CA2559077A1 - Process for extraction of diterpenes and triterpenes from biomaterial - Google Patents

Abstract

The present invention relates to a process for selectively extracting desired diterpenes or triterpenes and derivatives thereof by treatment of plant material with ultrasound combined to specific solvents into which tree parts are treated to allow dissolution of diterpenes or triterpenes and compounds of the same family.

Description

PROCESS FOR EXTRACTION OF DITERPENES AND
TRITERPENES FROM BIOMATERIAL
BACKGROUND OF THE INVENTION
a) Field of the invention The invention relates to a process for obtaining diterpenes or triterpenes and related compounds from trees. Particularly, the invention relates to a process for selectively extracting the diterpene or triterpene compounds in a relatively. short period of time at temperatures non denaturing for the extracted compounds.
b) Description of the prior art Terpenes are constructed of multiples of the five-carbon hydrocarbon isoprene (2-methyl-1,3-butadiene). Terpene compounds of variable size are widely distributed in nature and they can also be alternatively synthetically produced. The monoterpenes are constituted of two isoprene units, the sesquiterpenes of three, while those containing four, six, and eight units are called diterpenes, triterpenes, and tetraterpenes, respectively. The C3o compounds, or triterpenes, are ubiquitous among the dicotyledons to which the hardwood species belong. The Betulaceae is one of the hardwoods families notorious for its triterpenoid content. Several nutraceutical or pharmaceutical properties were attributed to these compounds.
An interesting example is squalene, a viscous oil that is found in large quantities in shark liver oil. It may be isolated in smaller amounts from olive oil, wheat germ oil or rice bran oil.
This non-cyclic hydrocarbon triterpene is an intermediate in the biosynthesis of cyclic triterpenoids and steroids.
No selective technique for the extraction of triterpenoids has been reported yet in literature. The use of high temperature like in Soxhlet extraction may cause chemical modification of some molecules. Maceration type techniques do not use high temperatures but the extraction yields are-usually smaller than for Soxhlet extraction.
Sonication has proven to be useful to improve extraction yield of essential oils from fragrant herbs (mainly monoterpenes and their derivatives).
In addition, the cultivation of plant cells in bioreactors is a promising means for the production of commercially important phytochemicals, particularly those derived from naturally rare and difficult to cultivate plant species. The diterpenoid derivative paclitaxel (generic name for Taxol A), for example, was originally isolated from the bark of the Pacific yew tree (Taxus brevifolia), which is a slow-growing species and not abundant in the world.
Extraction of Paclitaxel from trees cannot meet the increasing drug demand for clinical use.
Cell cultures of various Taxus species have been studied as an alternative route and renewable source of paclitaxel supply.
Culture or in. situ product removal strategy has been exercised previously in many different plant cell cultures, or naturally originating plant parts. However, the product harvesting and yields are still limited.
It would be highly desirable to be provided with a new process allowing the extraction of triterpenoids from trees. More particularly, it would be highly useful to be provided with a method allowing to selectively extract the triterpenoids.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a process for selectively obtaining the diterpene derivative, paclitaxel, or triterpenes from plant material comprising the steps of:
a) irmnerging portion of plant material, as for example, but not limited to, tree part, wood meal, leaves, branches, barlc, or twigs, in an organic solvent at a non denaturing temperature for diterpenes, triterpenes or derivatives thereof;
b) submitting said portion in organic solvent to ultrasound waves for a period of time causing diffusion of diterpenes or triterpenes into said organic solvent;
and c) separating selected diterpenes or triterpenes from said solvent .

