biotechnological approaches to the production of shikonins: a critical review with recent updates

http://informahealthcare.com/btyISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, Early Online: 1–14! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.961003

REVIEW ARTICLE

Biotechnological approaches to the production of shikonins: a criticalreview with recent updates

Sonia Malik1,2, Shashi Bhushan1, Madhu Sharma1, and Paramvir Singh Ahuja1

1Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India and 2Department of Chemical

Biology and Genetics, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University, Olomouc, Czech Republic

Abstract

Shikonins are commercially important secondary compounds, known for array of biologicalactivities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds areusually colored and therefore have application in food, textiles and cosmetics. Shikonin and itsderivatives, which are commercially most important of the naphthoquinone pigments, aredistributed among members of the family Boraginaceae. These include different species ofLithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant-based natural products has made this group of compounds one of the enthralling targets fortheir in vitro production. The aim of this review is to highlight the recent progress in productionof shikonins by various biotechnological means. Different methods of increasing the levels ofshikonins in plant cells such as selection of cell lines, optimization of culture conditions,elicitation, in situ product removal, genetic transformation and metabolic engineering arediscussed. The experience of different researchers working worldwide on this aspect is alsoconsidered. Further, to meet market demand, the needs for continuous and reliable productionsystems, as well as future prospects, are included.

Keywords

Alkannin, boraginaceae, cell culture,in vitro culture, naphthoquinones, naturalproducts, pigments, secondary metabolites

History

Received 21 December 2013Revised 16 July 2014Accepted 23 July 2014Published online 9 October 2014

Introduction

Plants are capable of synthesizing a bewildering array of

chemical compounds, which are used as pharmaceuticals,

agrochemicals, flavors, fragrances, pigments, dyes, cosmetics

and food additives (Lubbe & Verpoorte, 2011). These

chemical compounds, commonly known as secondary metab-

olites or natural products are heterogeneous compounds,

generally not only required for normal growth and develop-

ment of plants but increase the plant’s ability to survive and

overcome local challenges by allowing them to interact with

their environment (Harborne, 1993). These are produced

during specific developmental stages of plant or under

specific (seasonal, stress or nutritional) conditions and their

production levels are often low (Malik et al., 2009, 2010a,

2013a; Verpoorte, 2000).

Secondary metabolites of plant origin are in demand due to

a resurgence of public interest in plant-based products. Every

year, the market for plant-based products is increasing at a

rate of 12–15% (Raskin et al., 2002). Table 1 summarizes the

source of plant species for various secondary metabolites,

their economic use and market value.

Secondary metabolites are mainly classified into three

main groups: polyphenols, alkaloids and terpenes.

Naphthoquinones (belong to a class of polyphenols formed

on a C6–C4 skeleton) have developed great interest due to

their broad range of activities (Babula et al., 2009). These are

widespread in nature as secondary metabolites of fungi,

micro-organisms as well as plants. Naphthoquinones which

include shikonin, alkannin and their derivatives, juglans and

many more present a larger group of quinone pigments

(Figure 1). The molecular formula of naphthoquinone is

C10H6O2. These metabolites range from simple structures

such as 5-hydroxy derivative juglone, to pigments with

isoprenyl attachment such as alkannin. Shikonin, and its

derivatives, are commercially the most important of the

naphthoquinone pigments and this review is focused on

the production of shikonin and their derivatives in plants. The

main objective of this review is to highlight the reproducible

methods for in vitro production of shikonins and to illustrate

the different production systems for these important classes

of compounds.

Shikonins: their occurrence, distribution andeconomic value

Shikonin and its derivatives are the commercially most

important of the napthoquinone pigments, distributed among

members of the family Boraginaceae. These include different

species of Lithospermum, Arnebia, Alkanna, Anchusa,

Address for correspondence: Sonia Malik, Department of ChemicalBiology and Genetics, Centre of the Region Hana for Biotechnologicaland Agricultural Research, Palacky University, Slechtitelu 11, 783 71Olomouc, Czech Republic. Tel:+420 585 632 173. Fax: +420 585 634870. E-mail: [email protected]

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Echium and Onosma. Shikonin, alkannin and their derivatives

are known to possess a wide range of pharmaceutical

properties and are also used as natural dyes for coloring

silk, cosmetics, as well as food additives (Andujar et al.,

2013; Babula et al., 2009; Lu et al., 2013).

