Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2000-03-28
2003-09-16
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S119000, C435S118000, C435S117000, C435S155000
Reexamination Certificate
active
06620599
ABSTRACT:
FIELD
The present invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula 1 from
Taxus wallichiana
tissue cultures:
BACKGROUND
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 drug was first isolated from the stem bark of
Taxus brevifolia
. Thereafter, it has also been isolated from other Taxus species including
Taxus wallichiana.
Catechins, the basic structural unit of condensed tannins, belong to flavan-3-ol derivatives and are found in a wide variety of plant sources such as vegetables, herbs and teas (Phytochem (1981) 20:869). Considerable interest has been expressed regarding the various pharmacological functions of catechins, which have been proved to be antibacterial, antiviral, anti-tumour, antioxidant and radical scavengers (Phytochem (1998) 49:2379-82).
The direct manipulation of plant cell and tissue culture systems has resulted in an enhanced production of various secondary metabolites. In vitro production of catechins, mainly (−) epicatechin-3-O-gallate accompanied by (+) catechin and (−) epicatechin have been reported in
Fagophyrum 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) 18:659-662),
Crataegus monogyna
(Phytochem (1994) 37:1273),
Uncaria elliptica
(Phytochem (1998) 28:1099-1 100) and
Polygonum hydropiper
(Phytochem (1998) 49:1935-39).
Several approaches have been used for the establishment of in vitro cultures of various Taxus species (Plant Cell, Tissue and Organ Cult (1996) 46:59-75). Different explants and various basal media have been used for the initiation and maintenance of Taxus callus and cell suspension cultures. The culture media are frequently supplemented with organic substances, such as casein hydrolysate, polyvinylpyrrolidone, ascorbic acid and others. Several growth regulators are used for stimulation of cell proliferation.
Taxus wallichiana
, known as Himalayan yew is available in India. Suspension and callus cultures of
Taxus wallichiana
are found capable of producing taxol (Planta Med. (1998) 64:270-72) and some important taxanes, namely 2-deacetoxy-taxinine J and 2-deacetoxy austrospicatin (Planta Med. (1996) 62:333-35). The Applicants have been screening different callus lines of
Taxus wallichiana
induced from different explants of trees collected from different geographical regions of India. The protocol standardized for in vitro callus production is dependent on media composition (viz. Murashige and Skoog's, Gambrog's, White's, Nitsch and Nitsch's), hormonal regime combinations of different concentrations of cytokinins 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 source (preferably from needles, twigs, stems devoid of needles and seeds) and culture conditions (light or dark conditions).
The callus line developed from a specific explant on different media compositions having definite hormonal combinations resulted in the production of a phenolic compound. The compound, having a molecular formula of C
15
H
14
O
6
, mp 94-95° C., was isolated as an amorphous solid. The compound has been characterized as (+) catechin. The yield was noted to be 0.3% in six months old callus. The (+) catechin is very important precursor for the synthesis of other catechin derivatives, eg. Gallocatechin, epigallocatechin and epigallocatechin-3-O-gallate. It is 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-gallate and (−) epigallocatechin, (Phytochem (1989) 28:1099-1100, (1992) 31:1239-1241 (1993) 32:924-931, (1998) 49:1935-1939), whereas in the present invention only (+) catechin is expressed. Furthermore, in the present invention 0.3% concentration of (+) catechin has been detected in 6 months old callus while the expression of (+) catechin in
Polygonum hydropiper
occurred after one year of culture initiation (Phytochem (1998) 49(7):1935-39).
OBJECTS
Thus the main object of the present invention is to provide a process for In vitro production of a biologically active phenolic compound (+) catechin of formula I from
Taxus wallichiana
tissue cultures.
Another object of the invention is to provide a process for isolation of (+) catechin from cell cultures of
Taxus wallichiana
without the use of any cumbersome chromatographic separations.
SUMMARY
The present invention consisting of the in vitro production of (+) catechin from
Taxus wallichiana
constitute the first ever report of production of (+) catechin in the genus Taxus. Moreover, the yield of (+) catechin obtained in the invention is 0.3% which is significantly better to the so far reported ones from cell culture sources taking into consideration of the reported culture period (Phytochemistry (1998) 49(7):1935-1939).
DETAILED DESCRIPTION
Accordingly the present invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula I from
Taxus wallichiana
tissue cultures which comprises: (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 solvents to obtain a residue, and (e) treatment of the residue with a chlorinated solvent and isolation of the compound (+) catechin by filtration.
In an embodiment, the explants for induction of callus may be selected from needles, twigs, stems devoid of needles and seeds of
Taxus Wallichiana.
In another embodiment, the culture media for callus induction and multiplication are selected from Murashige and Skoog, (1962) (MS) medium, containing the following (in mg/l)—NH
4
NO
3
(1,650), KNO
3
(1,900), CaCl
2
.2H
2
O (400), MgSO
4
.7H
2
O (370), KH
2
PO
4
(170), Na
2
EDTA.2H
2
O (7.2), FeSO
4
.7H
2
O (27.8), MnSO
4
.4H
2
O (22.3), ZnSO
4
.7H
2
O (8.6), H
3
BO
3
(6.2), KI (0.83), Na
2
MoO
4
.2H
2
O (0.25), CuSO
4
.5H
2
O (0.025), CoCl
2
.6H
2
O (0.025), Glycine (2.0), Nicotinic acid (0.5), PyridoxineHCl (0.5), ThiamineHCl (0.1); Gamborg's; (1968) (B5) medium, containing the following (in mg/l)—KNO
3
(3,000), (NH
4
)2SO
4
(134), MgSO
4
.7H
2
O (500), CaCl
2
.2H
2
O (150), NaH
2
PO
4
.H
2
O (150), MnSO
4
.H
2
O (10.0), KI (0.75), H
3
BO
3
(3.0), ZnSO
4
.7H
2
O (2.0), CUSO
4
(0.025), NaMoO
4
.2H
2
O (0.25), CoCl
2
.6H
2
O (0.025), Na
2
EDTA.2H
2
O (37.2) FeSO
4
.7H
2
O (27.8), White's (1963) medium, consisting of the following (in mg/l) —Ca(NO
3
)
2
(142.0), KNO
3
(81.0), MgSO
4
.7H
2
O (70.0), KCl (65.0), KH
2
PO
4
(12.0), (FeSO
4
)
3
(2.46) and Nitsch and Nitsch; (1969) medium, containing the following (in mg/l)—NE
4
NO
3
(20.0), KNO
3
(950), H
3
BO
3
(10.0), KH
2
PO
4
(68.0), Na
2
MoO
4
.2H
2
O (0.143), CaCl
2
(41.5), MgSO
4
.7H
2
O (185), M
N
SO
4
.4H
2
O (15.0), ZnSO
4
.H20 (10.0), CuSO
4
(0.14), FeSO
4
(111.4), Na
2
EDTA (149), Biotin (0.05), Glycine (2.0), Nicotinic acid (5.0), Pyridoxine HCl (0.5), Thiamine HCl (0.5), Folic acid (5.0).
In yet another embodiment, the auxins may be selected from Indole acetic acid (IAA), naphthalene 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 following range (1.0-5.0 mg/l).
In still another embodiment, the cytokinins may
Agarwal Shipra
Banerjee Suchitra
Chattopadhyay Sunil Kumar
Kulshrestha Manish
Kumar Sushil
Council of Scientific and Industrial Research
Jenkens & Gilchrist
Marx Irene
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