CA2303550A1 - A process for the production of stable (+) catechin penta acetate useful as a precursor for the production of (+) catechin - Google Patents
A process for the production of stable (+) catechin penta acetate useful as a precursor for the production of (+) catechin Download PDFInfo
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Abstract
The invention relates to a process for the production of a stable compound (+) catechin penta acetate of formula (2) from T. wallichiana tissue cultures and production of compound (+) catechin of formula (1), said process comprising the steps of inoculation of explants on culture media supplemented with auxins and cytokinins, incubation of the cultures for callus initiation followed by subculturing at 4-6 weeks intervals, extraction of fresh pulverized calli with polar solvents, evaporating the polar solvent to give a residue, treating t:he residue with a chlorinated solvent to isolate (+) catechin by filtration or treating the residue with water and extracting with a chlorinated solvent and evaporating the solvent to semi-solid mass, subjecting the resultant mass to column chromatography over suitable adsorbents, eluting with organic solvent or mixtures of organic solvent to obtain(+)catechin penta acetate.
Description
31-MRR-2000 20:09 FROM KU~1ARAN & SAGAR TO 0014163647910 P.04 Field:
'The invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula (1) and a :stable compound (+) catechin yenta acetate of formula (2). More particularly, the invention relates to a process for the production of (+) catechin and a stable compound (+) catechin yenta acetate from Taxus wallichiana tissue cultures. (+) c_atechin and the sr_able compound(+) catechin yenta acetate are represented formulae (1) and (2) :respectively hereunder:
/ O'R
R
O
R
Formula ( 1 ) : R-H
OR
Formula ( 2 ) : R=Ac (+) Catechin yenta acetate of formula (2) can be prepared only from (+) catechin of formula (1) through ace~ylation. Therefore, the only source to get (+) caLerhin yenta acetate consists of two processes-first to isolate (+) catec:hin and then to convert it into (+) catechin yenta acetate through acecylation. However, in 31-MRR-2000 20-09 FROM KUMARAN & SAGAR TO 0014163647910 P.05 th:.s invention, the precursor (+) catechin penta acetate can be directly obtained from the cell cultures of 2axus wallichiana following the process of the process of the present invention. Moreover, (+) catechin of formula (7.) is a polyphenolic compound and is susceptible to aerial oxidation and forms mixture of compounds on exposures to air. However, (+) catechin pe:nta acetate of formula (2) is a stable molecule and ca:n withstand aerial oxidation. Thus, it has a high shelf life and can be useful as a precursor for the production of (+) catechin.
HACRGROUND
In recent years, different Taxus species have attracted world wide attention due to the presence of taxol or its analogues in the bark or needles of the trees.
Taxol, a highly oxygenated diterpenoid molecule and a potent anticancer dru<~ was first isolated from the stem bark of Taxus brevifclia. Thereafter, it has also been i:~olated from other Taxus species including T.
wallichiana.
Catechins, the basic structural unit of condensed tannins, belong to flavan-3-of derivatives and are found in a wide variety of plant sources such as vegetables, herbs an<i teas (Phytochem (1981) 20:869).
Considerable interest have been expressed regarding the various pharmacological functions of catechins, which have been proved do be antibacterial, antiviral, antitumour, antioxidant and radical scavengers (:2hytochem (1998) 49:2379-82) .
'The invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula (1) and a :stable compound (+) catechin yenta acetate of formula (2). More particularly, the invention relates to a process for the production of (+) catechin and a stable compound (+) catechin yenta acetate from Taxus wallichiana tissue cultures. (+) c_atechin and the sr_able compound(+) catechin yenta acetate are represented formulae (1) and (2) :respectively hereunder:
/ O'R
R
O
R
Formula ( 1 ) : R-H
OR
Formula ( 2 ) : R=Ac (+) Catechin yenta acetate of formula (2) can be prepared only from (+) catechin of formula (1) through ace~ylation. Therefore, the only source to get (+) caLerhin yenta acetate consists of two processes-first to isolate (+) catec:hin and then to convert it into (+) catechin yenta acetate through acecylation. However, in 31-MRR-2000 20-09 FROM KUMARAN & SAGAR TO 0014163647910 P.05 th:.s invention, the precursor (+) catechin penta acetate can be directly obtained from the cell cultures of 2axus wallichiana following the process of the process of the present invention. Moreover, (+) catechin of formula (7.) is a polyphenolic compound and is susceptible to aerial oxidation and forms mixture of compounds on exposures to air. However, (+) catechin pe:nta acetate of formula (2) is a stable molecule and ca:n withstand aerial oxidation. Thus, it has a high shelf life and can be useful as a precursor for the production of (+) catechin.
HACRGROUND
In recent years, different Taxus species have attracted world wide attention due to the presence of taxol or its analogues in the bark or needles of the trees.
Taxol, a highly oxygenated diterpenoid molecule and a potent anticancer dru<~ was first isolated from the stem bark of Taxus brevifclia. Thereafter, it has also been i:~olated from other Taxus species including T.
wallichiana.
Catechins, the basic structural unit of condensed tannins, belong to flavan-3-of derivatives and are found in a wide variety of plant sources such as vegetables, herbs an<i teas (Phytochem (1981) 20:869).
Considerable interest have been expressed regarding the various pharmacological functions of catechins, which have been proved do be antibacterial, antiviral, antitumour, antioxidant and radical scavengers (:2hytochem (1998) 49:2379-82) .
31-MAR-2000 20:09 FROM KUhIRRAN R. SAGAR TO 0014163647910 P.06 The direct manipulation of plant cell and tissue culture systems hae resulted in an enhanced production of various secondary metabolites. In vitro production of catechins, mainly (-)epicatechin-3-0-gallate accompanied by (+)catechin and (-)epicatechin have been reported in Fagophys~um esculentum calli and hairy root cultures (Phytochem (1992) 31:1239-1241; Phytochem (1993) 32:929), suspension cultures of Camellia sinensis (Chayekexue (1995) 15:111-116) and Vitis vinifera (Biotech Lett. (1996) 659-662). Crataegus monogyna (Phytochem (1994) 3?:1273), Uncaria elliptica (Phytochem (1998) 28:1099-1100) and Polygonum hydropiper (Phytochem (1998) 49:1935-39).
Several approaches have been used for the establishment of in vitro cultures of various Taxes species (Plant Cell, Tissue and Organ Cult. (1996) 46:59-~5).