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. The idea was that shikonin-containing

lipstick is not only biologically prepared but it also protects

against bacteria and has other anti-microbial activities

(Renneberg, 2008).

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 (DiCosmo & Misawa, 1996). 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. In this context, the in vitro production of commer-

cially important secondary metabolites by various biotechno-

logical means offers an attractive proposition (Malik et al.,

2013b; Rao & Ravishankar, 2002).

Biosynthesis of shikonins

In L. erythrorhizon, it is well documented that shikonin is

derived from two precursors originating from different

pathways (Figure 2). The aromatic precursor 4-hydroxyben-

zoic acid (4HB) formed via the shikimate and the phenyl-

propanoid pathway, while the isoprenoid precursor,

geranyldiphosphate (GPP) is derived from the mevalonate

pathway (Li et al., 1998). The 4HB geranyltransferase

reaction links the aromatic precursor 4HB to the isoprenoid

precursor GPP and yields 3-geranyl-4-hydroxybenzoate

(GBA), which is the first specific intermediate in shikonin

biosynthesis (Heide & Tabata, 1987b).

It has been reported that hydroxyl-3-methylglutaryl-

coenzymeA reductase (HMGR) and 4HB geranyltransferase

Table 1. Useful secondary metabolites from plants, their uses and market value.

Plant species Family Secondary product Economic use Price* (US$/Kg)

PharmaceuticalsRauwolfia serpentina Apocynaceae Ajmaline Sedative 75 000Artemisia annua Asteraceae Artemisinin Anti-malarial 400Atropa belladonna Solanaceae Atropine Anti-cholinergic n/aCatharanthus roseus Apocynaceae Ajmalicine Anticancer 37 000

Vinblastine Anticancer 1 000 000Vincristine Anti-leukemic 3 000 000

Coptis japonica Ranunculaceae Berberine Antiseptic 3250Papaver somniferum Papaveraceae Codeine Sedative 17 000

Morphine Analgesic 340 000Colchicum speciosum Colchicaceae Colchicine Antitumor 35 000Digitalis lanata Scrophulariaceae Digoxin Cardiatonic 3000Dioscorea deltoidea Dioscoreaceae Diosgenin Steroid, anti-fertility agent 1000Orchrosia elliptica Apocynaceae Ellipticine Antitumor 240 000Podophyllum sp., Linum sp. Berberidaceae Podophyllotoxin Viral diseases and skin cancer 800 000Cinchona ledgeriana Rubiaceae Quinine Anti-malarial 500Sanguinaria canadensis Papaveraceae Sanguinarine Antibiotic, expectorant, analgesic 4800Datura stramonium Solanaceae Scopolamine Anti-cholinergic n/a

PigmentsMorinda citrifolia Polygonaceae Anthraquinones Natural dye, purgative, cathartic n/aLithospermum erythrorhizon,

Arnebia sp.Boraginaceae Shikonin derivatives Cosmetics, natural dye,

anti-cancer, anti-bacterial4500

AgrochemicalsAzadirachta indica Meliaceae Azadirachtin Insecticide n/aJuniperus viriginiana Cupressaceae Cederene Repellant n/aNicotiana tabaccum Solanaceae Nicotine Insecticide n/aChrysanthemumcinerariaefolium

Asteraceae Pyrethrins, Pyretheroids Insecticide 413

Flavors and fragrancesCapsicum frutescens Solanaceae Capsicin Condiment n/aCymbopogon winterianus Gramineae Citronellal Food flavor n/aCrocus sativus Iridaceae Crocin Saffron n/aJasminum spp. Oleaceae Jasmine (oil) Perfume 350Citrus sp. Rutaceae Limonene Food flavor n/aMentha sp. Labiatae Menthol Food flavor n/aVanilla planifolia Orchidaceae Vanillin Flavor 1000

n/a, not available.