It will be easily recognized to those skilled in the art that the plant material can be obtained for example, but not limited to, any lignocellulosic plants, trees, or endovascular plants.
The diterpene or triterpene can be at least one of the compound selected from the group consisting of paclitaxel, non cyclic triterpenoids, squalene, lupane and norlupane type triterpenoids, lupeol and betulonic acid, oleane and ursane type triterpenoids, (i-amyrin, and a,-amyrin.
The trees are preferencially, but not limited to, a birch or a yew from the genus Betula or Taxus, and more preferentially from species Betula alleglZafaieyasis, Betula papy~ife~a or Taxus canadefZSis.
The portion can be under different form, but will be preferentially ground while performing the process invention.
Among organic solvent that can be chosen to in performing the process of the invention, it can be selected from the group consisting of methanol, dichloromethane, hexane, or ethyl acetate.
Also, the sound wave that is applied in the process of the present invention can be ultrasound, or can have an intensity of between 2kHz to 50kHz, preferentially between lOkHz to 20kHz, and can be pulsed at between 0.1 to 2 seconds separated by time periods between 0.1 to 2 second. The sound wave step may last between 10 to 90 minutes The process make use of non denaturing temperature which is preferentially between -20 to 40°C .
The separation of diterpenoids or tripernoids from the solvent can be performed by GC or HPLC.
In accordance with another aspect of the invention the tree portion can be under form of sawdust, with a granulometry of between 20 to 100 mesh, preferentially between 20 to 40 mesh.

Another aim of the present invention is to provide a process that allow to selectively extract diterpenoids or triterpenoids by varying temperature.
BRIEF DESCRIPTION
OF THE
DRAWINGS

Fig. 1 illustrates the chromatogram of the wood extract according to the method of Soxhlet;

Fig. 2 illustrates the effect of ultrasound intensity on extraction efficiency;

Fig. 3 illustrates the effect of the sonication time on the extraction efficiency;

Fig. 4 illustrates variations of extraction rate with temperature;

Fig. 5 illustrates the chromatogram from the wood extract sonicated at -15C;

Fig. 6 illustrates the chromatogram from the wood extract sonicated at 35C;

Fig. 7 illustrates the effect of granulometry on extraction efficiency;

Fig. 8 illustrates the effect of successive extractions;
using the same solvent system until saturation.

Fig. 9 illustrates the chromatogram of the triterpene extracts from birch leaves with the method of Sohxlet;

Fig. 10 illustrates the chromatogram of the extract obtained from birch leaves with the ultrasound process of the present invention;
' Fig. 11 illustrates the gas chromatogram of the extraction from birch twigs with the Sohxlet method;
Fig. 12 illustrates the gas chromatogram of the extract from birch twigs obtained by ultrasound of the present invention;
Fig. 13 illustrates gas chromatogram of birch bark extracts obtained by the Soxhlet method;
Fig. 14 illustrates the gas chromatogram of birch bark extracts obtained by the ultrasound process of the present invention;
Fig. 15 illustrates a Soxlhlet extraction system;
Fig. 16 illustrates an ultrasonic extraction system according to one embodiment of the present invention;
Fig. 17 illustrates an extract purification system;
Fig. 18 illustrates an high performance liquid chromatography (HPLC);
Fig. 19 illustrates the chemical structure of paclitaxel;
Fig. 20 illustrates a liquid chromatogram of paclitaxel in acidified methanol;
Fig. 21 illustrates a liquid chromatogram of Taxus extract in methanol;
Fig. 22 illustrates a liquid chromatogram of Taxus extract in methanol after performing Cass protocol (Cass et al. 1999, Phytochemical, Analysis, 10:88-92);
Fig. 23 illustrates a liquid chromatogram of Taxus extract in methanol after performing Mroczek protocol (Mroczek et al., 2000, Chromatography, 14:516-529);
Fig 24 illustrates a liquid chromatogram of Taxus extract in methanol after sonication at -20°C;

