6. Production of secondary metabolites from plants

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Plant Based Medicines, 2014: 93-109 ISBN: 978-81-308-0547-4 Editor: Remya Mohanraj
6. Production of secondary metabolites
from plants
Rahini.V.
Assistant Professor, Dept. of Biotechnology, Aarupadai Veedu Institute of Technology,
Paiyanoor, Kancheepuram 603 104.
Abstract. Plants are potent biochemical factories. They have been
components of phytomedicine since time immemorial. Plant
products of commercial interest are secondary metabolites which
can be derived from different parts of the plant like, bark, leaves,
roots, flowers, seeds, fruits etc. This review focuses on the various
techniques available for the isolation and purification of secondary
metabolites from plants. The significance of plant cell culture in
the production of secondary metabolites has also been reviewed.
1. Introduction
Medicinal plants are the most exclusive source of life saving drugs for
majority of the world’s population. The utilization of plant cells for the
production of natural or recombinant compounds of commercial interest has
gained increasing attention over past decades (Canter, et al., 2005).
In recent years, traditional system of medicine has become a topic of
global importance. Although modern medicine may be available in developed
countries, herbal medicines [phytopharmaceuticals] have often maintained
popularity for historical and cultural reasons.
Correspondence/Reprint request: Ms. Rahini. V, Assistant professor, Dept. of biotechnology, Aarupadai Veedu
Institute of Technology, Vinayaka Missions University, Rajeev Gandhi Salai (OMR Road), Paiyanoor603104, Kancheepuram (Dt), Tamil Nadu, India. E-mail: [email protected]
94
Rahini. V
The history of the extraction of natural products dates back to
Mesopotamian and Egyptian times, where production of perfumes or
pharmaceutically - active oils and waxes was a major business. In
archeological excavations 250 km south of Baghdad extraction pots from
about 3500 BC were found (Levey, 1959), made from a hard, sandy material
presumably air - dried brick earth. It is supposed that in the circular channel
was the solid feed, which was extracted by a Soxhlet - like procedure with
water or oil. The solvent vapors were condensed at the cap, possibly cooled
by wet rags. The condensate then did the leaching and was fed back through
holes in the channel to the bottom.
Generally, the plant products of commercial interest are secondary
metabolites, which in turn belong to three main categories: essential oils,
glycosides and alkaloids. The essential oils consist of mixture of terpenoids,
which are used as flavoring agents, perfumes and solvents. The glycosides
include flavanoids, saponins, phenolics, tannins, cyanogenic glycosides and
mustard oils, which are utilized as dyes, food colors and medicinals (e.g.,
steroid hormones, antibiotics, digitalis). The alkaloids are a diverse group of
compounds with over 4000 structures known; almost all naturally occurring
alkaloids are of plant origin. Alkaloids are physiologically active in humans
(e.g., cocaine, nicotine, morphine, strychnine) and therefore of a great interest
for pharmaceutical industry. (Shuler et al., 1981)
Figure 1. Secondary metabolites.
Production of secondary metabolites from plants
95
2. Extraction and purification of plant secondary metabolites
The production chain with natural products has three major parts to
consider. The first is agricultural, followed by, for example, extraction to get
a concentrated raw extract and in pharmaceutical applications (not with
cosmetics and nutraceuticals) a final purification step is necessary in order to
get ultrapure product. All the steps combined contribute to the overall yield
and determine the final economics.
2.1. Extraction
After harvesting the next step in the process chain is the extraction of the
desired substance. However, there are several regulations to be considered. If
the products are to be used with foods then there are regulations in the use of
appropriate solvents. According to European Union and governmental
regulations the following solvents are allowed:
•
•
•
water (with admixture of acids or base),
other foodstuffs with solvent properties and
solvents like propene, butane, ethylacetate, ethanol, CO 2 , N2O, acetone
(the latter not with olive oil).
In any case, for all these solvents the maximum content of arsenic or lead is
1 mg and no toxicologically critical additives are allowed. The use of a mixture of
hexane and ethylmethyl ketone is forbidden. (Hans - Jörg Bart, 2011).
