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stem cellsbiotechnology
Botanical extracts and plants have
always been a major source of food and
health ingredients, providing a wide range
of biologically active substances used widely
not only in nutrition but also for cosmetic
and pharmaceutical applications.
Despite a number of agricultural and
technological improvements in plant
cultivation and product manufacturing
procedures over the centuries, the required
safety, availability and purity standards of
natural ingredients cannot be totally
guaranteed because of the intrinsic nature of
the traditional production process itself.
Indeed numerous uncontrolled
environmental factors can deeply modify the
levels of active substances in the plant.
Furthermore the widespread industrial use of
ingredients derived from slow growing or
rare and endangered plant species poses
serious risks of over-harvesting, thus
underscoring the need for ecological
biodiversity protection and sustainable
production.
New means to cope with these issues
come from an innovative green
biotechnological approach using cultures of
plant cells that offers new possibilities in the
production of plant derived ingredients.[1]
While plant cell culture biotechnology is
well known in academic communities, it has
not yet been fully developed on an industrial
scale, despite the numerous advantages over
conventional methods, due to the high and
long-term investments required. As well as
being a totally environmentally friendly
technology, it is able to ensure a high degree
of safety, reproducibility and standardisation
in the composition of the final product.
Primary metabolites, such as proteins,
nucleic acids, lipids and carbohydrates, are
the essential components for plant life,
growth and reproduction and many basic
molecules are structurally and functionally
conserved in most plants; eg DNA,
phospholipids and cellulose. However, during
biological evolution, as an inevitable response
to a challenging environment and ever
changing climate, plants have developed an
enormous number of survival strategies
including secondary metabolites whose main
function is to protect the plant. Plants, as well
as all other living organisms, need to be
protected in very similar ways: from oxygen
toxicity, from UV light, from exceedingly
high or low temperatures, from viral and
bacterial colonisation. Secondary metabolites
are the major components shielding plants
from all these environmental stresses. Some
of these molecules appear to be specialised as
antioxidants, such as ascorbic acid or
astaxanthin; others appear more effective in
pathogen control as phytoalexins (eg
pterocarpans and coumarins); still other
secondary metabolites appear to provide a
protective shield against several
environmental threats. Among these latter
multitasking substances, there are a variety of
polyphenolic compounds of plant origin,
classified as phenylpropanoids (PP), which
are reported to possess significant
antioxidant, free radical scavenging, UV
protecting and antibacterial properties.[2]
PLANT CELL TECHNOLOGY
In vitro cell culture growth is strictly
dependent on the biological capability of
cells to divide and generate new replicas of
the original cell. In plants this proliferation
capability is also associated with the potential
to differentiate in cells with different features
as compared with the parent cells, up to
generating a whole new plant. Embryologists
term totipotency the ability of single cells to
generate all the tissues of a completely
differentiated organism and cultured plant
cells. The opportunity to control plant cell
growth and differentiation has been one of
the major driving forces for the development
of plant biotechnologies since the pioneering
attempts of Gottlieb Haberlandt more than a
century ago.
Plant biotechnology has made much
progress by basically defining the essential
nutrients required in the culture medium
and identifying auxins and cytokinins as the
major growth regulators controlling plant
cells. The plant cells can grow on a solid
surface as friable, pale brown lumps (called
callus), or as small clusters of cells in a liquid
medium called a suspension culture. These
cells can be maintained indefinitely provided
they are sub-cultured regularly into fresh
growth medium.
Tissue culture cells generally lack the
distinctive features of most plant cells. They
have a small vacuole, lack chloroplasts and
photosynthetic pathways and the structural
or chemical features that distinguish so many
cell types within the intact plant are absent.
They are very similar to the undifferentiated
Division
of the
spoils
Roberto dal Toso &
Francesca Melandri
introduce the use of safe,
standardised meristem cells
as highly concentrated
actives respecting
biodiversity for use in
cosmetic applications
cells found in meristematic regions which
become committed to develop into each
different cell type as the plant grows. Thanks
to totipotency, tissue cultured cells can also
FIGURE 1
CALLOGENESIS FROM PLANT EXPLANT
April 2011 SPC 35
biotechnologystem cells
be induced to re-differentiate into whole
plants by proper modifications of the growth
media.