Fig. 25 illustrates variations of paclitaxel concentration in relation with temperature;
Fig. 26 illustrates a liquid chromatogram of Taxus extract in methanol after soucation at -20°C and superposed on paclitaxel standard;
Fig. 27 illustrates a liquid chromatogram of Taxus extract in methanol after sonication at 40°C; and Fig. 28 illustrates a liquid chromatogram of Taxus extract in methanol after sonication at 0°C.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention will be described more fully hereinafter with reference to the accompanying figures, in which preferred embodiments of the invention are shown. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby incorporated herein by reference.
hi accordance with the present invention, the method for extraction of diterpemes, paclitaxel, triterpenes and related compounds is provided. The method of the present invention particularly provides the possibilities to selectively extract or separate a group of products from the family of diterpenes, paclitaxel, triterpenes or derivatives thereof.
In one embodiment of the present invention, the method or process for extraction of diterpenes, paclitaxel, or triterpene compounds uses sound waves, preferably ultrasounds, to cause a relatively rapid dissolution of the diterpenes, paclitaxel, or triterpenes into a solvent.

_7_ The maximum frequency of the ultrasonic probe that was used in this experiment, with a tip diameter of 0,5 inches, is 20 kHz. Best extraction yields were obtained at 14 kHz but more than 1,5 fold increase compared to the classical Soxhlet extraction was achieved for the frequencies between 10 kHz and 20 kHz.
I11 another embodiment of the present invention, parameters of the process can be chosen in order to selectively extract one particular compound from the paclitaxel, diterpenes, or triterpene family, such as for example, but not limited to paclitaxel, non cyclic triterpenoids (as squalene), lupane and norlupane type triterpenoids (as lupeol), oleane type triterpenoids (as (3-amyrin) and ursane type triterpenoids (as a-amyrin). For example, parameters that could be varied such as temperature, duration of ultrasound treatment, ultrasound frequency, solvent nature, or granulometry could vary in order to carry out the extraction of one specific diterpenes, paclitaxel, triterpene compounds, or derivatives thereof, while one parameter or more can vary to favor extraction of another diterpene, paclitaxel, or triterpene compound.
The extraction technique can be applied on most of the plant tissues under the conditions described herein. Variation, for example but not limited to, of the solvent for the extraction gives better extraction yield in most of the cases observed for yellow birch. We observed the same increase on the extraction yield upon ultrasonic extraction for all solvents studied.
Temperature has an effect on the selectivity of the extraction without significantly affecting the global extraction yield. The wood extracts obtained in the temperature range of -20 to 5°C are composed of over 80% of betulonic acid. Temperature between 15 and 40°C, the wood extracts contain about 60% betulonic acid and more than 30% of squalene.
Squalene is not present in the extracts obtained at a low temperature. This could be used to obtain the extracts rich in betulonic acid at low temperature. This low temperature extraction can be followed by a high temperature extraction of squalene.

