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SECONDARY METABOLITIES
Introduction
• Secondary metabolites are organic compounds that are
not directly involved in the normal growth, development
or reproduction of a plant/an organism.
• A common role of secondary metabolites in plants is
defence mechanism i.e. used to fight from herbivores,
pests and pathogens.
• The potential is so great that the about 1500 new
molecules are reported from plants each year of which up
to 30% may exhibit some degree of biological activity.
• In humans or plants, secondary metabolites are of great
importance as they are used in many fields such as
medicine, flavouring agent, cosmetics, food additives,
antimicrobial, dyes, etc.
• It is believed that production of secondary
metabolites is linked to the induction of
morphological differentiation.
• Cultures are initiated by simply placing freshly
cut sections from surface sterilized plant organs
on a nutrient medium containing suitable
hormones.
• Generally auxins and cytokinins are used for
this purpose.
• On such a medium explants exhibit callusing
which usually starts at the cut ends and
gradually extends over the entire surface of
tissue.
SECONDARY METABOLITIES ARE
MAINLY CLASSIFIED INTO THREE MAIN
GROUPS
1.
• There are three ways for the production of
secondary metabolite by plant tissue culture
which are as follows:-
i) Cell suspension culture
ii) Hairy root culture
iii) Immobilized cell culture
Advantages
• Compounds can be produced under controlled
condition as per market demands
• Culture systems are independent of
environmental factors, seasonal variation, pest,
microbial diseases, geographical variations, etc.
• Cell growth can be controlled to provide
improved products formation.
• Quality of products will be consistent (remain
the same) as it is produced by a specific cell line.
• Recovery of the product will be easily
• Plant culture are particularly useful in case of
plant which are difficult to be grown in fields
• Mutants cells lines can be developed for the
production of novel compounds of commercial
important which are normally not found in
plants
• Production time is less and labour costs are
minimal
• 25-30% of medicines for human use and
various chemical materials are obtained from
plant tissue culture
Disadvantages
• Non-differentiation can produces little amount
of compounds
• Culture cells are genetically unstable & may
undergo mutation
• Vigorous stirring is necessary to prevent
aggregation of the cells. it may damage the cells.
• Aseptic condition is very important
• Any infection during the culture growth can
adversely effects production of secondary
metabolites.
Through CELL SUSPENSION CULTURES
• Mostly in production of secondary metabolite we
follows Cell suspension culture which is more
suitable and convenient technique to handle.
Various stages involved:
A) Selection of suitable cell & Callus formation:
• The first step in plant tissue culture is to develop a
callus culture from the whole plant. For this
purpose we should select a cell having ability to
generate a whole plant cells. Such cells are known
as totipotent cells. An undifferentiated mass of
cells on culture plate is known as callus. A callus
must be obtained from a totipotent dividing cells.
B) Development of cell suspension culture
• The important factor count in culturing cell is precise, good
and sterilized medium. A suspension culture is developed by
transferring small amount of a callus into liquid medium and
is maintained under suitable conditions of aeration, agitation,
light, temperature and other physical factors.
C) Mechanism/media components
• Cell suspension cultures components include, Fresh friable
fragments of callus into 50 ml of MS liquid media in 250 ml
Erlenmeyer flasks. Regulators includes kinetin (0.5, 1.4 or 2.3
μM) and 2, 4-D- 2,4-Dichlorophenoxyacetic acid (2.2, 4.4 or
6.6 μM), and their combinations Benzyl adenine
combinations with kinetin contained NAA (0.54 μM) in one
treatment. Macro and micro nutrients. Precursor feeding
exogenous supply of a biosynthetic precursor to culture
medium is applied to increase the yield of the desired
product.
• According to Roberts and Shuler 1997, elicitation, is
one of the most effective strategies for improving the
productivity of bioactive secondary metabolites.
Elicitors are signals triggering the formation of secondary
metabolites. Elicitors are molecules that stimulate any of
a number of defense responses in plants. Research over
the past decade has focused on the mechanisms by
which plant cells perceive and transduce these biological
signals to activate defense responses.
