Stable high ginsenoside-yielding callus line of Panax...

Details Stable high ginsenoside-yielding callus line of Panax... Stable high ginsenoside-yielding callus line of Panax...

1999-10-19

2001-12-04

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Plant cell or cell line, per se ; composition thereof;...

C435S420000

Reexamination Certificate

active

06326202

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a stable high ginsenoside-yielding callus line of
Panax quinquefolium
(American ginseng) developed from root explants and a process for the development of these callus lines. More particularly, the invented callus line has a distinct morphological marker of purple pigmentation and saponin yield comparable in quantity and quality to that of normal roots.
The invention provides a viable alternate option to boosting the industrial production of ginseng saponins (ginsenosides) which are in high demand in market as important ingredients of health tonics and anti-aging drug preparations.
BACKGROUND
Ginsenosides (triterpene glycosides) extracted from roots of 4-7 years old plants of ginseng (common name for Panax species) are important constituents of herbal health care products today. Owing to their strong immuno-modulatory, adaptogenic and aphrodisiac actions, ginseng saponins are widely prescribed in several conditions of health disorders such as anaemia, diabetes, asthma, neuroaesthemia, dyspepsia, convulsion and even in cancer and AIDS. Priced at 750-1000 US $ per kg and with an annual global production of 35-40 thousand tons, Panax roots are the fourth largest selling herbal healthcare product today. Korea, China and Japan have the major share in the global supply of ginseng roots. [Indian pharmaceutical companies import about 400-500 tons of Panax root powder annually.] The chief source of ginseng roots are
P. ginseng
(Korean panax),
P. quinquefolium
(American panax) and
P. notoginseng
(Chinese panax). The Indian congeners i.e.
P. pseudoginseng
and
P. sikkimensis
Ban., growing wild in the sub-Himalayan zones (Darjeeling, Sikkim, Arunachal Pradesh etc.) though found to be on par in saponin quality and content with their oriental counterparts, have not yet been commercially exploited. Traditional field cultivation of Panax sp. is very slow and labour intensive. It takes 18-22 months for the seed to germinate (following 2-3 stratification cycles to break seed dormancy) and an extended gestation period of 3-5 years for the crop to mature and provide economic root biomass yield and quality of saponins. Tissue culture based strategies for rapid propagation (micro-cloning) and in vitro ginsenoside production in Panax, therefore, hold immense promise and potential.
PRIOR ART REFERENCES
These are many reports on tissue culture studies particularly, in vitro ginsenoside production in cell suspension cultures, of Korean ginseng—
P.ginseng
[Boitechnology in Agriculture & Forestry Vol. 4(1) (Ed) Y. P. S. Bajaj pp 484-500 (1988); Cell Culture and Somatic Cell Genetics of Plants Vol. 5 (Ed) I. K. Visil (1988)pp. 213-234, J. Biotechnol 52:121-126 Process Biochem, 33:69-74 (1998)]. The maximum ginsenoside level detected in cell culture of ginseng has been reported to be 16 mg/g. dry wt. (Agri Cell Rep., February, 1994). Possibility of ginsenoside production in genetically transformed hairy roots has also been indicated in
P.ginseng
[P1. Cell Rep. 12: 681-686; (1993), Ibid 15: 555-560) (1996), Phytochemistry 49:1929-1934 (1998)]. In contrast there have been very few reports concerning
P. quinquefolium
cell and tissue cultures [Phytochemistry 35: 1221-1225, (1994), Process Biochem., 33:69-74 (1998)]. The applicants, during their research, had earlier shown that callus and cell suspension cultures of
P. quinquefolium
are also capable of producing characteristic ginsenosides in vitro [Phytochemistry 35:1221-1225 (1994)]. The crude ginsenoside content (i.e. 0.56% and 0.65% for callus and cell cultures, respectively) and ratio of Rb and Rg group of ginsenosides of these wild line cultures were found to vary with their age (days after subculture) during a 5 week culture cycle. The possibility of isolating high ginsenoside yielding lines of
P. quinquefolium
, specifically rich in different ginsenoside fractions, was first hypothesized by the applicants in this report. The present invention is an outcome of the continued efforts made by the applicants in this direction and accordingly, the applicants have now developed a high-yielding callus line with a crude ginsenoside level as high as 1.21% f.wt. that matches well with that in 3-4 years old roots of field grown plants [Shoyakugaku Zosshi 32:96 (1978), J. Herb Spices & Med. P1. 3:41-50 (1995)]. Recent market trends show that because of extremely high price of wild roots of
P. ginseng
, the demand for
P. quinquefolium
has increased dramatically and is 5-10 times more expensive than its oriental counterparts [P1. Med. 61:466-469 (1996)].
The novelties of the inventions are as follows:
(a) The invention for the first time, provides a stable high-ginsenoside yielding callus line of
P. quinquefolium
whose saponin content is comparable in yield and quality to that of field grown plants,
(b) The procedure outlines the formulation of a callus culture maintenance medium [modified MS medium with double the amount of organic adjuvants+200 mg/l myoinositol+2, 4-D (1.0 mg/l)+Kinetin (0.25 mg/l)] for callus maintenance and multiplication and incubation environment such as continuous light (3000 Lux) and temperature 28±2° C. that support sustained growth and stable ginsenoside yield for over four and half years,
(c) The invention has resulted in identifying the parameters, such as inoculum age, inoculum to medium ratio, tissue harvesting schedule, media pH, and extraction, TLC densitometry and HPLC analysis of the crude ginsenoside, that are required for further scaling up of the isolated line,
(d) The isolated callus line has a morphologically distinguishable feature, characteristic DNA profile and a stable chemical fingerprint.
FIG. 1
depicts the characteristic purple pigmentation in the callus cells of the isolated line in comparison to pale white non-pigmented wild line counterpart. The morphological appearance is as nearly true as is reasonably possible to make the same in coloured illustration of this marker character,
(e) The isolated callus line has been shown to grow and accumulate ginsenoside in amounts and quality on medium having cheap market grade sugar as energy source in comparison to the conventionally employed costly analytical grade sucrose. This is a vital step towards cost reduction strategies for commercial utilisation of such tissue culture lines,
(f) The isolated callus line has all the desired attributes that enable it to be exploited on a commercial basis.
Objects
The main object of the present invention is to provide a stable high ginsenoside-yielding callus line of
Panax quinquefolium
(American ginseng), developed from root explants and a process for the development of said callus lines, obviating the drawbacks of the earlier methods.
Another object of the present invention is to isolate a callus line with saponin yield comparable in quantity and quality to that of normal roots so as to devise an in vitro process for the production of these health care compounds on a commercially feasible scale.
Still another object of the present invention is to identify in vitro growth conditions and other experimental parameters for commercialisation and in vitro production of
Panax quinquefolium.
Yet another object of the present invention is to reduce the cost of ginsenoside production in vitro by use of cheaper carbohydrate sources.
One more object of the present invention is to identify morphological marker(s), genetic marker (DNA fingerprint) and chemical fingerprint of the isolated high-yielding line.


REFERENCES:
Tsutomu Furuya, “Saponins (Ginseng Saponins)”, Chapter 12 ofCell Culture and Somatic Cell Genetics of Plants, vol. 5, 1988, Academic Press, Inc., pp. 213-234.
Shinji Inomata et al., “Growth pattern and ginsenoside production of Agrobacterium-transformedPanax ginsengroots”,Plant Cell Reports, 1993, pp. 681-686.
Lakafumi Yoshikawa et al., Saponin production by cultures ofPanax ginsengtransformed withAgrobacterium rhizogenes,Plant Cell Reports, 1987, p

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