Figure 1. Chemical structure of Naphthoquinone.

2 S. Malik et al. Crit Rev Biotechnol, Early Online: 1–14

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are the important enzymes for regulation of shikonin biosyn-

thesis (Gaisser & Heide, 1996; Heide et al., 1989; Lange

et al., 1998). Srinivasan & Ryu (1992) observed high

activities of the biosynthetic enzymes, phenylalanine ammo-

nia lyase (PAL) and 3-hydroxyl-3-methylglutaryl-CoA-reduc-

tase (HMGR) in shikonin producing cells of L. erythrorhizon.

Biotechnological approaches for the productionof shikonins

Biotechnological advancements provide us the opportunity to

make use of cells, tissue or organs of economically important

plants by growing them under in vitro conditions and to

genetically manipulate them to obtain the desired compounds.

Further, in vitro cultures could supersede the extraction of

valuable compounds from wild or cultivated plants (Kumar

et al., 2011; Malik et al., 2011a, 2014; Ravishankar & Rao,

2002).

In vitro production of secondary metabolites by cell and

tissue culture systems could be helpful in understanding the

biosynthesis of these compounds. Table 2 summarizes the

commercial or semi-industrial production of secondary com-

pounds from different plant species by in vitro means.

In vitro production of shikonin and its derivatives

Micropropagation and regeneration in shikoninbearing plants

Plant cells or tissues exhibit totipotency under appropriate

conditions and this potential can be exploited by the selection

of suitable media and culture conditions. Ji & Wang (2001)

obtained direct regeneration from excised leaves of Arnebia

euchroma on a modified MS (Murashige & Skoog, 1962)

medium with 0.4 mg/l Naphthalene Acetic Acid (NAA) and

1.5 mg/l Kinetin (Kn). In another report, Jiang et al. (2005)

used Thidiazuron (TDZ, 4.5 lM) for direct regeneration from

cotyledons and hypocotyls in same plant species. The

importance of pre-culture time and concentration of TDZ

for direct regeneration from in vitro leaves (attached to

shoots) of A. euchroma was studied by our group (Malik

et al., 2010b). Direct regeneration could be obtained from

shoots, which were initially pre-cultured for 40 days on

medium having higher TDZ concentration (20.0 lM) followed

by transfer to lower concentration (5.0 lM; Figure 3a–c). The

identity of shoot buds was confirmed by histological studies

(Figure 3d–f). Regenerated shoots were cultured on Kn

(5.0 lM) containing media for proliferation and rooted

plantlets were transferred to greenhouse (Figure 3g and h).

The mode of regeneration depends mainly on the species,

type of organ used and the level of growth regulating

substances in the medium. Manjkhola et al. (2005) achieved

shoot regeneration and somatic embryogenesis from leaf

derived callus on MS medium supplemented with 2.5 mM

Benzylaminopurine (BAP) + 2.5 mM Indole-3-butyric acid

(IBA) and 2.5 mM BAP + 1.0 mM IBA, respectively, while

root derived callus did not show any sign of organogenesis.

Later on, Ji & Wang (2001) reported regeneration from bud

derived callus from the same plant species after 3 months

culture on Linsmaier & Skoog (LS) (1965) medium contain-

ing NAA in combination with Kn or BAP.

Figure 2. Shikonin biosynthesis pathway(Singh et al., 2010).