• Agitation and Suspension cultures is incubate on rotary
shaker (100 rpm) for agitation under the dark in the
growth room.
• Each treatment consisted of four completely randomized
replicates without sub samples and growth assessment
was performed by recording data every five days during
a 30 day period for cells Fresh Weight (FW).
Steps involved
• Appropriate nutrients, their concentrations and
environmental factors are known to enhance the
yield and productivity of metabolites in plant cell
suspension cultures.
• Optimum aeration-agitation condition with respect
to capacity of oxygen supply and intensity of
hydrodynamic stress effects on the plant cells.
• Control of temperature, pH and nutrient
concentration inside the bioreactor.
• Control of aggregate size (which may be important
to enhance secondary metabolite production).
• Maintenance of aseptic conditions for relatively
longer cultivation period.
(iii) Hairy root culture: plant tissue culture that is
used to study plant metabolic processes or to
produce secondary metabolites or
recombinant proteins, often with plant genetic
engineering. culture produced after the
infection of the explants or cultures by the
gram negative soil bacterium Agrobacterium
rhizogenes (contain Ri plasmids) can infect
plant roots and cause them to produce a food
source for the bacterium, opines and to grow
abnormally.
PRODUCTION (IN-VIVO)
• Agrobacterium recognizes some signal molecules
exuded by wounded plant cells and become
attached to it.
• The bacteria contain the root inducing plasmid
(Ri-plasmid).
• The bacteria genetically transfer the part of the
Riplasmid called the transfer DNA(t-DNA) to the
genome. Proliferate by increasing the rate of cell
division(cytokinin expression) and cell
elongation(auxin expression) to produce the
hairy roots.
• Production of the opines which is the type
unusual amino acids (octopine, mannopine
etc) which is used by the bacterium as a
carbon, nitrogen and energy source.
• Explants are wounded and then inoculated with
Agrobacterium rhizogenes.
• After 2 or 3 days, the explants can be transferred
into solid media with antibiotics such as
ceftoaxime, vancomycin etc to kill or eliminate
redundant (no longer needed) bacteria.
• The hairy roots will be induced within a short
period of time which varies from 1 week to over
a month varying on different plant species.
• The decontaminated hairy root cultures can be
sub-cultured on phyto-hormone free medium.
APPLICATIONS
• Functional analysis of genes.
• Expressing foreign proteins.
• Production of secondary metabolites.
• May change the composition of metabolites.
• Can be used to regenerate a whole plant.
• May produce compounds which is not found in
untransformed roots.
Eg.(i) Quinine for malaria.
(ii)Shikonin used for anti bacterial and anti ulcer
agent.
(iii)Berberine, etc.
Shikonin & its production
• Shikonin, a red naphthoquinone pigment or
derivative extracted from a traditional Japanese or
Chinese medical perennial herb (mainly from root
part), Lithospermum erythrorhizon (family-
Boraginaceae).
• It has been found to posses a variety of biological
activities including strong wound healing,
antibacterial, anti-inflammatory, and anti-tumor
effects.
• Production of a red pigment- (Shikonin)used for
flavors, fragrances, pigments, dyes, cosmetics and
food additives.
• The biolipstick, a lipstick with biological colorant
‘‘shikonin’’ was a big hit in the cosmetics market
in 1985. In spite of its high price (US $ 30 a stick),
2 million pieces (sticks) were sold within a few
days by Kanebo, Japanese Cosmetics Company.
• Shikonin was the first product produced from
cell cultures of Lithospermum erythrorhizon by
Mitsui Petrochemical Industries Ltd. (now
Mitsui Chemicals Inc.), Tokyo, Japan in 1984.
Why In-vitro production
• The production of these secondary metabolites
in its natural habitat is hampered due to poor
germination, non-availability of planting
material, over-exploitation and geographical
limitation as well as lack of support for R&D
work by the industries.
• Also, the plant requires three to four years for
noticeable production of these pigments
Production of shikonin
• In L. erythrorhizon , it is well documented that
shikonin is derived from two precursors
originating from different pathways.