Different explants and various basal media have been used for initiation, and maintenance of Taxes callus and cell suspension cultures. The culture media are frequently supplemented with organic substances, such as casein hydrolysate, polyvinylpyrrolidone, ascorbic acid and others. Se~reral growth regulators are used for stimulation of cell proliferation.
Taxes wallichiana, known as Himalayan yew is available in India. Suspension and callus cultures of T.
wallichiana are found capable of producing taxol (Planta Med. (199,9) 64:2?0-72) and some important taxanes, namely 2-d;eacetoxy-taxinine J and 2-deacetoxy autrospicatin (Planta Med. (1996) 62:333-35). We have been screening diff~arent callus lines of T. wallichiana 31-11RR-2000 20 10 FROM KUMRRRN 8 SRGRR TO 0014163647910 P.07 induced from different. explants of tress collected from different geographical regions of India. The protocol standardized for in vitro callus production is dependent on media <:omposition (viz. Murashinge and Skoog's Gamborg's, white's, Nitsch and Nitsch's), hormonal regime combinations of different concentrations of cyt:okinins and auxins, (such as 6-benzyl aminopurine, TDZ, 2-ip, Kinetin, 6-methylamino purine, Zeatin with NAA, IAA, IBA, 2,4-D, 2,4-T, Picloram), explant siource (preferably from needles, twigs, stems devoid of needles and seeds) and culture conditions (light dark conditions).
T'he callus line developed from specific explanta on different media compositions having definite hormonal combinations resulted in the production of a phenolic compound of formula ClsFil,Os mp 94-95°C, as an amorphorus solid. Th~~ yield Was noted to be 0.3~ in six months old callus. The (+) catechin is very important precursor for the semisynthesis of other c:atechin derivatives, eg. Gallocatechin, epigallocatechin and epigallocatechin-3-0-gallate. It i.s worth mentioning here that most of the earlier reports of in vitro catechin production revealed production of a mixture of catechins, the majority of which were (-) epicatechin, (-) eipcatechin-3-O-c~allate and (-) ep~.gallocatechin, (Phytochem (1989) :?e:1099-1100, (1992) 31:1239-1241 (1993) 32:924-931, (1998) 49:1935-1939), whereas in the present invention only (+) catechin i;s expressed. Furthermore, in the present invention 0.3% concentration of (+1 catechin has been detected in 6 months old callus while the 31-MRR-2000 20 10 FROM KUMARAN & SHGRR TO 0014163647910 P.08 expression of (+) catechin in Polygonum hydropiper occurred after one year of culture initiation (Phytochem (1998) 490):1935-39).
This phenolic compaund is further converted into a stable (+) catechin pants acetate having molecular formula of CZSH=a~m mp 131 to 132°C, was isolated as an amorphorus solid. This compound Was isolated as a crystalline solid with a yield of 0.05%. The compound has been characteri;.ed as (+) catechin pants acetate.
The (+)catechin pants acetate is a very important precursor for the synthesis of catechin which in turn can be converted into its other biologically active derivatives, eg. gallocatechin, epigallocatechin and epigallocatechin-3-o-gallate.
There has not been any process reported in the literature for direct isolation of (+) catechin pants acetate either from higher plants, microbes or from cell cultures of plants.
Thus, the drawbacks of the previous processes for the preparation of (+) catechin pants acetate are as under:
~ These pro<:esse9 require isolation of (+) catechin and the conversion thereof to catechin ' pants acetate through acetylation process.
~ (+) catechi.n can not be preserved for a longer period of time due to its instability towards aerial oxidation. However, (+) catechin pants acetate c:an be preserved Without its 31-MAR-2000 20:10 FROM KUIhRRAN & SAGAR TO 001416364'7910 P.09 decomposition for a longer period of time and it easily can be converted into (+) catechin with a quantitative yield.
OBJECTS
Accordingly, the main object of the present invention i.s to provide a procea s for the production of a stable precursor (+) catechin penta acetate of formula 2 from ~!'axus wallichiana tissue cultures.
Another object is to provide a process for in vitro production of a biologically active phenolic compound (+) catechin from T. wallichiana tissue cultures employing the precursor (+) catechin penta acetate.
'Yet another object of the invention is to provide a ;process for isolation of (+) catechin and its precursor compound from cell cultures of Taxes wallichiana without the use o:E any cumbersome chromatographic separations.
Still another object. is to provide a process for the production of (+) catechin penta acetate from the cell cultures of T. walli.chiana which can then be converted into (+) catechin qu~~.ntitatively.
SUI~iARY
The present provides a novel process for in vitro production of compound (+) catechin of formula (1) and a stable(+) catechin penta acetate of formula (2), which constitutes the first ever report of production of (+) catechin penta acetate in the genus Taxes. In 31-MAR-2000 20:11 FROM KUI"'ARAN & SAGAR TO 001416364?910 P.10 contrast to prior art processes for the production of the precursor (+) catechin penta acetate, which comprise, first i:>olation of the (+) catechin (which is susceptible to aew~ial oxidation and polymerization) and its subsequent conversion into catechin penta acetate, the invention provides a process for the direct production of a stable precursor (+) catechin penta acetate from the cell cultures of T. wallichiana. Also, this stable precursor (+) catechin penta acetate can be converted into (+) catechin quantitatively.
Thus, the novelt5r of the present invention is that it provides a process for the in vivo production of a stable precursor (+) catechin penta acetate in the cell cultures of T. wa.llichiana, which can be converted into (+) catechin quantitatively.
Detailed description:
Accordingly the present invention relates to a process for the production of a stable compound (+) catechin penta acetate of formula (2) from T. wallichiana tissue cultures and production of compound (+) catechin of formula (1), said process comprising the steps of:
a) inoculation of explants on culture media supplemented with combinations of auxins (1-5 mg/1) and cytokinins (0.1-1.0 mg/1) ;
b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals:
c) extraction of fresh pulverized calli with polar solvents at rcom temperature;
T
31-MRR-2000 20:11 FROM KUhIFiRAN & SRGRR TO 0014163647910 P.11 d) evaporating the polar solvent to give a residue, treating the residue with a chlorinated solvent to isolate (+) catechin by filtration or treating the residue with water and extracting with a chlorinated solvent and evaporating the solvent to semi-solid mass;
e) subjecting the resultant mass to column chromatography o~~er suitable adsorbents;
f) eluting with organic solvent or mixtures of organic solvent to obta:~n (+) catechin penta acetate, and isolating (+) cat:echin penta acetate by filtration.