_$_ Better total extraction yields are obtained from samples with granulometry of between 60 mesh to fine powder. However, suitable extraction yields are ~btained from samples with granulometry of 40 to 60 mesh.
Extraction of foliage with ultrasounds yields a and (3-amyrin as the major triterpene components of the extracts which are accompanied by squalene. Extraction of the twigs produces extracts with high concentration. of lupeol (80% and +). Extractions of the inner and outer bark produce a wide range of lupane type triterpenoids, and lupeol is also the major component at more than 60% of the extracts. Extracts from wood meal are rich in betulonic acid (60-80%) and in squalene (5-35%).
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
Assessment of parameter combination for triterpene extraction.
Each parameter was fixed at an arbitrary value while only one of them was varied.
Once the parameter has been optimized, it was then fixed at the optimum value while the others were varied one at the time. Time of extraction has been optimized between 10 and 90 minutes and the best yields of extraction have been found for the extraction times between 30 and 50 minutes. The effect of sound wave with frequency between 4 and 20kHz was studied. The best extraction yields have been obtained for the intensities varying between 12 and 16 kHz. The pulsation rate has been evaluated first against the constant ultrasound flux, then by decreasing the active time as well as the passive time. Therefore, the better extraction yields were achieved using a succession of 0,1 passive and between 0,4 and 0,2 seconds active. The temperature did not affect directly the total extraction yields but affected the molecular composition of the extracts, demonstrating that the temperature parameter can be modulated to selectively extract a desired triterpene product. It has been observed that low temperature extraction (between -20°C and-5°C) produced extracts rich in betulonic acid and that temperature over 0°C favored an increase of squalene concentration in the wood extracts. The granulometry of the samples was also important for the total extraction yield. It has been found that the smallest particles were the most effective ones. The optimized version of the extraction technique was tested for all examined tissues of yellow birch with dichloromethane, hexane and ethyl acetate. We compared the yields obtained with classical Soxhlet extraction for all solvents and tree tissues studied here, to those achieved by application of the ultrasonic device. In all cases, we obtained a maximum of a twofold yield increase with ultrasonic probe.
EXAMPLE II
Extraction of paclitaxel Materials and methods Moisture determination Three samples of plant tissue were dried at 100°C for 24 hours. The samples were kept in desiccators before determination of the water content. The dried mass of the tissues was used for the calculations and all results presented here are expressed on an oven dry mass of the studied tissue.
Extraction of the tissues The extraction of the tissues can be performed by three comparable techniques.
First of these techyiques has been applied in a reference of Mroczek (Mroczek et al., 2000, Chromatography, 14:516-529) and utilizes the Soxhlet technique. The second was applied by Cass et al., (Cass et al. 1999, Phytochemical, Analysis, 10:88-92) and consist of long time maceration. The third one implies the application of ultrasound for the extraction of plant tissues. This technique has been developed in our laboratories. It is important to precise that techniques 1 and 2 are used for comparison with the third one. The extracts obtained through performing the techniques was submitted to pre-purification before HPLC
analysis. It is important to mention that the solvents used were of ACS grade and were purchased with EMD.
The green plant ground into powder was placed in Soxhlet system (Fig.lS) in which it has been extracted in methanol for a maximum period of 6 days. The volume of the methanolic extracts obtained was reduced under vacuum to the 1/IO of its initial value. The concentrated solution was then pre-purified with a 10% solution of lead acetate. Boiling water was then added to the methanolic solution in a quantity required to obtain 50%
aqueous solution.
The extracts were then filtered to separate the sediments and the supernatant was extracted with methylene chloride.
The extraction was performed on l Og of dried yew foliage, with 150m1 of methanol under agitation, then left to sit overnight. The sample was again agitated next morning. for several hours and then filtered with a Buchner funnel and a Whatman filtering paper no.l . The methanolic extract was then mixed with 1 S Oml of dichloromethane and the mixture divided into equal parts. Each portion of 60m1 was transferred together with 30m1 of distilled water in a 250m1 decantation funnel. Several 2,m1 aliquots of methanol have been added in order to see more clearly the separation of the two phases and the organic phase was removed from the mixture. Each of the 5 aliquots has been partitioned three times with SOmI of methylene chloride. The organic phases collected from five portions are then combined and evaporated to dry residue under vacuum. The residue 'was then dissolved in 100m1 of acetone and 125m1 of hexane and the mixture was then evaporated in order to eliminate residual water. The remaining product was finally dissolved in 40-65m1 of methylene chloride.