• The aromatic precursor 4-hydroxybenzoic acid
(4HB) formed via the shikimate and the
phenylpropanoid pathway, while the isoprenoid
precursor, geranyldiphosphate (GPP) is derived
from the mevalonate pathway (Li et al., 1998).
• One of the key enzymes for the shikonin
biosynthesis of cells, p-hydroxylbenzoic acid
(PHB) geranyltransferase.
Factors effecting the production of
Shikonin
1. Optimization of Cultural Conditions:
1.1 Medium: These factors include media
components, phytohormones (growth
regulators), pH, temperature, aeration,
agitation, light, etc.
• IAA = Indole-3-acetic acid
NAA = 1-Naphthalene acetic acid
2,4-D = 2,4-Dichlorophenoxy acetic acid
Kin = Kinetin
BA = Benzyladenine
1.2 Temperature, pH, Light and Oxygen:
• The effects of temperature, pH, light and
oxygen are all parameters that must be
examined in the studies secondary
metabolites production.
• A temperature of 17- 25° C is normally used
for induction of callus tissues and growth of
cultured cells.
• The medium pH is usually adjusted to
between 5 and 6 before autoclaving and
extremes of pH are avoided.
1.3 High Cell Density Culture:
• To increase the productivity of secondary
metabolites, high cell density cultures have been
investigated. Using a newly designed fermentor
and optimized culture medium, cells were grown
up to 75 g/L of cell mass.
1.4 Absorption of Products:
addition of active charcoal in the medium
stimulated the yield, XAD-7.
1.5 Selection of High-Producing Strains:
The physiological characteristics of individual
plant cells are not always uniform. For example,
pigment producing cell aggregates typically
consist of producing cells and non-producing
cells.
1.6 Addition of Precursors:
• Addition to the culture media of appropriate
precursors or related compounds sometimes
stimulates secondary metabolite production.
• Phenylalanine is one of the biosynthetic
precursors of rosmarinic acid (67).
• Addition of this amino acid
to Salvia officialis suspension cultures
stimulated the production of rosmarinic acid
and shortened the production time as well.
1.7 Biotransformation:
• This approach has been extensively applied in
the fermentation industry using
microorganisms and their enzymes. For
example, L-aspartic acid and L-malic acid are
being manufactured commercially from
fumaric acid, respectively using
microorganisms.
• And various steroids are also produced by
microbial biotransformations.
1.8 Elicitor Treatment
• Elicitors are molecules that stimulate any of a
number of defence responses in plants.
Research over the past decade has focused on
the mechanisms by which plant cells perceive
and transduce these biological signals to
activate defence responses.
Protocol
• By systematically accessing of all the component
medium, two stages culture systems are involved.
1. In first stage: Cells of high producing strain M-18
were propagated in a growth medium MG-5
(Modified LS Medium) without producing shikonin
derivatives. The volume of the culture will be 200L
in small tanks and culture for 9 days.
2. In 2nd Stage:- The cells from the growth medium
are transferred to M-9 medium for shikonin
production. The components of LS & M-9 medium
are more suitable for shikonin production. The
shikonin yield also increased when both the initial
cell density and medium concentration were
doubled. The volume of the culture will be 750L in
large tanks and culture for 14 days.
• The concentration of dissolved oxygen is also vital. It
should be adjusted to 6.4 and 6.0 ppm for the first
and the second culture stage.
• Cells of the stock culture are grown in a jar
fermenters are inoculated in the first stage tank (200
L) with MG-5 medium for cell growth and incubated
for 9 days and subsequently transferred to filtering
apparatus.
• The production medium M-9 is poured into the tank
and cell in this medium are pumped into 2nd tank
(750 L) for shikonin production.
• After incubating the cells for 14 days in the 2nd tank,
the pigmented cells are harvested by filtering off the
medium.
• The extracted and purification can be carried by
treating the cells with n-hexane from the
cultured cells and subsequently hydrolysis with
2% KOH and recrystallized to give a pure
shikonin.