In embodiment. the: calli are harvested at different growth phases ranging from 1 to 36 months, n another embodiment, the calli are harvested at different growth phases preferably, 12 to 36 months, Tn yet another embodiment, the explants for induction of callus may be selected from needles, twigs, stem devoid of needles <ind seeds.
In another embodiment of the present invention the culture media for callus induction and multiplication may be selected from Murashige and Skoog(1962)(MS) medium, containing the following (in mg/1)- NH, NO:
(1, 650) , KNO,; (1, 900), CaCl~.2H~0 (400) , MgS0~.7HZ0 ( 37 0 ) , KH=PO4 ( 1'7 0 ) , Na2EDTA. 2H20 ( 7 . 2 ) , FeSO~ . 7H20 (27.8) , MnS0a.4Hz0 (22.3) , ZnS0~.7H10 (8.6) , H,BO, (6.2) , kI ( 0 . 83 ) , NazMoOa . 2H~0 ( 0 . 25 ) , CuSOa . 5H20 ( 0 . 025 ) , CoCl,.6H20(0.025), Glycine (2.0), Nicotinic acid (0.5), Pyridoxine HCL (0.5), Thiamine HCl (0.1); Gamborg's 31-MRR-2000 20 11 FROM KUI'1ARRN & SAGAR TO 0014163647910 P.12 CoC12.6Hz0(0.025), Glycine (2.0), Nicotinic acid (0.5), Pyridoxine HCL (0..5), Thiamine HC1 (0.1); Gamborg's (1968) (B5) medium, containing the following (in mg/1)-KN03 (3, 000) , (NH,~) ZSO, (134) , MgS0,.7Hz0 (500) , CaCla.2H=O (150), NaH=POa.HzO (150), MnS04.H20 (10.0), KI
( 0 . 75 ) . H3B03 ( 3 . 0 ) , ZriS04 . 7Ha0 ( 2 . 0 ) , CuSOa ( 0 . 025 ) , NaMoO, . 2Hz0 ( 0 . 25 ) , CoCl2 . 6Hz0 ( 0 . 025 ) , NazEDTA. 2HZ0 (37.2), FeS04.7H20 (27.8); White's (1963) medium, consisting of the following (in mg/1)- Ca(N03)z (142.0), KN03 (81.0), MgS04.7Hz0 (70.0), KC1 (65.0), KH2P0q (12.0), Fe(S04)3 (2.46) and Nitsch and Nitsch; (1969) medium, containing the following tin mg/1)- NH, NO;
(20.0) , KN03 (950) , H3B03 (10.0), KHzPO, (6B.0) , NazMoOa . 2H=0 ( 0 .143 ) , CaClz ( 41 . 5 ) , MgSO, . 7Hz0 ( 18 5 ) , MnSO, . 4Hz0 ( 15 . 0 ) , ZnS04 . Hz0 ( 10 . 0 ) , CuS04 ( 0 . 14 ) , FeSOa (111.4), Na2EDTA (1491, Biotin (0.05), Glycine (2.0), Nicotinic acid (5.c)), Pyridoxine HC1 t0.5), Thiamine HC1 (0.5), Folic acid (5.0).
In yet another embodiment, the auxins may be selected from indole acetic acid (IAA) , napthelene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D). 2,4,6-trichlorophenoxy acetic acid (2,4-T) and piclorarn within the following range (0.2-20 mg/1) .
In an embodiment, the auxins may be selected from indole acetic acid (IAA), napthelene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D),, 2,4,6-trichlorophenoxy acetic acid (2,4-T) and picloram within the preferred range of about 0.2-20 mg/1.
31-MHR-2000 20-12 FROM KUI'1RRRN & SRGRR TO 0014163647910 P.13 I:n still another embodiment, the cytokinins may be selected from 6-benzyl amino purine (HAP), 6-methyl aminopurine (MAP), k.inetin (Kn), zeatin, thiadiazuron ITDZ) and 2-isopentenyl amino purine (2-ip) within the following range (0.02-2 mg/1) .
7:n another embodiment, the cultures may be incubated under continuous lught of 300-300 lux or under continuous dark conditions.
7:n yet another embod:lment, harvesting time get maximum product may be from one to thirty six months.
In another embodiment, the preferred harvesting time get maximum product may be from twenty four to thirty ;~ ix months .
..n an embodiment of the present invention, the polar ;solvents may be selected from methanol, ethanol, propanol and butanol.
zn still another embodiment, the ratio of auxins and <:ytokinins used ranges between 5 to 20:1.
:~n still another embodiment, the preferred ratio of auxins and cytokinins used ranges between 6 to 20:1.
:Cn still another embodiment, the medium may be supplemented with casein hydrolysate ranging between :~00 to 400 mg/1.
31-MAR-2000 20:12 FROM KUMARAN & SAGAR TO 0014163647910 P.14 In still another embodiment, ascorbic acid used ranges between 10-50 mg/1.
In still another embodiment, the chlorinated solvents used for partitioning may be selected from chloroform, c.ichloromethane.
In still another embodiment, the adsorbents used for column chromatography for isolating catechin penta acetate may be sele~~ted from silica gel, alumina and florosil.
In still another w_mbodiment, the organic solvent, mixtures of organic: solvents used for eluting the column may be selected from ethyl acetate, chloroform, dichloromethane, hexane-ethyl acetate mixtures, pet. ether-ethyl acetate mixtures, hexane-chloroform mixtures, pet. ether-chloroform mixtures.
Repeated experimentations have proved that use of particular explants and specific ratio of auxins and cytokinins are the critical factors for in vitro expression of (+) cat:echin penta acetate production.
Z'he invention is described in detail in the examples given below which are provided to illustrate the invention and therefore should not be construed to limit the scope of the invention.
31-~"l~iR-2000 20:12 FROM KU~~RAN 8 SRGAR TO 0014163647910 P.15 Preparation of +catechin:
Needle explants of mature trees of Taxus wallichiana were collected from different geographical regions of India, and callus cultures were initiated on Murashige and Skoog's (MS) basal medium supplemented with 2, 4-D (5mg/1), kinetin (0:25 mg/1), ascorbic acid (40 mg/1), sucrose (3%) and agar (0.8%).
The cultures were maintained at 2512°C under continuous light condition (3000 lux).
After initiation, the callus was maintained on the same medium with sub-culturing at every 4 weeks. The calli were harvested at different growth phases and extracted with methanol (Sg/20m1).
The methanol extract was concentrated under reduced pressure to a semisolid residue. The residue was treated with chloroform with stirring to obta:ln solids. The solids were filtered to give (+) catechin as amorphorus solid (yield 0.3%).