In order to adequately perform the extraction of yew foliage by our technique previously developed on yellow birch the same ratio tissueaolvent (lg in 150m1) was applied.
The extraction was selectively adjusted in function of the extraction temperature.
Variation of temperature during extraction.
The yew foliage to be extracted was dried for 24 hours in an oven at 70°C, then ground in a hammer mill. The moisture content was then determined on the ground samples before the extraction. The samples were dried for 24 hours at 100°C for moisture content determination. The ultrasonic probe was inserted into a beaker placed into a isotherm bath (filled with ethylene glycol) the temperature of which was set at -20°C. The extraction was performed for 30 minutes with ultrasound pulsations consisting of 0,2 active seconds. There was no agitation. The solvent for the extraction was methanol. The extractions were performed at each of the following temperatures: -20, -10, 0, 10, 20, 30, 40.
Purification of the extracts The purification of the extracts was performed according to the purification applied by Castor (Castor and Tyler, 1993, J. Liq. Chromato., 16:723-731) as it has been confirmed as rapid and efficient (Cass .et al. 1999, Phytochemical, Analysis, 10:88-92). A
sample of lml was diluted in a volume of 10m1 in dichloromethane. The cartridge (1cc) was purchased from Aldrich and was filled with lcm3 of silica gel (granulation 70-230 mesh, Silicagel 40) of EM
Science. This cartridge was then pre-conditioned with 5 ml of methanol followed by Sml of methylene chloride, then filled with 8m1 of the diluted extract. The column was then washed with 4m1 of methylene chloride in order to elute a pale yellow oily liquid.
Twelve millilitres of an acetone-dichloromethane mixture (4/96 v/v) was added into the cartridge in order to elute a dark green band at the bottom of the recipient. The final extract was obtained by washing the cartridge with a 10m1 of a mixture of acetone-dichloromethane (20/80 v/v) and the obtained extract was then evaporated. The residue is dissolved in 2ml of methanol using a pipette for the transfer.
HPLC standardisation The HPLC (Agilent serie 1100) is presented in Fig. 18. The degasification apparatus was a G1379A type, the quaternary pump a G1311A type, the ALS a G1329A type, the ALS
temperature control apparatus a 61130 type, the DAD a G1315B type and the Colcom a 61316 type. The chemicals used for the standard deviation curves were purchased from the following company: Paclitaxel (Fig.l9) at Aldrich, acetonitril HPLC grade at EMD, acetic acid HPLC
grade at Fisher Scientifics. The HPLC grade water was obtained from a filtration system type Sybron Barnstead Nanopure II. The standard deviation curve on the HPLC was performed following the work of Dolfmger (Dolfinger et al. 2003, Anal. Chemistry 75:1355-1364) on a C~
type analytical column. The taxane standards were previously dissolved in an ACN/water/Acetic acid (70/30/0.1) (v/v/v) at an approximate 40ug/ml concentration. The initial temperature of the column is 21°C and the elution was at this point 45/55 ACN/water (v/v). A SuL sample of taxane was then injected in the column. The solvent stays isocratic for a 8 minutes period then a gradient leads to a X0/20 ACN/water (v/v) in 10 minutes. The temperature was fixed at 2loC, the pump flow adjusted to l,Oml/min. The UV
detection was made at 227nm. Before injecting the samples obtained from the extractions of the yew tissues, the potential of the method in relation to the available instruments was checked. Therefore, a Paclitaxel standard was injected in the HPLC in an order to assess the elution time of the targeted molecules as well as the trace amount of acetic acid which was used to dissolve the standard. A total of SuL of a 40ug/ml of methanol was injected on the column.
The liquid chromatogram of this injection is presented in Fig. 20.
Acetic acid (CH3COOH) Fig. 20 shows that the analytical technique developed by Dolfmger (Dolfinger et al.
2003, Anal. Chemistry 75:1355-1364) was accurate for the apparatus available in our laboratory. The injection of this standard was also useful to allow a quantitative evaluation of the amount of Paclitaxel in the yew needles using a simple mathematical relation.
Maceration of the green tissues In order to be able to compare the effect of the moisture content of the tissues, analysis of both green and dried tissues has been performed.
This part of the experiment has been performed to see if there were any advantages of a long drying process when the natural resource is used at its initial state. Contrarily to the maceration technique used on the dried tissues, this procedure was carried out on a 6 day basis to be sure that all extractives were dissolved in the solvent. The chromatogram obtained following the injection of this extract is presented in Fig. 21.