FACTORS INFLUENCING SHIKONIN
BIOSYNTHESIS
• Temperature: It should be between 20–25 ˚C is
favourable for shikonin production in callus cultures.
The production must be carried out in the dark.
• Growth hormones – The synthetic auxins 2,4-D & 1-
nepthalacetic acid are highly inhibitory to the
biosynthesis of shikonin derivatives. Contrarily, the
natural auxin- indole acetic acid (IAA) not only
promotes the growth, but also induces shikonin
synthesis.
• Carbon Source – sucrose 5% in LS medium is the best
sucrose for maintaining the callus cultures. (Glucose,
fructose, maltose or lactose inhibits the production).
• Nitrogen Source: it is used in the LS medium and should
contain 20 mM NH4+ 40 mM NO3- . Any variation in the
total nitrogen tends to inhibit the shikonin in callus
cultures. Inclusion of L-phenylalanine (10-4M) as a
precursor increases the level of products.
• Rosmarinic acid – The endogenous level of rosmarinic acid
is important in the cellular control of shikonin synthesis.
• Polysaccharides – shikonin production can be induced in
cell suspension culture by adding agar powder to the LS
medium.
• Ammonium tends to inhibits shikonin synthesis in the
absence of agar. Higher concentration of Ca2+, NO3- ,
Fe2+, BO33- , SO42- , and sucrose increases the shikonin
yield.
Application
• The annual market value of vincristine, vinblastine
and are ranged about $1,000,000–3,500,000 per kg.
(Source-https://www.thieme-
connect.com/products/ejournals/abstract/10.1055/
s-0036-1578706)
• In vitro production of secondary metabolites by cell
and tissue culture systems could be helpful in
understanding the biosynthesis of these
compounds.
• Azadirachtin from Azadirachta indica- Insecticidal
• Berberine from Coptis japonica- Antibacterial, anti-
inflammatory, anti-cancer
• Capsaicin from Capsicum annum cures rheumatic
pain, etc.

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Secondary metabolities, Shikonin production

  • 2. Introduction • Secondary metabolites are organic compounds that are not directly involved in the normal growth, development or reproduction of a plant/an organism. • A common role of secondary metabolites in plants is defence mechanism i.e. used to fight from herbivores, pests and pathogens. • The potential is so great that the about 1500 new molecules are reported from plants each year of which up to 30% may exhibit some degree of biological activity. • In humans or plants, secondary metabolites are of great importance as they are used in many fields such as medicine, flavouring agent, cosmetics, food additives, antimicrobial, dyes, etc.
  • 3. • It is believed that production of secondary metabolites is linked to the induction of morphological differentiation. • Cultures are initiated by simply placing freshly cut sections from surface sterilized plant organs on a nutrient medium containing suitable hormones. • Generally auxins and cytokinins are used for this purpose. • On such a medium explants exhibit callusing which usually starts at the cut ends and gradually extends over the entire surface of tissue.
  • 4.
  • 5. SECONDARY METABOLITIES ARE MAINLY CLASSIFIED INTO THREE MAIN GROUPS 1.
  • 6.
  • 7.
  • 8.
  • 9.
  • 10.
  • 11.
  • 12. • There are three ways for the production of secondary metabolite by plant tissue culture which are as follows:- i) Cell suspension culture ii) Hairy root culture iii) Immobilized cell culture
  • 13. Advantages • Compounds can be produced under controlled condition as per market demands • Culture systems are independent of environmental factors, seasonal variation, pest, microbial diseases, geographical variations, etc. • Cell growth can be controlled to provide improved products formation. • Quality of products will be consistent (remain the same) as it is produced by a specific cell line. • Recovery of the product will be easily
  • 14. • Plant culture are particularly useful in case of plant which are difficult to be grown in fields • Mutants cells lines can be developed for the production of novel compounds of commercial important which are normally not found in plants • Production time is less and labour costs are minimal • 25-30% of medicines for human use and various chemical materials are obtained from plant tissue culture
  • 15. Disadvantages • Non-differentiation can produces little amount of compounds • Culture cells are genetically unstable & may undergo mutation • Vigorous stirring is necessary to prevent aggregation of the cells. it may damage the cells. • Aseptic condition is very important • Any infection during the culture growth can adversely effects production of secondary metabolites.