Water, solvents from natural sources (limonene etc.), organic solvents
and liquefied gases are used in the food industry. Here liquefied CO 2
dominates the market and is used for decaffeination of green coffee beans or
tea, preparation of leaf extracts, extraction of spices, herbs, essential oils,
pungent constituents, natural colorants and antioxidants as well as production
of high - value fatty oils (Lack & Simandi, 2001). Criteria for solvent
selection includes
Solubility
Selectivity
Recoverability of Solvent
Viscosity and Melting point
Surface Tension
Toxicity and flammability
Corrosivity
Thermal and chemical stability
Availability and costs
Environmental impact
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Rahini. V
Figure 2. General procedure for preparing extracts representing a range of
polarities,including a virtually tannin-free chloroform extract.
Production of secondary metabolites from plants
97
Figure 3. General fractionation procedure to obtain a precipitate of crude saponin
from plants.
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Rahini. V
Figure 4. General procedure to obtain alkaloidal extracts from crude plant material.
2.2. Purification
The purification methods rely on chromatography and the final product
is then obtained by crystallization. In applications with nutraceuticals,
cosmetics and fragrances there is no need for ultrapure products, which is
in strong contrast to the pharmaceutical field. Chromatographic methods are
very flexible due to their separation principles. Various Chromatographic
methods include
Adsorption Chromatography
Partition Chromatography
Ion exchange Chromatography
Size exclusion Chromatography
Production of secondary metabolites from plants
99
Bio-Affinity Chromatography
True Moving Bed (TMB) Chromatography
Simulated Moving Bed (TMB) Chromatography
Annular Chromatography
Isolation of secondary metabolites by solvent extraction from the
naturally grown whole plants leads to the continued destruction of plants has
posed a major threat to the plant species getting extinct over the years.
Alternative methods for the production of these compounds by the use of
plant cell culture technologies have gained particular interest (DiCosmo et al.,
1989).
3. Plant cell culture
In plant cell culture, the isolated cells from the whole plant (or parts
derived thereof) are cultivated under appropriate physiological conditions and
the desired product is extracted from the cultured cells.
The large scale plant cell and tissue cultures have been considered as an
alternative source of biochemicals over the last 40 years. Routien and Nickel
received the first patent for the cultivation of plant tissue in 1956
(Routien et al., 1956) and suggested its potential for the production of
secondary metabolites (Scragg, 1991). Shortly after that time, the National
Aeronautics and Space Administration (NASA) started to support research of
plant cell cultures for regenerative life support systems. Since early 1960s,
experiments with plants and plant tissue cultures were performed under
various conditions of microgravity in space (one-way spaceships,
biosatellites, space shuttles and parabolic flights, the orbital stations Salyut
and Mir) and accompanied by ground studies using rotating clinostat vessels
(http://www.estec.esa.nl./spaceflights). Growth, development and metabolism
of plant cells and tissues have been studied to improve our understanding of
plant cell biology and tissue physiology, and derive criteria for bioprocess
design (Kordym, 1997).
The concept of using plant cell cultures is confined to the production of
valuable natural products such as pharmaceuticals, flavors and fragrances,
and fine chemicals – over 20000 different chemicals are produced from
plants, with about 1600 new plant chemicals added each year.
A number of plant species have been used for generation and propagation
of cell-suspension cultures, ranging from model systems like Arabidopsis,
Catharanthus and Taxus, to important monocotyledon or dicotyledonous crop
plants like rice, Soya bean, alfalfa and tobacco. The secondary metabolites
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Rahini. V
are known to play a major role in the adaptation of plants to their
environment, but also represent an important source of pharmaceuticals
(Ramachandra Rao & Ravishankar, 2002). The scheme of production of
some important plant pharmaceuticals produced in cell cultures (Vanisree &
Tsay, 2004) has been presented in Table 1.
Table 1. Bioactive secondary metabolites from plant cell culture.
Production of secondary metabolites from plants
Table 1. Continued
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102
Table 1. Continued
Rahini. V
Production of secondary metabolites from plants
103
The production of pharmaceuticals by the large scale plant tissue culture
is an attractive alternative to the traditional methods of plantation, as it offers
the following advantages (Fowler et al., 1985; Saurabh et al., 2002;
Vijaya Sree, et al., 2010)
Control of supply of product independent of the availability of the
plant itself.