Plant tissue cultures can be initiated from
almost any part of a plant. The physiological
state of the plant does have an influence on
its response to attempts to initiate tissue
culture. The source, termed explant, is
obtained from the fresh plant tissue by
mechanical incision which mimics a wound,
thus initiating the tissue repair process that
stimulates callus formation by meristem cell
growth and somatic cell dedifferentiation
(figure 1).Younger tissues contain a higher
proportion of actively dividing cells and are
more responsive to a callus initiation
programme.
The exact conditions required to initiate
and sustain plant cells in culture, or to
regenerate intact plants from cultured cells,
are different for each plant species. Each
variety of a species will often have a
particular set of cultural requirements.
Despite all the knowledge that has been
obtained about plant tissue culture during
the 20th century, these conditions have still
to be identified for each variety through
experimentation.
Once the callus has grown, a proper
selection programme based on the most
interesting biochemical and morphological
features of the cells begins. This is a long and
time consuming activity but can eventually
yield a stable cell line which maintains
relatively constant metabolic traits. With time
however, as occurs for all living and
replicating organisms, some of the cell
features will change due to ageing or natural
gene mutation. To avoid these changes,
cryopreservation of the most interesting
strains is the only way to properly store the
cell line for long-term maintenance and
industrial back-up, and IRB, an Italian green
biotech company and manufacturer of active
ingredients, has established all the techniques
to achieve this goal.
BIODIVERSITY & SUSTAINABILITY
A very important advantage of the indefinite
growth of the plant cell cultures is that they
provide a source of active substances with no
further need to breed, cultivate and collect
plants in fields any more.
This provides the opportunity to
produce active substances, particularly
phenylpropanoids, from rare, endangered
and protected plants, such as Leontopodium
alpinum (edelweiss), thus preserving natural
biodiversity and dramatically improving
environmental sustainability. Indeed
considering the amount of soil and water
required of produce 1kg of echinacoside
from Echinacea angustifolia obtained from
plant roots cultivated in open field as
36 SPC April 2011
TABLE 1: AVERAGE CONSUMPTION FOR
THE PRODUCTION OF 1KG OF
ECHINACOSIDE FROM ECHINACEA
ANGUSTIFOLIA
Water (t)
Area (m2)
Solvents (kg)
Traditional
method
1.379
1.149
500
HTN(tm)
technology
1
3
100
compared with the biotechnological
approach, there is over a 1000-fold
reduction in the requirement for land and
water and a more than fivefold reduction in
the use of solvents during the extraction
procedure. The overall benefits for the
environment also include the elimination
of pesticide use as well as soil fertilization,
which is one of the major sources of water
pollution (table 1).
THE PHENYLPROPANOIDS
As mentioned, phenylpropanoids (PPs) are
one of the most interesting groups of
compounds present in plant cell cultures.
From a chemical point of view, all PPs derive
from cinnamic acid, which can undergo a
number of hydroxylation, methylation and
dehydration reactions producing p-coumaric
acid, caffeic and ferulic acid. In newly
formed plant tissues, as well as in cell
cultures, one or more caffeic acid residues are
then covalently linked to a carbohydrate
backbone to maintain solubility. In more
differentiated plant tissues, PPs also conjugate
with each other to from insoluble lignins and
tannins that make the tissue resistant to
environmental stresses. PPs are part of the
plant cell defensive system and are induced
by a number of environmental stresses.[3]
They are very effective scavengers of free
radicals due to high reactivity of their caffeic
hydroxyl substituents. Both free radical
scavenging and anti-inflammatory activity are
apparently responsible for the inhibitory
effects of PPs towards an array of superoxide
producing enzymes and lipid peroxidation
and could also protect human skin against
the deleterious effects of various types of
environmental stress.[4,5,6]
PPs were initially isolated from plant cell
cultures of different plants such as Buddlejia
davidii and Leontopodium alpinum. Plant cell
cultures allow for several advantages over
plant extracts, such as higher purity of active
principles, seasonless standardisation of
quality and patent protection. A number of
PPs (verbascoside, leontopodic acids and
dicaffeoyl-quinic acids etc) were isolated,
identified by chemical/structural analysis and
used for further biological testing.
LEONTOPODIUM ALPINUM
(EDELWEISS) MERISTEM CELLS
A particularly important example to
highlight the usefulness of the plant cell
culture technology is its application to
Leontopodium alpinum, more commonly
known as edelweiss. This is a rare plant
growing in the Alps and is a protected
species because it is at risk of extinction.