  • 16.
  • 17. Through CELL SUSPENSION CULTURES • Mostly in production of secondary metabolite we follows Cell suspension culture which is more suitable and convenient technique to handle. Various stages involved: A) Selection of suitable cell & Callus formation: • The first step in plant tissue culture is to develop a callus culture from the whole plant. For this purpose we should select a cell having ability to generate a whole plant cells. Such cells are known as totipotent cells. An undifferentiated mass of cells on culture plate is known as callus. A callus must be obtained from a totipotent dividing cells.
  • 18. B) Development of cell suspension culture • The important factor count in culturing cell is precise, good and sterilized medium. A suspension culture is developed by transferring small amount of a callus into liquid medium and is maintained under suitable conditions of aeration, agitation, light, temperature and other physical factors. C) Mechanism/media components • Cell suspension cultures components include, Fresh friable fragments of callus into 50 ml of MS liquid media in 250 ml Erlenmeyer flasks. Regulators includes kinetin (0.5, 1.4 or 2.3 μM) and 2, 4-D- 2,4-Dichlorophenoxyacetic acid (2.2, 4.4 or 6.6 μM), and their combinations Benzyl adenine combinations with kinetin contained NAA (0.54 μM) in one treatment. Macro and micro nutrients. Precursor feeding exogenous supply of a biosynthetic precursor to culture medium is applied to increase the yield of the desired product.
  • 19. • According to Roberts and Shuler 1997, elicitation, is one of the most effective strategies for improving the productivity of bioactive secondary metabolites. Elicitors are signals triggering the formation of secondary metabolites. Elicitors are molecules that stimulate any of a number of defense responses in plants. Research over the past decade has focused on the mechanisms by which plant cells perceive and transduce these biological signals to activate defense responses. • Agitation and Suspension cultures is incubate on rotary shaker (100 rpm) for agitation under the dark in the growth room. • Each treatment consisted of four completely randomized replicates without sub samples and growth assessment was performed by recording data every five days during a 30 day period for cells Fresh Weight (FW).
  • 20.
  • 22. • Appropriate nutrients, their concentrations and environmental factors are known to enhance the yield and productivity of metabolites in plant cell suspension cultures. • Optimum aeration-agitation condition with respect to capacity of oxygen supply and intensity of hydrodynamic stress effects on the plant cells. • Control of temperature, pH and nutrient concentration inside the bioreactor. • Control of aggregate size (which may be important to enhance secondary metabolite production). • Maintenance of aseptic conditions for relatively longer cultivation period.
  • 23.
  • 24. (iii) Hairy root culture: plant tissue culture that is used to study plant metabolic processes or to produce secondary metabolites or recombinant proteins, often with plant genetic engineering. culture produced after the infection of the explants or cultures by the gram negative soil bacterium Agrobacterium rhizogenes (contain Ri plasmids) can infect plant roots and cause them to produce a food source for the bacterium, opines and to grow abnormally.
  • 25. PRODUCTION (IN-VIVO) • Agrobacterium recognizes some signal molecules exuded by wounded plant cells and become attached to it. • The bacteria contain the root inducing plasmid (Ri-plasmid). • The bacteria genetically transfer the part of the Riplasmid called the transfer DNA(t-DNA) to the genome. Proliferate by increasing the rate of cell division(cytokinin expression) and cell elongation(auxin expression) to produce the hairy roots.
  • 26.
  • 27. • Production of the opines which is the type unusual amino acids (octopine, mannopine etc) which is used by the bacterium as a carbon, nitrogen and energy source.