Table 1 shows the quantification and identification of the Paclitaxel A in the freshly grinded tissues of Taxus Cahadertsis when extracted using the maceration technique.
Table 1 Net extraction yields (on a dry tissue basis) in milligram of Paclitaxel A per gram of tissues for a maceration type extraction in methyl alcohol of the green tissues of Taxus cacraadehsis.
Identification Mass Peak area traction field ,; + 0 0001 TvIAm =, +'0 ~;'m 1 ain le,~l ~ v0 0600.. 110.~g~ 0 066 Saiizple 2 '10.220'0 67 -47450 ' 0 040 Sain le 3 .;:~ 1,0,2600 9~ 4()166 0 0~8:
'~

": ~ve~a a ~ . ;_ It is possible to observe that the value obtained for Sample 2 is the lowest.
This extraction yield diminution could be related to many events and probably to a' simple experimental mistake during the numerous purification steps, or maybe because the tissues used for the different extraction were containing noticeably important variations as for their needle/twigs ratio.
Maceration of the dried tissues In the case of this maceration and contrarily to the preceeding one, Canadian yew tissues were extracted following strictly the original protocol applied by Cass (Cars et al. 1999, Phytochemical, Analysis, 10:88-92). It was noted that the extracts from the dried tissues were of the color dark brown, while the extracts of the green tissues were dark green. Drastic change of an extract's colour usually indicates the change of the chemical composition. An example of the chromatograms obtained for the dry tissues maceration is presented in Fig.
22. Comparison of Fig. 21 and 22 confirmed this point.

The first observation to be made concern the net decrease in the amount of Paclitaxel on the chromatogram in comparison to Fig.21 as observed before. In order to be able to efficiently compare the two batch macerations made during this experiment, it was essential to consider their extraction yields. Table 2 shows an evaluation of the Paclitaxel extraction rates observed in the three dried tissues extracted by maceration.
Table 2 Net extraction yields (on a dry tissue basis) in milligram of Paclitaxel A per gram of tissue for a methyl alcohol maceration type extraction of the dry tissues of Taxus cauadeusis.
Identification. Mass ' Peak'area ' Extraction yld +_ 0:'0001 MAm 0 00001 rn . E' Sarri le 1 v 9.57.01 46.50301 0.036 am le 2 8:2154 4'6.85365 0.043E
" ' Sarn le 3 . -=9.8635: 48:'59$8. 0.037 k Avera a The results shown in Table 2 indicate a low variation between the samples, which could mean that the extracted tissues were homogeneous in content, which could explain why the extraction yields do not change significantly between extractions.
Soxhlet extraction of the ~,reen tissues The second extraction technique evaluated here is a commonly applied technology for the extraction of plant tissues. Protocol elaborated by Mroczek et al.
(Mroczek et al., 2000, Chromatography, 14:516-529) was applied directly in order to be able to compare the results obtained with green 'tissues when macerated at room temperature (Mroczek et al., 2000, Chromatography, 14:516-529).
One thing to consider concerning the Soxhlet apparatusis that the technique implies higher temperatures than the one used for the maceration which were performed at room temperature. It was therefore possible that some of the targeted molecules have been partially altered due to relatively high temperatures. An example of the chromatograms obtained for extracts of Canadian yew using the Soxhlet technique is presented in Fig. 23.
The individual rate of Paclitaxel did not seem to be affected by the use of higher extraction temperature. It is otherwise interesting to see the concentrations of the molecules eluted at 7.682 and 9.122 minute in comparison to the concentrations of the same compounds obtained following the maceration of the green and dried tissues. The amount of compounds eluted in the chromatograms let us suppose of a wider diversity of constituent in the extracts when green tissues are used. Table 3 shows the Paclitaxel extraction rates when using the Soxhlet technique.
Table 3 Net extraction yields (on a dry basis) in milligrams of Paclitaxel A per gram of Taxus canadensis green tissues when extracted in methyl alcoliol by the Soxhlet technique.
Identification 'r ~ v"' Peak airea Ext~aeti'on ,~ field .

. , + 0 001 '";MAm '+ 0'00001.
' Sale l a.. x.9454 , 760~968~ ' 0.. 077 .

