Note: Descriptions are shown in the official language in which they were submitted.
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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
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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) .
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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
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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
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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
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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
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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
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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
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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.
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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.
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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.
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Preparation of +catechin:
EXAMPLE - 1
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%).
EXAMPLE -2
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%).
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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
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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 ) .
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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
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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.
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