Glucose Glyceraldehyde 3-phosphate

Chorismic acid

Phenylalanine

Coumaric acid

Dimethylallyl pyrophosphate

Cinnamic acid

p-hydroxybenzoic acid

SHIKIMATE PATHWAY

Diphosphomevalonate Decarboxylase

Deoxyshikonin Shikonin

m-geranyl-p-hydroxybenzoic acid

m-geranylhydroxy quinone

Mevalonate pyrophosphate

HMGCoA synthase

Pyruvate

Acetoacetyl CoA

3-Hydroxy 3-methylglutaryl CoA

Mevalonate

Mevalonate phosphate

Isopentenyl pyrophosphate

Geranylpyrophosphate

Mevalonate kinase

4-Coumaroyl Co A ligase IPP Isomerase

Acetoacetyl CoA

PHB geranyl transferase

Phenylalanine ammonia lyase

Phosphomevalonate kinase

Acetyl CoA

HMG CoA reductase

Geranyl diphosphate synthase

MEVALONATE PATHWAY

Cinnamic acid-4 hydroxylase

DOI: 10.3109/07388551.2014.961003 Biotechnological approaches to the production of shikonins 3

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Tissue and cell culture

The first successful attempt for in vitro production of shikonin

derivatives was made from callus cultures of L. erythrorhizon

by Tabata et al. (1974). The induction, growth and color of

callus depend upon the type of explant used and presence of

plant growth regulators (PGRs). In A. euchroma, friable callus

was obtained from in vitro leaves within 20–25 days of culture

on MS medium fortified with 10.0 mM BAP and 5.0 mM IBA

(Figure 4).

Tabata et al. (1974) observed that callus culture derived

from seedlings of L. erythrorhizon accumulated shikonin

derivatives on LS medium supplemented with IAA (1.0 mM)

and Kn (10.0mM). The derivatives were similar to that of root

bark of the plant. Exposure to light and presence of 2,4-D in

the medium suppressed the synthesis of these compounds.

Callus cultures of L. erythrorhizon were able to produce

shikonin derivatives in agar gelled LS media but cell

suspension cultures in the liquid medium of same compos-

ition did not show the production of these derivatives (Fujita

et al., 1981a).

It has been reported that production of shikonin derivatives

from cells of L. erythrorhizon is affected by the supply of

nutrients, availability of oxygen and the presence of agar in

culture medium (Hara et al., 1987). Fujita et al. (1981b)

established the cell suspension cultures of L. erythrorhizon in

a modified WH medium with the possibility of large scale

production of these compounds under in vitro conditions. The

cell suspension cultures were shown to produce all the

shikonin derivatives as found in the roots of the intact plant

(Fujita et al., 1983). The annual consumption of shikonin

derivatives is 150 kg (Nosov, 2012). To meet the demand of

shikonin derivatives in Japan, almost 10 000 kg roots of L.

erythrorhizon are used annually. This plant is almost extinct

in Japan due to uninhibited collection from the wild (Fujita,

1988b). Even cultivation of these plants failed to provide

sufficient raw materials for commercial production of this

valuable product. Moreover, shikonin derivatives reached

only a maximum of 1–2% in their roots after 5–7 years growth

(Fujita, 1988a,b). Therefore, Japan had to import ten metric

tons of raw material (valued app. 4500 US$ per kg) annually

from South Korea and China. However, supply of plant

material from countries other than Japan could not meet the

increasing demand of this valuable product due to geographic

reasons as well as the high price associated with import of the

plants (Renneberg, 2008). Hence, Japanese researchers

focused on in vitro production of these compounds. Mitsui

Chemicals Inc., Japan became successful in producing

shikonin from cell culture and presented the first example

of commercial production of a secondary metabolite from cell

culture. Chemical synthesis of shikonin is not economically

Table 2. Commercial and emerging/semi-emerging large-scale in vitro production of plant secondary metabolites.