  • 28. • Explants are wounded and then inoculated with Agrobacterium rhizogenes. • After 2 or 3 days, the explants can be transferred into solid media with antibiotics such as ceftoaxime, vancomycin etc to kill or eliminate redundant (no longer needed) bacteria. • The hairy roots will be induced within a short period of time which varies from 1 week to over a month varying on different plant species. • The decontaminated hairy root cultures can be sub-cultured on phyto-hormone free medium.
  • 29.
  • 30. APPLICATIONS • Functional analysis of genes. • Expressing foreign proteins. • Production of secondary metabolites. • May change the composition of metabolites. • Can be used to regenerate a whole plant. • May produce compounds which is not found in untransformed roots. Eg.(i) Quinine for malaria. (ii)Shikonin used for anti bacterial and anti ulcer agent. (iii)Berberine, etc.
  • 31.
  • 32.
  • 33.
  • 34.
  • 35.
  • 36. Shikonin & its production • Shikonin, a red naphthoquinone pigment or derivative extracted from a traditional Japanese or Chinese medical perennial herb (mainly from root part), Lithospermum erythrorhizon (family- Boraginaceae). • It has been found to posses a variety of biological activities including strong wound healing, antibacterial, anti-inflammatory, and anti-tumor effects. • Production of a red pigment- (Shikonin)used for flavors, fragrances, pigments, dyes, cosmetics and food additives.
  • 37.
  • 38.
  • 39.
  • 40.
  • 41. • The biolipstick, a lipstick with biological colorant ‘‘shikonin’’ was a big hit in the cosmetics market in 1985. In spite of its high price (US $ 30 a stick), 2 million pieces (sticks) were sold within a few days by Kanebo, Japanese Cosmetics Company. • Shikonin was the first product produced from cell cultures of Lithospermum erythrorhizon by Mitsui Petrochemical Industries Ltd. (now Mitsui Chemicals Inc.), Tokyo, Japan in 1984.
  • 42. Why In-vitro production • The production of these secondary metabolites in its natural habitat is hampered due to poor germination, non-availability of planting material, over-exploitation and geographical limitation as well as lack of support for R&D work by the industries. • Also, the plant requires three to four years for noticeable production of these pigments
  • 43. Production of shikonin • In L. erythrorhizon , it is well documented that shikonin is derived from two precursors originating from different pathways. • The aromatic precursor 4-hydroxybenzoic acid (4HB) formed via the shikimate and the phenylpropanoid pathway, while the isoprenoid precursor, geranyldiphosphate (GPP) is derived from the mevalonate pathway (Li et al., 1998). • One of the key enzymes for the shikonin biosynthesis of cells, p-hydroxylbenzoic acid (PHB) geranyltransferase.
  • 44. Factors effecting the production of Shikonin 1. Optimization of Cultural Conditions: 1.1 Medium: These factors include media components, phytohormones (growth regulators), pH, temperature, aeration, agitation, light, etc.
  • 45. • IAA = Indole-3-acetic acid NAA = 1-Naphthalene acetic acid 2,4-D = 2,4-Dichlorophenoxy acetic acid Kin = Kinetin BA = Benzyladenine
  • 46. 1.2 Temperature, pH, Light and Oxygen: • The effects of temperature, pH, light and oxygen are all parameters that must be examined in the studies secondary metabolites production. • A temperature of 17- 25° C is normally used for induction of callus tissues and growth of cultured cells. • The medium pH is usually adjusted to between 5 and 6 before autoclaving and extremes of pH are avoided.
  • 47. 1.3 High Cell Density Culture: • To increase the productivity of secondary metabolites, high cell density cultures have been investigated. Using a newly designed fermentor and optimized culture medium, cells were grown up to 75 g/L of cell mass. 1.4 Absorption of Products: addition of active charcoal in the medium stimulated the yield, XAD-7. 1.5 Selection of High-Producing Strains: The physiological characteristics of individual plant cells are not always uniform. For example, pigment producing cell aggregates typically consist of producing cells and non-producing cells.