Young twigs were collected from mature trees of Taxus wallichiana and inoculated on Gamborg's (B~, medium supplemented with NAA (5mg/1), Kn o.25 mg/1), ascorbic acid (40 mg/1), casein hydrolysate (250mg/1) sucorse (30) and agar (0.8%). The cultures w~sre maintained at 25a2°C under continuous dark condition. After initiation the callus was maintained on the same medium as well a;s on medium supplemented With 2, 4-D and kinetin of the r~aspective concentrations with sub-culturing at every 4 weeks interval. The calli were harvested at different growth phases and extracted with ethanol ( 5 g/20 ml ) . The ethanol extract was concentrated under reduced pressure to a semi-solid residue. The residue was treated w.th dichloromethane with stirring to obtain solid. The solids were filtered to give (+) catechin as amorphorus solid (yield (0.3%).
31-~MRR-2000 20:13 FROM KUMRRRN & SRGRR TO 0014163647910 P.16 EXAbIPLE - 3 Suspension cultures were raised from the callus initiated on B5 solid medium supplemented with NAA (1-5 mg/1) and kinetin (0.1-1.0 mg/1). These cultures were grown and maintained in MS liquid medium supplemented with similar hormonal combinations, incubated on rotary shaker at 100-120 rpm under dark conditions.
The suspensions were harvested at different growth phases. For the chemical analysis, cell suspension was filtered through celite powder in order to separate the cells from the broth.
Cells were extracted with polar solvent which was followed by evaporation of the solvent under reduced pressure to obtain a semisolid residue. 'The residue was treated with dichloromethane with stirring to a solid which was filtered to give (+) catechin as amorphous solid (yield 0.2?%).
Preparation of +catechin penta-acetate Example 4 Needle explants of mature trees of T. wallichiana were collected from different geographical regions of India, and callus cultures. were initiated on Murashinge and Skoog's (MS) basal medium supplemented with 2,4-D (5 mg/1), kinetin (0.:?5 m/1), ascorbic acid (40 mg/1), sucrose (3%), agar (0.8%). The cultures were maintained at 25t 2°C under continuous light condition (3000 lux).
After initiation, t:he callus maintained on the same medium with sub-culturing at every 4 weeks. The calli were harvested at different growth phases and extracted with methanol (5 g/20 ml). The methanol extract was concentrated under reduced pressure to a semisolid 31-htRR-2000 20:13 FROM KUMARAN 8 SRGAR TO 0014163647910 P.17 residue. The residues was treated with water and extracted with chloroform. -The chloroform extract: was column chromatographed over silica gel (60-120 mesh): The column was first eluted with pet. ether and then with 10% ethyl acetate in pet.
Ether. The eluant of the latter fraction on concentration gave catechin penta acetate (yield 0.05%) .
Example S
'i~oung twigs were col:Lected from mature trees of Taxus wallichiana and inoculated on Gamborg's (B5 medium supplemented with 2,4~-D (2mg/1) , picloram (3 mg/1) , Kn (~~.25 mg/1), casein hydrolysate (25o mg/1), sucrose ( :3 0 % ) and agar ( 0 . 8 % ) . The cul tures were maintained at 2:St2°C under continuous dark condition. After initiation the callus was maintained on the same medium as well as on medium supplemented with 2,4-D and kinetin of the respective concentrations with sub-culturing at every 4 weeks interval. The c:alli were harvested at different growth phases and extracted with ethanol (5 g/20 ml).
The ethanol extract was concentrated under reduced pressure ~to a semi-solid residue. The residue was treated with water and extracted with dichloromethane.
The dicholoromethane extract was column chromatographed over florosil. The column was first eluted with hexane a:nd then with hexane-chloroform mixtures. The eluant of the latter on concentration gave catechin penta acetate (yield 0 . 05 v ) .
31-~MAR-2000 20:13 FROM KUMRRAN & SRGAR TO 0014163647910 P.18 Example 6 Catechin penta acetate (500 mg) was dissolved in 1N
methanolic NaOH solution (20 ml) with stirring. It was then diluted with water and extracted with EtoAc (3 x 50 ml), ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate and concentrated to give (+) catechin (285 mg).
Advantages 1) Large scale production of (+) catechin through in vitro callus arid suspension cultures with yield of 0.3% which is :significantly better to the reported yield per unit time.
2) The above process of the invention provides an alternative source of the compound thereby sparing the mature trees from being exploited.
3) Following the process of the present invention (+) catechin can be: obtained as a single entity in the cell cultures of Taxus wallichiana in contrast to earlier reports of production of a mixture of many related phenolic compounds (e. g. (-) epicatechin-3-0-gallate, (~-) epicatechin, gallocatechin) along with (+) catechin from cell cultures of other plants.
4) Taxus wallich:Lana generally grows at the high altitudes of ~Iimalayan belt, hence to obtain the compound from such geographical locations is really difficu:Lt while the in vitro production and extraction protocol ensures convenient source of supply.
Several approaches have been used for the establishment of in vitro cultures of various Taxes species (Plant Cell, Tissue and Organ Cult. (1996) 46:59-~5).
Different explants and various basal media have been used for initiation, and maintenance of Taxes callus and cell suspension cultures. The culture media are frequently supplemented with organic substances, such as casein hydrolysate, polyvinylpyrrolidone, ascorbic acid and others. Se~reral growth regulators are used for stimulation of cell proliferation.
Taxes wallichiana, known as Himalayan yew is available in India. Suspension and callus cultures of T.
wallichiana are found capable of producing taxol (Planta Med. (199,9) 64:2?0-72) and some important taxanes, namely 2-d;eacetoxy-taxinine J and 2-deacetoxy autrospicatin (Planta Med. (1996) 62:333-35). We have been screening diff~arent callus lines of T. wallichiana 31-11RR-2000 20 10 FROM KUMRRRN 8 SRGRR TO 0014163647910 P.07 induced from different. explants of tress collected from different geographical regions of India. The protocol standardized for in vitro callus production is dependent on media <:omposition (viz. Murashinge and Skoog's Gamborg's, white's, Nitsch and Nitsch's), hormonal regime combinations of different concentrations of cyt:okinins and auxins, (such as 6-benzyl aminopurine, TDZ, 2-ip, Kinetin, 6-methylamino purine, Zeatin with NAA, IAA, IBA, 2,4-D, 2,4-T, Picloram), explant siource (preferably from needles, twigs, stems devoid of needles and seeds) and culture conditions (light dark conditions).