Bioreactor

Product Plant species Type Max. Volume (L) Producer(s) Country

Anthocyanins (c) Euphorbia milliiAralia cordata

Rotary culture system – Nippon Paint Company Japan

Arbutin (c) Catharanthus roseus – – Mitsui PetrochemicalIndustries

Japan

Berberine (c) Thalictrum minus,Coptis japonica

Batch and continuousflow; impeller driven

4000 Mitsui PetrochemicalIndustries Ltd.

Japan

Betacyanins (c) Beta vulgaris – – Nippon Shinyaku JapanCamptothecin (h) Camptotheca acuminate – – ROOTec bioactives GmbH SwitzerlandCarthamin (c) Carthamus tinctorius – – Kibun JapanDigoxin (c) Digitalis lanata Airlift system 300 Boehringer GermanyGinsenosides (c, r) Panax ginseng – 20 000 Nitto Denko Corp.

CBN BiotechJapanRepublic of Korea

ImmunostimulatingPolysaccharides (c)

Echinacea purpurea,E. auguslifolia

– – Diversa Germany

Podophyllotoxin (c,h) Podophyllum sp. – – Nippon OilROOTec bioactives GmbH

JapanSwitzerland

Purpurin (c) Rubia akane – – Mitsui PetrochemicalIndustries Ltd., NittoDenko Corp.

Japan

Rosmarinic acid (c) Coleus blumei – – Nattermann GermanySanguinarine (c) Papaver somniferum Airlift system 300 Vipont Research Labs USAScopolamine (c) Duboisia sp. – – Sumitomo Chemical

IndustriesJapan

Shikonin (c) Lithospermum erythrorhizon Batch 750 Mitsui PetrochemicalIndustries Ltd.

Japan

Taxol (Paclitaxel) (c) Taxus brevifolia,T. cuspidata

Impeller driven and air-lift reactor systems

75 000 ESCAgenetics Phyton Inc.Nippon Oil CompanySamyang, Genex Co. Ltd.Bristol-Myers Squibb Co.

USAJapanKoreaGermany

Vanillin(Phyto vanilla) (c)

Vanilla planifolia Impeller driven reactor 72 ESCAgenetics USA

Large-scale commercial production of secondary compounds from in vitro culture is recognized for shikonin, ginsenosides, berberine and taxol. c: cellculture; r: root culture; h: hairy root culture.

Data collected from Malik et al. (2011a) and Nosov (2012).


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feasible as it involves twelve step reactions and the final yield

is only 0.7% (Terada et al., 1983). In order to find an

alternative plant source to meet the still emerging demand

of this product, other members of the family Boraginaceae

were also exploited for their potential to produce shikonin

derivatives.

Pietrosiuk et al. (1999) isolated shikonin derivatives and

pyrrolizidine alkaloids from callus and cell suspension

cultures of A. euchroma. The presence of acetylshikonin

and b-acetoxyisovalerylshikonin in cell suspension cultures of

A. euchroma has been reported by our group (Sharma et al.,

2008). The cell culture of A. hispidissima was found to

produce arnebins (Jain et al., 1999). There are reports on the

production of shikonin derivatives in callus cultures of

Echium lycopsis (Fukui et al., 1983; Inouye et al., 1981).

Isolation procedures for isohexenylnaphthazarin compounds

from callus and cell suspension cultures of A. euchroma have

been demonstrated by Damianakos et al. (2012).

Methods for improving productivity

Although plant cells are capable of producing secondary

metabolites under in vitro conditions, until now only a few

plant metabolites have been commercialized or reached at

industrial scale through cell culture. In order to improve the

yield for commercial exploitation, efforts were focused on

Figure 3. Regeneration in A. euchroma. (a) Shoot cultures with vitrified leaves (arrow marked) on 20.0 lM TDZ supplemented medium. (b) Shoot budregeneration (arrow marked) from intact leaves (attached to shoots) on 5.0 lM TDZ. (c) Growth of shoot buds on 5.0 lM TDZ. (d) A part of swollenleaf with protuberances (arrow marked) and scattered vascular bundles. (e) Initiation of shoot bud (continuation of vascular strand with the motherexplant). (f) Shoot bud showing shoot apex (SA), subtending leaves (SL) and vascular strand (VS) in continuity with the mother explant (arrowmarked). (g) Rooted plantlet on 0.25mM IBA after 20 days. (h) Hardened plants under greenhouse conditions. Reprinted from Malik et al. (2010b)Copyright (2010) International Federation for Cell Biology, with permission from John Wiley and Sons.