  • 48. 1.6 Addition of Precursors: • Addition to the culture media of appropriate precursors or related compounds sometimes stimulates secondary metabolite production. • Phenylalanine is one of the biosynthetic precursors of rosmarinic acid (67). • Addition of this amino acid to Salvia officialis suspension cultures stimulated the production of rosmarinic acid and shortened the production time as well.
  • 49. 1.7 Biotransformation: • This approach has been extensively applied in the fermentation industry using microorganisms and their enzymes. For example, L-aspartic acid and L-malic acid are being manufactured commercially from fumaric acid, respectively using microorganisms. • And various steroids are also produced by microbial biotransformations.
  • 50. 1.8 Elicitor Treatment • Elicitors are molecules that stimulate any of a number of defence responses in plants. Research over the past decade has focused on the mechanisms by which plant cells perceive and transduce these biological signals to activate defence responses.
  • 51. Protocol • By systematically accessing of all the component medium, two stages culture systems are involved. 1. In first stage: Cells of high producing strain M-18 were propagated in a growth medium MG-5 (Modified LS Medium) without producing shikonin derivatives. The volume of the culture will be 200L in small tanks and culture for 9 days. 2. In 2nd Stage:- The cells from the growth medium are transferred to M-9 medium for shikonin production. The components of LS & M-9 medium are more suitable for shikonin production. The shikonin yield also increased when both the initial cell density and medium concentration were doubled. The volume of the culture will be 750L in large tanks and culture for 14 days.
  • 52. • The concentration of dissolved oxygen is also vital. It should be adjusted to 6.4 and 6.0 ppm for the first and the second culture stage. • Cells of the stock culture are grown in a jar fermenters are inoculated in the first stage tank (200 L) with MG-5 medium for cell growth and incubated for 9 days and subsequently transferred to filtering apparatus. • The production medium M-9 is poured into the tank and cell in this medium are pumped into 2nd tank (750 L) for shikonin production. • After incubating the cells for 14 days in the 2nd tank, the pigmented cells are harvested by filtering off the medium.
  • 53. • The extracted and purification can be carried by treating the cells with n-hexane from the cultured cells and subsequently hydrolysis with 2% KOH and recrystallized to give a pure shikonin.
  • 54.
  • 55. FACTORS INFLUENCING SHIKONIN BIOSYNTHESIS • Temperature: It should be between 20–25 ˚C is favourable for shikonin production in callus cultures. The production must be carried out in the dark. • Growth hormones – The synthetic auxins 2,4-D & 1- nepthalacetic acid are highly inhibitory to the biosynthesis of shikonin derivatives. Contrarily, the natural auxin- indole acetic acid (IAA) not only promotes the growth, but also induces shikonin synthesis. • Carbon Source – sucrose 5% in LS medium is the best sucrose for maintaining the callus cultures. (Glucose, fructose, maltose or lactose inhibits the production).
  • 56. • Nitrogen Source: it is used in the LS medium and should contain 20 mM NH4+ 40 mM NO3- . Any variation in the total nitrogen tends to inhibit the shikonin in callus cultures. Inclusion of L-phenylalanine (10-4M) as a precursor increases the level of products. • Rosmarinic acid – The endogenous level of rosmarinic acid is important in the cellular control of shikonin synthesis. • Polysaccharides – shikonin production can be induced in cell suspension culture by adding agar powder to the LS medium. • Ammonium tends to inhibits shikonin synthesis in the absence of agar. Higher concentration of Ca2+, NO3- , Fe2+, BO33- , SO42- , and sucrose increases the shikonin yield.
  • 57.
  • 58.
  • 59.
  • 61. • The annual market value of vincristine, vinblastine and are ranged about $1,000,000–3,500,000 per kg. (Source-https://www.thieme- connect.com/products/ejournals/abstract/10.1055/ s-0036-1578706) • In vitro production of secondary metabolites by cell and tissue culture systems could be helpful in understanding the biosynthesis of these compounds. • Azadirachtin from Azadirachta indica- Insecticidal • Berberine from Coptis japonica- Antibacterial, anti- inflammatory, anti-cancer • Capsaicin from Capsicum annum cures rheumatic pain, etc.