T'he callus line developed from specific explanta on different media compositions having definite hormonal combinations resulted in the production of a phenolic compound of formula ClsFil,Os mp 94-95°C, as an amorphorus solid. Th~~ yield Was noted to be 0.3~ in six months old callus. The (+) catechin is very important precursor for the semisynthesis of other c:atechin derivatives, eg. Gallocatechin, epigallocatechin and epigallocatechin-3-0-gallate. It i.s worth mentioning here that most of the earlier reports of in vitro catechin production revealed production of a mixture of catechins, the majority of which were (-) epicatechin, (-) eipcatechin-3-O-c~allate and (-) ep~.gallocatechin, (Phytochem (1989) :?e:1099-1100, (1992) 31:1239-1241 (1993) 32:924-931, (1998) 49:1935-1939), whereas in the present invention only (+) catechin i;s expressed. Furthermore, in the present invention 0.3% concentration of (+1 catechin has been detected in 6 months old callus while the 31-MRR-2000 20 10 FROM KUMARAN & SHGRR TO 0014163647910 P.08 expression of (+) catechin in Polygonum hydropiper occurred after one year of culture initiation (Phytochem (1998) 490):1935-39).
This phenolic compaund is further converted into a stable (+) catechin pants acetate having molecular formula of CZSH=a~m mp 131 to 132°C, was isolated as an amorphorus solid. This compound Was isolated as a crystalline solid with a yield of 0.05%. The compound has been characteri;.ed as (+) catechin pants acetate.
The (+)catechin pants acetate is a very important precursor for the synthesis of catechin which in turn can be converted into its other biologically active derivatives, eg. gallocatechin, epigallocatechin and epigallocatechin-3-o-gallate.
There has not been any process reported in the literature for direct isolation of (+) catechin pants acetate either from higher plants, microbes or from cell cultures of plants.
Thus, the drawbacks of the previous processes for the preparation of (+) catechin pants acetate are as under:
~ These pro<:esse9 require isolation of (+) catechin and the conversion thereof to catechin ' pants acetate through acetylation process.
~ (+) catechi.n can not be preserved for a longer period of time due to its instability towards aerial oxidation. However, (+) catechin pants acetate c:an be preserved Without its 31-MAR-2000 20:10 FROM KUIhRRAN & SAGAR TO 001416364'7910 P.09 decomposition for a longer period of time and it easily can be converted into (+) catechin with a quantitative yield.
OBJECTS
Accordingly, the main object of the present invention i.s to provide a procea s for the production of a stable precursor (+) catechin penta acetate of formula 2 from ~!'axus wallichiana tissue cultures.
Another object is to provide a process for in vitro production of a biologically active phenolic compound (+) catechin from T. wallichiana tissue cultures employing the precursor (+) catechin penta acetate.
'Yet another object of the invention is to provide a ;process for isolation of (+) catechin and its precursor compound from cell cultures of Taxes wallichiana without the use o:E any cumbersome chromatographic separations.
Still another object. is to provide a process for the production of (+) catechin penta acetate from the cell cultures of T. walli.chiana which can then be converted into (+) catechin qu~~.ntitatively.
SUI~iARY
The present provides a novel process for in vitro production of compound (+) catechin of formula (1) and a stable(+) catechin penta acetate of formula (2), which constitutes the first ever report of production of (+) catechin penta acetate in the genus Taxes. In 31-MAR-2000 20:11 FROM KUI"'ARAN & SAGAR TO 001416364?910 P.10 contrast to prior art processes for the production of the precursor (+) catechin penta acetate, which comprise, first i:>olation of the (+) catechin (which is susceptible to aew~ial oxidation and polymerization) and its subsequent conversion into catechin penta acetate, the invention provides a process for the direct production of a stable precursor (+) catechin penta acetate from the cell cultures of T. wallichiana. Also, this stable precursor (+) catechin penta acetate can be converted into (+) catechin quantitatively.
Thus, the novelt5r of the present invention is that it provides a process for the in vivo production of a stable precursor (+) catechin penta acetate in the cell cultures of T. wa.llichiana, which can be converted into (+) catechin quantitatively.
Detailed description:
Accordingly the present invention relates to a process for the production of a stable compound (+) catechin penta acetate of formula (2) from T. wallichiana tissue cultures and production of compound (+) catechin of formula (1), said process comprising the steps of:
a) inoculation of explants on culture media supplemented with combinations of auxins (1-5 mg/1) and cytokinins (0.1-1.0 mg/1) ;
b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals:
c) extraction of fresh pulverized calli with polar solvents at rcom temperature;
T
31-MRR-2000 20:11 FROM KUhIFiRAN & SRGRR TO 0014163647910 P.11 d) evaporating the polar solvent to give a residue, treating the residue with a chlorinated solvent to isolate (+) catechin by filtration or treating the residue with water and extracting with a chlorinated solvent and evaporating the solvent to semi-solid mass;
e) subjecting the resultant mass to column chromatography o~~er suitable adsorbents;
f) eluting with organic solvent or mixtures of organic solvent to obta:~n (+) catechin penta acetate, and isolating (+) cat:echin penta acetate by filtration.
In embodiment. the: calli are harvested at different growth phases ranging from 1 to 36 months, n another embodiment, the calli are harvested at different growth phases preferably, 12 to 36 months, Tn yet another embodiment, the explants for induction of callus may be selected from needles, twigs, stem devoid of needles <ind seeds.