Figure 4. A. euchroma callus (a) induction and (b) proliferation on MSmedium with BAP (10.0 mM) + IBA (5.0mM).


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isolating the biosynthetic activities of cultured cells, by

selecting high yielding cell lines, optimizing the nutrient

media and the culture conditions (Dornenburg & Knorr,

1995).

Selection of high yielding cell lines

Several techniques have been employed to isolate secondary

metabolite producing cells from non-producing ones (Dix,

1986). Colored cells are visually selected at subculture by

colorimeteric tests, fluorescence (Chaprin & Ellis, 1984) or

optical microspectrophotometry (Hall & Yeoman, 1986).

Such a strategy resulted in stable and high production of

shikonin derivatives in L. erythrorhizon (Tabata et al., 1974).

Using cell-aggregate cloning techniques, Mizukami et al.

(1978) obtained two stable cell lines in L. erythrorhizon with

improved growth and pigment production. In A. euchroma, we

screened eight cell lines showing either a higher growth rate

or pigmentation over a period of 2 years. Amongst colour

producing lines, the stable cell line was selected which was

used for further studies (Figure 5).

Bulgakov et al. (2001) used PFP for selection of high

pigment producing cell lines in A. euchroma and reported

two-fold higher shikonin content in the selected p-fluorophe-

nylalanine resistant callus lines. Zakhlenjuk et al. (1993) also

selected PFP-resistant cell lines in A. euchroma where the

biosynthesis of shikonins was found to be inhibited in this

selected cell line.

Optimization of nutrient media

The composition of the culture medium had a pronounced

effect on cell growth and shikonin derivatives production.

Fujita et al. (1981b) studied the effect of different basal

media and reported the optimum cell growth in Linsmaier &

Skoog (1965) (LS) medium. However, shikonin derivatives

were obtained only in the White (1954) (WH) medium. In cell

suspension cultures of A. euchroma, a dark red pigment was

observed when WH medium was modified by varying the

concentration of different macro nutrients (designated as

Arnebia Production Medium, APM; Figure 6, Malik et al.,

2008). The red pigment extracted from cells growing in APM

turned blue when dissolved in 2.5% KOH solution, indicating

the presence of shikonins (Figure 7). Spectrophotometric

analysis showed maximum absorbance at 620 nm. The

pigment was confirmed to be a shikonin derivative as

compared with the standard (Malik, 2009).

In Echium italicum, the maximum induction of callus

producing shikonin acetate was reported in the WH medium

(Zare et al., 2011). On the basis of a requirement for each

component in the culture medium for the optimal production

of shikonin derivatives from cell culture of L. erythrorhizon,

M-9 medium has been devised and the yield of shikonin

derivatives from cells was enhanced from 120 mg/l (2% DW),

as found in WH medium, to 1400 mg/l (12% DW; Fujita

et al., 1981a).

Carbon source

The growth and production of secondary compounds in cell

culture depends greatly on the type and concentration of

Figure 7. (a) Cells of A. euchroma showing pigment in APM and(b) qualitative reaction of red pigment with 2.5% KOH solution.

Figure 6. Cell suspension cultures of A. euchroma on (a) MS and(b) APM.

Figure 5. (a) Non-pigmented and (b) pigmented callus line ofA. euchroma obtained after selection.


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carbon source, and the biosynthetic capacity of cells. The

optimum concentration for such factors varies with respect to

plant species. Mizukami et al. (1977) observed maximum

yield of shikonin derivatives from callus cultures of

L. erythrorhizon on raising the sucrose (5%, w/v) concentra-

tion in the culture medium. Similarly, 6% sucrose was found

to be optimal for high shikonin derivative production from

cell suspension cultures of A. euchroma as observed in this

laboratory (Malik et al., 2011b).