Sam' ' Ie 2 5.7911 ~ '' &652586 O.Of 8 .

S~ Ie 3 " '7.8121 ' 43.2 330 0.033 E=''" Avei~a 0:060 a It is possible to observe that the average extraction yields are higher that the one observed for the two preceding techniques. It is possible that variations in extraction yields be related to the grinding technique which is in this case closely related to the one used for the maceration technique.
Ultrasonic extraction of dried tissues The ultrasonic assisted extraction technique was elaborated and optimized for the extraction of triterpenoids in yellow birch (Betula alleghayaiensis) wood.
During the optimization process performed on yellow birch wood, it has been possible to observe a distinctive difference between the extracts obtained at lower temperatures compared to the ones obtained at the boiling point limit of the extraction solvent. It was therefore interesting to verify if the experimental conditions previously stated would have a direct effect on the extraction yields and on the nature of Taxus canadenszs extracts. Fig. 24 shows an example of the chromatograms obtained for the ultrasonic extraction.
Exactly as we did for the other extractions, it is important at this point to identify the different extraction yields obtained for the ultrasonically assisted extractions. Each of the values stated in Table 4 are an average of three distinct extractions.
Table 4 Net extraction yields (on a dry basis) in milligrams of Paclitaxel A per gram of Tcrxus cauadensis green tissues when extracted in methyl alcohol by the ultrasound technique at various temperatures.
'Identification ~f a s ~~, ' Peak area EXtracftori'' ' field ~", ~' 0:0001 ' .MAm +-0 00001 - ~ ~ ' F'.
,: ' 'E
-20~ << ' ~ 0157 E ' ' 21r o37g v " o i' i 1 ~15C 1~ . ' _281',6445" 0 144 ' ' -1.0C ~ 1 0005 '' X41 9373 "' 0125 ~

' 1 029 . ' 76 9956 0 039 . 0C ' _ 1 0;160 82 407$ ' ~ 0 042 '.

5~~, ' ' :< 0'' g 0,3 '.~'. ' 59 499 ' ~ 0 031 .
:. ' ' :

_ ' t ': ' 11, 095 v' ~ '0 060 ' 10C " ;1 0311 ' r 1 SC'''' ' ~' 1~"' ' 89 5319' ..' 0 046 .
~'~

.1.03'3 8 .. ' '91x'247 '' 0.046 " ''' 25G 'E f 1 0137 ' v 89.1530 .: 0046 30C 1 009 ' 33 5138 0 0'1'~

1 026 50'2788' 0 026 40C 1 0286 ' 42 9508 '0 022 Fig. 25 illustrates a graphical comparison of the results stated in Table 4.
The identification of the targeted molecule in the extracts was completed by superposition of the liquid chromatograms obtained. It was observed that superposition of the chromatograms (Fig.26) on the same extract allowed to identify the impurities observed ~in Fig. 20. This confirmed that the products detected on the standard deviation curve were in fact compounds related directly to the isolation of the Paclitaxel. Furthermore, the company where the standards were bought clearly stated that the compounds were obtained by the isolation from the yew foliage extracts.
Furthermore, the identification of this impurity confirms the statement of the company which was that the compound was 95% pure meaning the presence of a 5%
amount of impurities.
Discussion Table 1 shows that green tissues allowed to obtain a O,OSSmg/g of Paclitaxel per gram of biomass, while 0,0035mg/g were obtained from dry tissues (Table 2).
The last point on this subject concerns the molecular variation of the extracts obtained by the ultrasonic probe extractions observed when the high and low temperature chromatograms are compared. The low temperature chromatograms are presented in Figs. 24, 25 and 26, and the 0°C extraction is presented in Fig. 27 and the 40°C extraction in Fig. 28.
From the chromatograms presented herein, it is possible to conclude that the low temperature extraction allowed a complete extraction of the involved tissues compared to the higher temperatures. As an example, the compound eluted at 9,142 minutes is one of the most important compounds at low temperature. The amount of this constituent decreases significantly at 0°C (Fig.28) then disappears completely at 40°C(Fig.27). It is also important to notice that this same compound was present in large amount in green yew tissues extracts obtained by maceration, at room temperature. As for the Soxhlet technique, it seems that the ultrasonic assisted extraction has a tendency to denaturise the molecules found in the extracts when applied at high temperature but not when temperature is low. The compounds found at 7,701 and 11,390 minutes show the same trend as the compound eluted at 9,142 min., proportionally lowering with extraction temperature.