In another embodiment of the present invention the culture media for callus induction and multiplication may be selected from Murashige and Skoog(1962)(MS) medium, containing the following (in mg/1)- NH, NO:
(1, 650) , KNO,; (1, 900), CaCl~.2H~0 (400) , MgS0~.7HZ0 ( 37 0 ) , KH=PO4 ( 1'7 0 ) , Na2EDTA. 2H20 ( 7 . 2 ) , FeSO~ . 7H20 (27.8) , MnS0a.4Hz0 (22.3) , ZnS0~.7H10 (8.6) , H,BO, (6.2) , kI ( 0 . 83 ) , NazMoOa . 2H~0 ( 0 . 25 ) , CuSOa . 5H20 ( 0 . 025 ) , CoCl,.6H20(0.025), Glycine (2.0), Nicotinic acid (0.5), Pyridoxine HCL (0.5), Thiamine HCl (0.1); Gamborg's 31-MRR-2000 20 11 FROM KUI'1ARRN & SAGAR TO 0014163647910 P.12 CoC12.6Hz0(0.025), Glycine (2.0), Nicotinic acid (0.5), Pyridoxine HCL (0..5), Thiamine HC1 (0.1); Gamborg's (1968) (B5) medium, containing the following (in mg/1)-KN03 (3, 000) , (NH,~) ZSO, (134) , MgS0,.7Hz0 (500) , CaCla.2H=O (150), NaH=POa.HzO (150), MnS04.H20 (10.0), KI
( 0 . 75 ) . H3B03 ( 3 . 0 ) , ZriS04 . 7Ha0 ( 2 . 0 ) , CuSOa ( 0 . 025 ) , NaMoO, . 2Hz0 ( 0 . 25 ) , CoCl2 . 6Hz0 ( 0 . 025 ) , NazEDTA. 2HZ0 (37.2), FeS04.7H20 (27.8); White's (1963) medium, consisting of the following (in mg/1)- Ca(N03)z (142.0), KN03 (81.0), MgS04.7Hz0 (70.0), KC1 (65.0), KH2P0q (12.0), Fe(S04)3 (2.46) and Nitsch and Nitsch; (1969) medium, containing the following tin mg/1)- NH, NO;
(20.0) , KN03 (950) , H3B03 (10.0), KHzPO, (6B.0) , NazMoOa . 2H=0 ( 0 .143 ) , CaClz ( 41 . 5 ) , MgSO, . 7Hz0 ( 18 5 ) , MnSO, . 4Hz0 ( 15 . 0 ) , ZnS04 . Hz0 ( 10 . 0 ) , CuS04 ( 0 . 14 ) , FeSOa (111.4), Na2EDTA (1491, Biotin (0.05), Glycine (2.0), Nicotinic acid (5.c)), Pyridoxine HC1 t0.5), Thiamine HC1 (0.5), Folic acid (5.0).
In yet another embodiment, the auxins may be selected from indole acetic acid (IAA) , napthelene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D). 2,4,6-trichlorophenoxy acetic acid (2,4-T) and piclorarn within the following range (0.2-20 mg/1) .
In an embodiment, the auxins may be selected from indole acetic acid (IAA), napthelene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D),, 2,4,6-trichlorophenoxy acetic acid (2,4-T) and picloram within the preferred range of about 0.2-20 mg/1.
31-MHR-2000 20-12 FROM KUI'1RRRN & SRGRR TO 0014163647910 P.13 I:n still another embodiment, the cytokinins may be selected from 6-benzyl amino purine (HAP), 6-methyl aminopurine (MAP), k.inetin (Kn), zeatin, thiadiazuron ITDZ) and 2-isopentenyl amino purine (2-ip) within the following range (0.02-2 mg/1) .
7:n another embodiment, the cultures may be incubated under continuous lught of 300-300 lux or under continuous dark conditions.
7:n yet another embod:lment, harvesting time get maximum product may be from one to thirty six months.
In another embodiment, the preferred harvesting time get maximum product may be from twenty four to thirty ;~ ix months .
..n an embodiment of the present invention, the polar ;solvents may be selected from methanol, ethanol, propanol and butanol.
zn still another embodiment, the ratio of auxins and <:ytokinins used ranges between 5 to 20:1.
:~n still another embodiment, the preferred ratio of auxins and cytokinins used ranges between 6 to 20:1.
:Cn still another embodiment, the medium may be supplemented with casein hydrolysate ranging between :~00 to 400 mg/1.
31-MAR-2000 20:12 FROM KUMARAN & SAGAR TO 0014163647910 P.14 In still another embodiment, ascorbic acid used ranges between 10-50 mg/1.
In still another embodiment, the chlorinated solvents used for partitioning may be selected from chloroform, c.ichloromethane.
In still another embodiment, the adsorbents used for column chromatography for isolating catechin penta acetate may be sele~~ted from silica gel, alumina and florosil.
In still another w_mbodiment, the organic solvent, mixtures of organic: solvents used for eluting the column may be selected from ethyl acetate, chloroform, dichloromethane, hexane-ethyl acetate mixtures, pet. ether-ethyl acetate mixtures, hexane-chloroform mixtures, pet. ether-chloroform mixtures.
Repeated experimentations have proved that use of particular explants and specific ratio of auxins and cytokinins are the critical factors for in vitro expression of (+) cat:echin penta acetate production.
Z'he invention is described in detail in the examples given below which are provided to illustrate the invention and therefore should not be construed to limit the scope of the invention.
31-~"l~iR-2000 20:12 FROM KU~~RAN 8 SRGAR TO 0014163647910 P.15 Preparation of +catechin:
Needle explants of mature trees of Taxus wallichiana were collected from different geographical regions of India, and callus cultures were initiated on Murashige and Skoog's (MS) basal medium supplemented with 2, 4-D (5mg/1), kinetin (0:25 mg/1), ascorbic acid (40 mg/1), sucrose (3%) and agar (0.8%).
The cultures were maintained at 2512°C under continuous light condition (3000 lux).
After initiation, the callus was maintained on the same medium with sub-culturing at every 4 weeks. The calli were harvested at different growth phases and extracted with methanol (Sg/20m1).
The methanol extract was concentrated under reduced pressure to a semisolid residue. The residue was treated with chloroform with stirring to obta:ln solids. The solids were filtered to give (+) catechin as amorphorus solid (yield 0.3%).
Young twigs were collected from mature trees of Taxus wallichiana and inoculated on Gamborg's (B~, medium supplemented with NAA (5mg/1), Kn o.25 mg/1), ascorbic acid (40 mg/1), casein hydrolysate (250mg/1) sucorse (30) and agar (0.8%). The cultures w~sre maintained at 25a2°C under continuous dark condition. After initiation the callus was maintained on the same medium as well a;s on medium supplemented With 2, 4-D and kinetin of the r~aspective concentrations with sub-culturing at every 4 weeks interval. The calli were harvested at different growth phases and extracted with ethanol ( 5 g/20 ml ) . The ethanol extract was concentrated under reduced pressure to a semi-solid residue. The residue was treated w.th dichloromethane with stirring to obtain solid. The solids were filtered to give (+) catechin as amorphorus solid (yield (0.3%).
31-~MRR-2000 20:13 FROM KUMRRRN & SRGRR TO 0014163647910 P.16 EXAbIPLE - 3 Suspension cultures were raised from the callus initiated on B5 solid medium supplemented with NAA (1-5 mg/1) and kinetin (0.1-1.0 mg/1). These cultures were grown and maintained in MS liquid medium supplemented with similar hormonal combinations, incubated on rotary shaker at 100-120 rpm under dark conditions.
