to a dose of C02 greater than 350 ppm. The invention also relates to the modified Taxus plant obtainable from said process, which comprises a concentration by weight of taxanes greater than the concentration by weight of taxanes in an unmodified Taxus plant. Advantageously, the Taxus plant obtainable according to said procedure has a higher concentration of secondary metabolites of great interest with therapeutic qualities for the treatment of cancer.

Description

Modified Taxus Plant and procedure for obtaining it.

Field of the Invention

The present invention relates to a modified Taxus plant and the method of obtaining it.

The modified plant, according to the present invention has levels in taxanes (especially paclitaxel, also known as taxol®) higher than those found in an unmodified plant.

Background of the invention

Carbon fertilizer in greenhouses is a technique quite known to be used in order to increase biomass in trees and, in agriculture, to increase the quality of fruit.

On the other hand, the compound known as paclitaxel (taxol®) is the only diterpene amide taxane that has antitumor and antileukemic properties. Therefore, it is of great interest to obtain this therapeutic chemical agent for the clinical treatment of patients presenting with breast cancer. However, taxol® exists only in small quantities, of the order of 0.01% inside the bark, in the Tejo ( Taxus ) plants. Until not many years ago, taxol could not be synthesized and even now, the cheapest source of this compound still comes from the Pacific Yew ( Taxus brevifolia ). For this reason, large areas of Pacific Tejo forests have been destroyed in the northeastern United States in order to obtain this anticancer drug.

The paclitaxel compound is thus a metabolite secondary of great interest for its therapeutic qualities in the cancer treatment and therefore there is a need for Increase its extraction without causing damage to the nature.

It is an object of the present invention to obtain a modified Taxus plant that is characterized by a concentration of taxanes and, in particular, taxol®, higher than that of an untreated plant.

In the same way, the present invention relates to the method of obtaining said modified Taxus plant.

Description of the invention

The present invention relates to a modified Taxus plant which, surprisingly, comprises a concentration of taxanes higher than that of an unmodified Taxus plant.

For the purposes of the present invention, `` plant '' means any of the vegetative states that plants present in their natural state or when they are grown in vivo in greenhouses, excluding intermediate growth or propagation states used in this definition. in in vitro culture techniques.

`` Modified Taxus plant '' means any plant of the genus Taxus that has been subjected to the conditions of the process of the invention.

More specifically, the invention relates to a modified Taxus plant having a bark with a concentration by weight of taxanes greater than the concentration by weight of said compounds in the bark of an unmodified Taxus plant.

Preferably, the concentration by weight of taxanes in the bark is equal to or greater, by more than 50%, to the concentration by weight of taxanes in the bark of an unmodified Taxus plant.

Even more preferably, the concentration by weight of taxanes in the bark is equal to or greater, by more than 100%, to the concentration by weight of taxanes in the bark of an unmodified Taxus plant.

The invention also relates to a modified Taxus plant having leaves with a concentration by weight of taxanes greater than the concentration by weight of said compound in the leaves of an unmodified Taxus plant.

Preferably, the concentration by weight of taxanes in the leaves is equal to or greater than, by more than 20%, the concentration by weight of taxanes in the leaves of an unmodified Taxus plant.

Even more preferably, the concentration by weight of taxanes in the leaves is equal to or greater, by more than 25%, to the concentration by weight of taxanes in the leaves of an unmodified Taxus plant.

The present invention also relates to the process for obtaining a modified Taxus plant.

Said procedure is characterized by following experimental conditions:

-

Water regime of 50-100% (400-800 mm.year -1); Y

-

Dose of CO2 upper 350.

Preferably, the dose of CO2 ranges between 500 and 700 ppm and the water regime, preferably, is 100%

It is evident to an expert in the field that the object of the present invention can be carried out with several modifications without departing from the scope of protection of the invention defined by the claims.

Description of the figures

- Figures 1 and 2: Relative growth rate (RGR) of the dry weight of leaves at 400 and 800 mm.year -1, respectively.

- Figures 3 and 4: Relative growth rate (RGR) of the dry weight of stems at 400 and 800 mm.year -1, respectively.

- Figures 5 and 6: Relative growth rate (RGR) of the dry root weight at 400 and 800 mm.year -1, respectively.

- Figures 7 and 8: Relative growth rate (RGR) of the total dry weight at 400 and 800 mm.year -1, respectively.

- Figure 9: Dry leaf weight distribution grown at three doses of CO2 and two of water. The values are the Average of five plants ± standard error.

Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = 0.007x + 1,165 \ cr \ + R2 = 0.8334 \ cr
Dashed line: \ + y = 0.0032x + 4.0342 \ cr \ + R2 =
0.8358 \ cr}
      

Figure 10: Dry root weight distribution grown at three doses of CO2 and two of water. The values are the Average of five plants ± standard error. ! n Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = 0.0042x + 5.2618 \ cr \ + R2 = 0.6816 \ cr
Dashed line: \ + y = 0.0154x + 0.7915 \ cr \ + R2 =
0.9519 \ cr}
      

Figure 11: Foliar area of Taxus plants grown at three doses of CO2 and two of water. Values are the average of five plants ± standard error.

Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = -0.0507x + 550.9 \ cr \ + R2 = 0.6401 \ cr
Dashed line: \ + y = 0.0154x + 0.7915 \ cr \ + R2 =
0.9933 \ cr}
      

Figure 12: Root / cup ratio of Taxus plants, grown at three doses of CO2 and two of water. Values are the average of 5 plants ± standard error.

Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = -0,0001x + 1.1577 \ cr \ + R2 = 0.5793 \ cr
Dashed line: \ + y = 0.0009x + 0.6029 \ cr \ + R2 =
0.4718 \ cr}
      

Figure 13: Concentration of taxanes in the leaves of the plants of the genus Taxus grown in three concentrations of CO2 and two doses of water. Values are the average of 2 values ± standard error.

Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = 0.0019x + 1.0841 \ cr \ + R2 = 0.3335 \ cr
Dashed line: \ + y = 0.0023x + 0.6103 \ cr \ + R2 =
0.7737 \ cr}
      

Figure 14: Concentration of taxanes in the bark of the stems of plants of the genus Taxus grown in three concentrations of CO2 and two doses of water. Values are the average of 2 values ± standard error.

Where:

         \ dotable {\ tabskip6pt # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 Continuous line: \ + y = 0.0027x-0.6655 \ cr \ +
R2 = 0.531 \ cr Dashed line: \ + y =
0.00834x-2.4862 \ cr \ + R2 =
0.9817 \ cr}
      

Next, the invention by preferred embodiments thereof.

Examples of embodiments of the invention

The following is described, by way of example, not limiting, an experimental procedure to increase levels of taxanes in bark and leaves.

Example 1 Obtaining a modified plant of the genus Taxus

In January 1997, seedlings were planted from a commercial seed in a 16 cm pot diameter, in a substrate composed of pine bark.

Yews are irrigated by spraying twice a day with a nutrient solution that includes nutrients N: P 2 O 5: K 2 O, in a ratio 1: 0.5: 1.5 and pH 6.5.

During the winter period (from January to May) CO2 was supplied at various levels (350, 500 and 700 ppm), and each level of CO2 was supplied with different water regimes (50 and 100%, 400 and 800 mm. year -1 respectively). In this way the yews were treated in six different conditions.

As of May, due to the need for ventilate the greenhouse because of the high temperatures, stopped the supply of CO2. And the water regime was applied by irrigation located with the same solution, with a few doses of water 116 l / m2 (100% treatment) and 58 l / m2 (treatment fifty%).

The application of CO2 was carried out by conventional polyethylene pipes. The source of CO2 was a liquid CO2 tank equipped with an evaporator to pass the CO2 at gas phase. The dosage of CO2 was carried out by a system composed of an infrared analyzer, a automaton and a series of solenoid valves that injected CO2 into function of the automaton orders.

Every May, after finishing the addition of CO2, biomass and taxanes were measured present in the leaves and bark.

Example 2 Effects of CO2 enrichment on yew

The procedure followed to analyze the Taxane levels comprise the following stages:

one-

Freeze leaves and stems

two-

Lyophilization

3-

Conservation in dry.

4-

Analysis of the amount of taxanes by an Elisa enzyme immunoassay (Heinstein and Chang, 1994).

The results obtained are shown in the Figures 1-8, from which the following follows:

-

The growth of yew due to enrichment with CO2 is more marked the first year, disappearing the second year (Callaway et. al. 1994).

-

 Carbon fertilizer does not promote significant differences in the growth rate relative (RGR) of the entire yew plant or its different fractions

-

 The tree shows a positive growth trend in the presence of levels greater than CO2 during the two years.

In this way it is verified that during the first year the increase in dry weight of leaves, stems and roots of plants grown at 500 ppm of CO2 is higher than grown at 350 ppm (27.8; 7, 2; 48.5%). When supplied CO2 at a dose of 700 ppm the increase in weight is smaller being even negative (-1.3; 9.2; 37.2%), as illustrated in the Figures 9-12.

The effect of water on yew growth does not shows significant differences although the trend is shown that dry weight values increase the more water be contributed

As for the production of taxanes, at contrary to what happens with biomass, it does show significant differences in the different treatments with CO2 and different water regimes, and presents a tendency to increase the amount of taxanes as the CO2 concentration; this is illustrated by figures 13 and 14.

In this way, plants grown at 700 ppm they contain 28.8% more taxanes in 400 mm.year -1 and 49.36% more of taxanes in 800 mm.year -1 on the leaves with respect to the subjected to 350 ppm.

In the bark, the plants that are given provided a dose of CO2 of 700 ppm presented 152% more of taxanes in 400 mm.year -1 and 517% more taxanes in 800 mm.year -1 in the cortex with respect to those subjected to 350 ppm.

Claims (6)

1. Procedure for obtaining a modified Taxus plant, characterized by the fact that the following stages are carried out:

-

said Taxus plant is subjected to a water regime 800 mm / year, and

-

then it subject said Taxus plant to a dose of CO2 comprised between 500 and 700 ppm.

2. Modified Taxus plant obtainable from the method according to claim 1, comprising a concentration by weight of taxanes in bark and leaves greater than the concentration by weight of taxanes in bark and leaves of an unmodified Taxus plant. 3. Plant according to claim 2, characterized in that said bark comprises a concentration by weight of taxanes equal to or greater than 50%, at the concentration by weight of taxanes in the bark of a non- Taxus plant modified 4. Plant according to claim 3, characterized in that in said bark the concentration by weight of taxanes is equal or greater, by more than 100%, to the concentration by weight of taxanes in the bark of a Taxus plant unmodified 5. Plant according to claim 2, characterized in that in said sheets of said modified Taxus plant the concentration by weight of taxanes is equal to or greater than, by more than 20%, the concentration by weight of taxanes in the leaves of an unmodified Taxus plant. 6. Plant according to claim 5, characterized in that in said sheets the concentration by weight of taxanes is equal to or greater than, by more than 25%, the concentration by weight of taxanes in the leaves of a Taxus plant unmodified