One of the most important part of this discussion concerns the compounds found conjointly with Paclitaxel (compounds eluted at 13,939; 14,204; 15,24 and 15,472 minutes) which are completely absent from the mixture when extraction temperature is increased.
Higher Paclitaxel yields are obtained here with the ultrasonic extraction at low temperature, followed by the Soxhlet extraction which is more efficient that the classical maceration. It has been found that the use of dry tissues and of high temperature for the extraction of green yew tissues has a negative impact on the chemical composition of the extracts.
It has been also demonstrated that the use of an ultrasonic probe on dry tissues at low temperature allows to obtain nearly twice the extraction yields than what is obtained by the maceration at room temperature, or by the Soxhlet technique. One advantage of the ultrasonic extraction applied at high temperature is the extraction time, which is much shorter (30 minutes compared to 6 days extractions for the other two techniques).
While the invention has been described in connection with specific embodiments therefore, it will be understood that it is prone to further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within knov~m or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (20)

1. A process for selectively extract diterpenes or triterpenes from plant material comprising the steps of:
a) immerging portion of plant material in an organic solvent at a non denaturing temperature for diterpenes or triterpenes;
b) submitting said portion in organic solvent to sound waves for a period of time causing diffusion of diterpenes triterpenes into said organic solvent; and c) separating selected diterpenes or triterpenes from said solvent.

2. The process of claim 1, wherein said plant material is obtained from at least one of a tree, an endovascular plant, or a lignocellulosic plant.

3. The process of claim 1, wherein said triterpene is at least one of the compound selected from the group consisting of non cyclic triterpenoids, such as squalene, cyclic triterpenoids of lupane and norlupane type, such as lupeol, oleane and ursane type triterpenoids, such as .beta.-amyrin and .alpha.-amyrin, or derivatives thereof.

4. The process of claim 1, wherein said diterpene is at least one of a paclitaxel, or derivatives thereof.

5. The process of claim 1, wherein said tree is a birch or from the genus of Betula, or Taxus.

6. The process of claim 5, wherein said tree is Betula alleghaniensis, Betula lutea or Taxus canadensis.

7, The process of claim 1, wherein said portion of tree is wood meal, leaves, branches, bark, or twigs.

8. The process of claim 1, wherein said portion is ground.

9. The process of claim 1, wherein said organic solvent is selected from the group consisting of methanol, dichloromethane, hexane, or ethyl acetate.

10. The process of claim 1, wherein said sound wave is ultrasound.

11. The process of claim 1, wherein said sound wave has intensity of between 2kHz to 50kHz.

12. The process of claim 1, wherein said sound wave has a frequency of 10kHz to 20kHz.

13. The method of claim 1, wherein said sound wave have pulsed at between 0.1 to 2 seconds separated by time periods between 0.1 to 2 seconds.

14. The process of claim 1, wherein said non denaturing temperature is between -20 to 40°C.

15. The process of claim 1, wherein said separation is performed by HPLC.

16. The process of claim 1, wherein submitting to sound waves of step b) is performed during 10 to 90 minutes

17. The process of claim 1, wherein said portion is under form of sawdust.

18. The process of claim 17, wherein said sawdust has a granulometry of between 20 to 100 mesh.

19. The process of claim 17, wherein said sawdust has a granulometry of between 20 to 40 mesh.

20. The process of claim 1, wherein said diterpenes or triterpenes or derivatives thereof are selectively extracted by varying temperature.