The suspensions were harvested at different growth phases. For the chemical analysis, cell suspension was filtered through celite powder in order to separate the cells from the broth.
Cells were extracted with polar solvent which was followed by evaporation of the solvent under reduced pressure to obtain a semisolid residue. 'The residue was treated with dichloromethane with stirring to a solid which was filtered to give (+) catechin as amorphous solid (yield 0.2?%).
Preparation of +catechin penta-acetate Example 4 Needle explants of mature trees of T. wallichiana were collected from different geographical regions of India, and callus cultures. were initiated on Murashinge and Skoog's (MS) basal medium supplemented with 2,4-D (5 mg/1), kinetin (0.:?5 m/1), ascorbic acid (40 mg/1), sucrose (3%), agar (0.8%). The cultures were maintained at 25t 2°C under continuous light condition (3000 lux).
After initiation, t:he callus maintained on the same medium with sub-culturing at every 4 weeks. The calli were harvested at different growth phases and extracted with methanol (5 g/20 ml). The methanol extract was concentrated under reduced pressure to a semisolid 31-htRR-2000 20:13 FROM KUMARAN 8 SRGAR TO 0014163647910 P.17 residue. The residues was treated with water and extracted with chloroform. -The chloroform extract: was column chromatographed over silica gel (60-120 mesh): The column was first eluted with pet. ether and then with 10% ethyl acetate in pet.
Ether. The eluant of the latter fraction on concentration gave catechin penta acetate (yield 0.05%) .
Example S
'i~oung twigs were col:Lected from mature trees of Taxus wallichiana and inoculated on Gamborg's (B5 medium supplemented with 2,4~-D (2mg/1) , picloram (3 mg/1) , Kn (~~.25 mg/1), casein hydrolysate (25o mg/1), sucrose ( :3 0 % ) and agar ( 0 . 8 % ) . The cul tures were maintained at 2:St2°C under continuous dark condition. After initiation the callus was maintained on the same medium as well as on medium supplemented with 2,4-D and kinetin of the respective concentrations with sub-culturing at every 4 weeks interval. The c:alli were harvested at different growth phases and extracted with ethanol (5 g/20 ml).
The ethanol extract was concentrated under reduced pressure ~to a semi-solid residue. The residue was treated with water and extracted with dichloromethane.
The dicholoromethane extract was column chromatographed over florosil. The column was first eluted with hexane a:nd then with hexane-chloroform mixtures. The eluant of the latter on concentration gave catechin penta acetate (yield 0 . 05 v ) .
31-~MAR-2000 20:13 FROM KUMRRAN & SRGAR TO 0014163647910 P.18 Example 6 Catechin penta acetate (500 mg) was dissolved in 1N
methanolic NaOH solution (20 ml) with stirring. It was then diluted with water and extracted with EtoAc (3 x 50 ml), ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate and concentrated to give (+) catechin (285 mg).
Advantages 1) Large scale production of (+) catechin through in vitro callus arid suspension cultures with yield of 0.3% which is :significantly better to the reported yield per unit time.
2) The above process of the invention provides an alternative source of the compound thereby sparing the mature trees from being exploited.
3) Following the process of the present invention (+) catechin can be: obtained as a single entity in the cell cultures of Taxus wallichiana in contrast to earlier reports of production of a mixture of many related phenolic compounds (e. g. (-) epicatechin-3-0-gallate, (~-) epicatechin, gallocatechin) along with (+) catechin from cell cultures of other plants.
4) Taxus wallich:Lana generally grows at the high altitudes of ~Iimalayan belt, hence to obtain the compound from such geographical locations is really difficu:Lt while the in vitro production and extraction protocol ensures convenient source of supply.
5) The isolation process to get the compound is very simple which does not need any chromatographic 31-MAR-2000 20 14 FROM KLIMRRAN & SAGAR TO 001416364'7910 P.19 column. Thus, the process would be cost effective and adaptable to large scale production.
6) The above processs of the invention provides for the first time a source of catechin penta acetate, a precursor of (-~) catechin.
Following the process of the present invention,(+) catechin penta acetate can be obtained as a single entity in the cell cultures of Taxus wallichiana which in turn can be converted into catechin and its other derivatives .
8) The isolation p;.~ocess of catechin penta acetate did not need any extreme condition, thus the process will be practicable. , , (+) catechin penta acetate is stable towards aerial oxidation and does not polymerize and hence it has a higher shelf life.
Following the process of the present invention,(+) catechin penta acetate can be obtained as a single entity in the cell cultures of Taxus wallichiana which in turn can be converted into catechin and its other derivatives .
8) The isolation p;.~ocess of catechin penta acetate did not need any extreme condition, thus the process will be practicable. , , (+) catechin penta acetate is stable towards aerial oxidation and does not polymerize and hence it has a higher shelf life.
Claims (20)
1. A process for the production of a stable compound (+) catechin penta acetate of formula (2) from T.
wallichiana tissue cultures and production of compound (+) catechin of formula (1)., said process comprising the steps of:
a) inoculation of explants on culture media supplemented with combinations of auxins (1-5 mg/l) and cytokinins (0.1-1.0 mg/l);
b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals;
c) extraction of fresh pulverized calli with polar solvents at room temperature:
d) evaporating the polar solvent to give a residue, treating then residue with a chlorinated solvent to isolate (+) catechin by filtration or treating the residue with water and extracting with a chlorinated solvent and evaporating the solvent to semi-solid mass;
e) subjecting the resultant mass to column chromatography over suitable adsorbents;
f) eluting with organic solvent or mixtures of organic solvent to obtain (+)catechin penta acetate, and isolating (+) catechin penta acetate by filtration.
wallichiana tissue cultures and production of compound (+) catechin of formula (1)., said process comprising the steps of:
a) inoculation of explants on culture media supplemented with combinations of auxins (1-5 mg/l) and cytokinins (0.1-1.0 mg/l);
b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals;
c) extraction of fresh pulverized calli with polar solvents at room temperature:
d) evaporating the polar solvent to give a residue, treating then residue with a chlorinated solvent to isolate (+) catechin by filtration or treating the residue with water and extracting with a chlorinated solvent and evaporating the solvent to semi-solid mass;
e) subjecting the resultant mass to column chromatography over suitable adsorbents;
f) eluting with organic solvent or mixtures of organic solvent to obtain (+)catechin penta acetate, and isolating (+) catechin penta acetate by filtration.