Cultivation under controlled and optimized conditions.
Strain improvements with programs analogous to those used for
microbial systems.
Pesticides and herbicides are not needed.
Possibility of synthesizing novel compounds, not present in nature
by feeding of compounds analogous to natural substrates.
No dependence on climate and geographical location etc.,
well defined production systems which result in higher yields and
more consistent quality of the product
Cultured cells would be free of microbes and insects
The cells of any plants, tropical or alpine, could easily be multiplied
to yield their specific Metabolites.
Automated control of cell growth and rational regulation of
metabolite processes would reduce of labor costs and improve
productivity.
Organic substances are extractable from callus cultures.
In order to obtain high yields suitable for commercial exploitation,
efforts have been focused on isolating the biosynthetic activities of cultured
cells, achieved by optimizing the cultural conditions, selecting high
producing strains and employing precursor feeding, transformation methods,
and immobilization techniques (Dicosmo & Misawa, 1995). Transgenic
hairy root cultures have revolutionized the role of plant tissue culture in
secondary metabolite production. They are unique in their genetic and
biosynthetic stability, faster in growth, and more easily maintained. Using
this methodology a wide range of chemical compounds have been
synthesized (Giri & Narasu, 2000). Advances in tissue culture, combined
with improvement in genetic engineering have led to a great increase in
pharmaceuticals, nutraceuticals and other beneficial substances (Hansen &
Wright, 1999). Recent advances in the molecular biology, enzymology and
fermentation technology of plant cell cultures suggest that these systems will
become a viable source of important secondary metabolites (Abdin, 2007).
Genome manipulation is resulting in relatively large amounts of desired
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Rahini. V
compounds produced by plants infected with an engineered virus, whereas
transgenic plants can maintain constant levels of production of proteins
without additional intervention (Abdin & Kamaluddin, 2006).
Large scale plant tissue culture is an attractive alternative to the
traditional methods of plantation, as it offers two advantages:
(1) Controlled supply of biochemicals independent of plant availability
(climate, pests, politics), and
(2) Well defined production systems which result in higher yields and more
consistent quality of the product (Fowler, 1985).
However, various problems associated with low cell productivity, slow
growth, genetic instability of high-producing cell lines, poor control of
cellular differentiation and inability to maintain photoautotrophic growth
have limited the application of plant cell cultures. Also, much of the research
in this field has been done by scientists associated with private industry, not
published and not available to academic institutions (Beachy. 1997). In
addition, plant cell culture is expensive, and the best product candidates are
those of high value (500–1000 US$ per kg) and low-volume, not synthesized
by microorganisms, and too complex for chemical synthesis to be a
reasonable alternative. As a rule of thumb, the process is commercially
feasible if it yields a revenue of about 12 c/l/day (Humphrey, 1991). In spite
of potential advantages of the production of secondary metabolites in plant
cell cultures, only shikonin, ginsenosides and berberine are presently
produced on a large scale, and all three process plants are located in Japan
(Hara, 1996). In addition, the anti-cancer drug Taxol (registered trademark of
Bristol-Myers Squibb) is currently under consideration for large-scale
production (Roberts et al., 1995; Seki et al., 1995; Seki et al., 1997).
Consequently, the ongoing research in plant cell culture is largely focused on
the identification of rate limiting steps in biosynthetic pathways. Several
approaches were investigated: elicitation followed by monitoring the
activities of pathway enzymes; measuring enzyme levels in cell lines of
different biosynthetic capacities or after an addition of precursors; and
determining the transformation and over-expression of pathway genes
(Roberts et al., 1995, Bisaria & Panda 1991, Vasil 1991, Taticek et al., 1994,
Knauf, 1995, Hahn et al 1997, Prince et al., 1997). However, the design of
biochemical reaction networks requires identification of the critical branch
point(s) and determination of the exact enzyme(s) that must be modified.
Although molecular biology has provided the means to conduct precise
genetic changes, the difficulties with targeting specific enzymes are still the
limiting factor of faster progress in this area (Stephanopoulous, 1994).