Leontopodium alpinum lives in a very
damaging environment, particularly exposed
to high levels of UV light, cold temperatures
and other abiotic stresses. Its survival depends
on the ability of the plant to synthesise a
number of defensive molecules that can
protect it from various types of
environmental stresses. Some of these
molecules have been recently identified and
most belong to the PP family.[7] Particularly
interesting are the leontopodic acids A and B
(figure 2) which provide an antioxidant
capacity at least three times more effective
than trolox (TEAC) (figure 3) taken as a
benchmark.
However, edelweiss plants are strictly
controlled for commercial use and are
available only in limited amounts. Thus one
of the few ways, if not the only option, of
providing such important substances for
commercial use is by plant cell cultures.
Leontopodic acids, together with other
interesting antioxidant molecules such as
dicaffeoyl-quinic acids, have been found in
Leontopodium alpinum cell cultures and are
made fully available as cosmetic ingredients
FIGURE 2
LEONTOPODIC ACIDS A AND B: MOLECULAR STRUCTURE
biotechnologystem cells
by a plant stem or meristem cell preparation
together with polysaccharides and lipids of
the same cells, thus providing a unique
opportunity to utilise a functionally rich and
well standardised phytocomplex. As a further
development, IRB is currently able to supply
the only edelweiss biotech extract with a
guaranteed minimum level of 0.1% of
leontopodic acid A (Leontopodium alpinum
stems G).
This complex of Leontopodium alpinum
meristem cells has also been shown to inhibit
both collagenase and hyaluronidase, two
enzymes of the dermal matrix linked to skin
ageing. Thus the use of the whole plant
meristem cells as a cosmetic ingredient is a
new possibility offered by the
biotechnological approach since the peculiar
properties of this rare plant could not
previously be utilised for industrial
applications.
Based on the in vitro testing, a clinical
anti-ageing and anti-wrinkle trial on 20 aged
subjects was performed with a topical cream
formulation with 1% of this biotech
ingredient. The treatment was made twice
daily to the eye contour area and results were
registered by digital scanning of the wrinkle
FIGURE 3
ANTIOXIDANT ACTIVITY
(TEAC METHOD) OF
MOLECULES PRESENT IN
LEONTOPODIUM ALPINUM
STEMS G COMPARED
WITH STANDARD
ANTIOXIDANTS
microprofile and depth at day 0, day 22 and
day 40 of use. The benefits are clear:
statistically significant (p<0.05) 15%
reduction after 22 days and further
improvement after 40 days of treatment
(figure 4).
Biotechnology applied to the production
of natural compounds from plant cell
cultures offers higher safety, availability and
standardisation levels over more conventional
processes of harvesting and extraction from
crops cultivated in the open field. This
approach enables the production of very
effective substances, such as verbascoside and
leontopodic acids, as well as a new complex,
a standardised ingredient with interesting
applications in the cosmetics industry.
The technology of plant cell cultures is
fully eco-sustainable and non-GM and
provides industrial availability of active
substances even from protected or
endangered plants such as Leontopodium
alpinum or from plants containing low
amounts of secondary metabolites, without
affecting the delicate natural ecosystem
balance and endangering biodiversity.
stem cellsbiotechnology
FIGURE 4
MICROPROFILE EVALUATION OF THE EYE CONTOUR AREA BY SKIN SCANNER
0 days
22 days
References
1.Wink M, Functions of plant secondary
metabolites and their exploitation in biotechnology,
Sheffield Academic Press, p1-17 (1999)
2. Korkina LG, Cell Mol Biol, 53,15-25 (2007)
3. Dixon R & Paiva (2005)
4. Aleo E, Ricci R, Passi S & Cataudella S, Progr
Nutr, 7, 154-182 (2005)
5. Korkina LG, Mikhal’chik E, Suprun MV,
Pastore S & Dal Toso R, Cell Mol Biol, 53, 8491 (2007)
6. Pastore S, Potapovich A, Kostyuk V, Mariani V,
Lulli D, De Luca C & Korkina L, Ann N Y
Acad Sci, 1171, 305-13 (2009)
7. Schwaiger S, et al, Phytochem Anal, 17, 291298 (2006)
40 days
Contact
Roberto dal Toso
R&D manager
IRB
Italy
tel +39 0444 371463
[email protected]
www.irbtech.com