2. A process as claimed in claim 1, wherein the different explants used to initiate calli may be selected from needles, twigs, stem segments devoid of needles and seeds of T. wallichiana.
3. A process as claimed in claim 1, wherein the media compositions used may be selected from Murashinge and Skoog (MS), Gamborg (85), White and Kitsch and Kitsch basal media.
4. A process as claimed in claim 1, wherein the auxin supplements used may be selected from IAA, NAA, 2,4-D, 2,4-T, picloram and IBA.
5. A process as claimed in claim 1, wherein the cytokinin supplements may be selected from BAP, MAP, kinetin, zeatin, 2-ip and TDZ.
6. A process as claimed in claim 1, wherein culture conditions used to maintain the cultures may be selected between continuous light (300-3000 lux) or dark conditions.
7. A process as claimed in claim 1, wherein harvesting time may range between one month to thirty six months.
8. A process as claimed in claim 1, wherein the preferred harvesting time may range between twelve month to thirty six months.
9. A process as claimed in claim 1, wherein the polar solvent used to extract the callus is selected from methanol, ethanol, propanol, butanol.
10. A process as claimed in claim 1, wherein the ratio of auxin and cytokinin used ranges between 12 to 20:1.
11. A process as claimed in claim 1, wherein the preferred ratio of auxin and cytokinin ranges between 6 to 20:1
12. A process as claimed in claim 1, wherein the medium may be supplemented with casein hydrolysate ranging between 100-400 mg/l.
13. A process as claimed in claim 1, the chlorinated solvent used for partitioning may be selected from chloroform and dichloromethane.
14. A process ae clammed in claim 1, the adsorbents used for column chromatography for isolating catechin acetate may be selected from silica gel, alumin, florosil.
15. A process as claimed in claim 1, wherein the organic solvent, mixtures or organic solvents used for eluting the column may be selected from ethyl acetate, chloroform dichloromethane, hexane-ethyl acetate mixture, Pet. ether-ethyl acetate mixtures, hexane-chloroform mixtures, Pet. Ether-chloroform mixtures.
16. A process as claimed in claim 3 wherein Murashige and Skoog; (MS) medium comprises NH4N03 1,650 mg/l, KNO3 1, 900 mg/l, CaCl2.2H2O 400 mg/l, MgSO4.7H2O 370 mg/l, KH2PO4 170 mg/l, Na2EDTA.2H2O 7.2 mg/l, FeSO4.7H2O 27.8 mg/l, MnSO4.4H2O 22.3 mg/l, ZnSO4.7H2O
8.6 mg/l, H3BO3 6.2 mg/l, KI 0.83 mg/l, Na2MoO4.2H2O
0.25 mg/l, CuSO4.5H2O 0.025 mg/l, CoCl2.6H2O 0.025 mg/l, Glycine 2.0 mg/l, Nicotinic acid 0.5 mg/l, PyridoxineHCl 0.5 mg/l, and ThiamineHCl 0.1 mg/l.
8.6 mg/l, H3BO3 6.2 mg/l, KI 0.83 mg/l, Na2MoO4.2H2O
0.25 mg/l, CuSO4.5H2O 0.025 mg/l, CoCl2.6H2O 0.025 mg/l, Glycine 2.0 mg/l, Nicotinic acid 0.5 mg/l, PyridoxineHCl 0.5 mg/l, and ThiamineHCl 0.1 mg/l.
17. A process as claimed in claim 3 wherein Gamborg's (B5) medium, comprising KNO3 (3,000) lmg/l, (NH4) 2504 134 mg/l, MgSO4.7H2O 500 mg/l, CaCl2.2H2O
150 mg/l, NaH2PO,.H2O 150 mg/l, MnSO4.H2O 10.0 mg/l, KI 0.75 mg/l, H3BO3 3.0 mg/l, ZnSO4.7H2O 2.0 mg/l, CuSO4 0.025 mg/l, NaMoO4.2H2O 0.25 mg/l, CoCl2.6H2O
0.025 mg/l, Na2EDTA.2H2O 37.2. mg/l, FeSO4.7H2O 27.8 mg/l.
150 mg/l, NaH2PO,.H2O 150 mg/l, MnSO4.H2O 10.0 mg/l, KI 0.75 mg/l, H3BO3 3.0 mg/l, ZnSO4.7H2O 2.0 mg/l, CuSO4 0.025 mg/l, NaMoO4.2H2O 0.25 mg/l, CoCl2.6H2O
0.025 mg/l, Na2EDTA.2H2O 37.2. mg/l, FeSO4.7H2O 27.8 mg/l.
18. A process as claimed in claim 3 wherein White's medium comprises Ca(NO3)2 (142.0 mg/l), KNO3 81.0 mg/l, MgSO4.7H2O 70.0 mg/l, KCl 65.0 mg/l, KH2PO, 12.0 mg/l, and (FeSO4)3 2.46 mg/l.
19. A process as claimed in claim 3 wherein Nitsch and Kitsch's medium comprises comprising NH4NO3
20.0mg/l, KNO3 950 mg/l, H3BO, 10.0 mg/l, KH2PO4 68.0 mg/l, Na2MoO4.2H2O 0.143 mg/l, CaCl2 41.5 mg/l, MgSO4.7H2O 185 mg/l, M NSO4.4H2O 15.0 mg/l, ZnSO4.H2O
10.0 mg/l, CuSO4 0.14 mg/l, FeSO4, 111.4 mg/l, Na2EDTA
149 mg/l, Biotin 0.05 mg/l, Glycine 2.0 mg/l, Nicotinic acid 5.0 mg/l, Pyridoxine HCl 0.5 mg/l, Thiamine HCl 0.5 mg/l, and Folic acid 5.0 mg/l.
10.0 mg/l, CuSO4 0.14 mg/l, FeSO4, 111.4 mg/l, Na2EDTA
149 mg/l, Biotin 0.05 mg/l, Glycine 2.0 mg/l, Nicotinic acid 5.0 mg/l, Pyridoxine HCl 0.5 mg/l, Thiamine HCl 0.5 mg/l, and Folic acid 5.0 mg/l.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102823498A (en) * | 2012-09-20 | 2012-12-19 | 重庆文理学院 | Culture medium for subculture multiplication of tissue cultured seedlings of red-flesh kiwifruits |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102823498A (en) * | 2012-09-20 | 2012-12-19 | 重庆文理学院 | Culture medium for subculture multiplication of tissue cultured seedlings of red-flesh kiwifruits |
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