Advances in molecular biology hold promise for new products and new
Production of secondary metabolites from plants
105
processes in plant biotechnology. Two major strategies for creating
transgenic plants have been devised: (1) genetic transformation of the plants
using Agrobacterium gene vectors or direct gene transfer by protoplast
fusion, microprojectile bombardment, or electroporation, and (2) genome
manipulation of plant pathogenic viruses. Genome manipulation is resulting
in relatively large amounts of desired compound produced by a plant infected
with an engineered virus, whereas transgenic plants can maintain constant
levels of production of proteins without additional intervention. Genetic
manipulation of plant cells poses several fundamental problems, including
the stability of inserted genes, the functional expression of the enzymes
involved, the localization of enzymes and products within the cellular
compartments, and the effects of an additional pathway on the growth and
physiology of plant cells. As a result, the productivity of only a few medicinal
plants has been increased in this way (Flores et al., 1987, Rhodes et al., 1990,
Wilson et al., 1990, Yun et al., 1992, Subroto et al., 1996).
Plant cell cultures can be designed to produce valuable therapeutic
proteins, including monoclonal antibodies, antigenic proteins that act as
immunogenes (edible subunit vaccines for rabies, cholera, hepatitis B,
malaria), human serum albumin, interferon, human enzyme for treating
Gaucher’s disease, immuno-contraceptive protein, ribosome inactivator
trichosantin,
antihypersensitive
drug
angiotensin,
leu-enkephalin
neuropeptide, and human hemoglobin (Hahn et al., 1997; Hiatt et al., 1989;
Manson & Arntzen et al., 1995; Wahl et al., 1995; Arntzen,1997; An &
Lee, 1997; Marden et al., 1997; Wongsamuth, 1997). Due to an increased
appeal of natural products for medicinal purposes, the metabolic engineering
can affect the production of pharmaceuticals and help design new therapies.
The candidate plant cell cultures are generally chosen by screening from
medicinal and aromatic plants already used in drug production. At present,
research and development are focused on plants producing substances with
immunomodulating, antiviral, antimicrobial, antiparasite, antitumor, antiinflammatory, hypoglycemic, tranquilizer and antifeedant activity (Yamada,
1991). The delivery of these drugs is often more complicated than that of
conventional drugs, necessitating gene therapies or controlled drug release
systemsbased on biodegradable polymer materials (Langer 1990; Langer &
Vacanti, 1993; Mulligan, 1993; Edwards et al., 1997). Advances in material
science, reactor design and integration of the existing and novel separation
processes into an overall processing scheme can further improve large scale
plant tissue engineering.
Current developments in tissue culture technology indicate that
transcription factors are efficient new molecular tools for plant metabolic
engineering to increase the production of valuable compounds (Gantet &
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Rahini. V
Memelink, 2002). In vitro cell culture offers an intrinsic advantage for
foreign protein synthesis in certain situations since they can be designed to
produce therapeutic proteins, including monoclonal antibodies, antigenic
proteins that act as immunogenes, human serum albumin, interferon,
immuno-contraceptive protein, ribosome unactivator trichosantin,
antihypersensitive drug angiotensin, leu-enkephalin neuropeptide, and human
hemoglobin (Doran, 2000). The appeal of using natural products for
medicinal purposes is increasing, and metabolic engineering can alter the
production of pharmaceuticals and help to design new therapies. At present,
researchers aim to produce substances with antitumor, antiviral,
hypoglycaemic, anti-inflammatory, antiparasitic, antimicrobial, tranquilizer
and immunomodulating activities through tissue culture technology.
Exploration of the biosynthetic capabilities of various cell cultures has been
carried out by a group of plant scientists and microbiologists in several
countries during the last decade. Most applications of plant-cell-suspension
cultures in biotechnology are aimed at the production of naturally occurring
secondary metabolites. This has included production of shikonin,
anthocyanins, and ajmalicine and, recently, important anti-tumor agents like
taxol, vinblastine and vincristine (Oksman-Caldentey & Inze, 2004). In the
last few years promising findings have been reported for a variety of
medicinally valuable substances, some of which may be produced on an
industrial scale in the near future. Today, the expression of recombinant anti
bodies and antibody fragments in plants is a well-established technique, and
the advantages of plants over bacterial or mammalian production systems
have been reviewed (Hiatt & Mostov, 1993).
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