PGRs and additives

The low concentration (1.0mM) of IAA favored the produc-

tion of shikonin derivatives, while Kn was inhibitory in cell

cultures of L. erythrorhizon (Fujita et al., 1981a).

Brassinolide, along with IAA and BAP, favored cell growth

and production of shikonin derivatives in Onosma panicula-

tum (Yang et al., 1999). Gibberellins and 2,4-D inhibited the

shikonin biosynthesis in cell cultures of L. erythrorhizon

(Yamamoto et al., 2002; Yoshikawa et al., 1986).

Yazaki et al. (1987) reported that inhibitory role of

glutamine for shikonin derivatives production in cell suspen-

sion cultures of L. erythrorhizon was not due to release of

ammonium from glutamine but by glutamine itself.

Role of pH

The pH is an important factor of any biochemical reaction, as

every enzyme/catalyst has its own pH optima and, therefore,

has pronounced effects on the outcome of any biosynthetic

process. In A. euchroma, acidic pH favors cell growth,

whereas alkaline pH exhibited higher shikonin derivatives

production. However, cell growth seems to be hampered at

extremes of pH (Malik et al., 2008; Figure 8). The higher

yield of these pigments at alkaline pH was correlated to

enhanced activity of geranyltransferase, a key regulatory

enzyme, which showed optimum activity at pH 7.1–9.3

(Heide & Tabata, 1987a,b).

Optimization of culture conditions

The biosynthesis and accumulation of secondary compounds

is influenced by the photoperiod, quality and intensity of light

and temperature. Under in vivo conditions, shikonins are

accumulated in underground parts of the plants. The in vitro

cultures of L. erythrorhizon also showed similar behavior, as

pigment formation was inhibited under light conditions

(Yazaki et al., 1997a, 1999).

Heide et al. (1989) observed the reversible effects of light

on shikonin biosynthesis and suggested that cell cultures of

L. erythrorhizon can act as a model to study the light

regulation of shikonin biosynthesis. Tabata et al. (1993)

suggested that light inactivates the flavoprotein necessary for

an enzymatic process leading to shikonin by decomposing the

cofactor flavin mononucleotide (FMN) into lumiflavin. Later,

Yazaki et al. (2001) characterized the dark inducible genes

from L. erythrorhizon (LEDI) and found the regulatory role

of LEDI-II for shikonin biosynthesis. The inhibitory effects

of light on shikonin biosynthesis were also elucidated in

E. lycopsis (Fukui et al., 1983), O. paniculatum (Liu et al.,

2006), O. echiodies (Lattoo, 2005) and A. euchroma (Malik

et al., 2011b; Figure 9). Optimum production of shikonin

derivatives from cell culture of L. erythrorhizon was observed

at 20–25 �C (Tabata & Fujita, 1985). Also, Malik et al.

(2011b) reported maximum pigment production in

A. euchroma at 25 �C (Figure 10).

Two-stage culture medium

Fujita et al. (1981a) found enhanced production of shikonin

derivatives in M-9 medium from L. erythrorhizon cell culture

with poor biomass yield. Therefore, a two-stage culture

process was employed using modified LS medium (MG-5) for

cell growth and modified WH (M-9) for shikonin derivatives

production (Tabata & Fujita, 1985). Also, in cell suspension

cultures of A. euchroma, a two stage culture media approach

was adopted due to non-linked behavior of cell growth and

0

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160

180

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0.300

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0.400

0.450

0.500

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Pigment

Figure 8. Effect of pH on shikonin derivatives production and average cell biomass yield during cultivation of A. euchroma cell suspension (±SD).Reprinted by permission from Malik et al. (2011b). Copyright (2011) International Federation for Cell Biology.


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shikonin derivatives production. Cells were first cultured

for 8 days in a liquid MS + BAP (10.0mM) + IBA (5.0 mM)

medium for cell growth (Growth Medium) and then

transferred to shikonin derivatives or pigment production

medium (APM; Malik et al., 2008).

Addition of precursors and adsorbents

The addition of a precursor, phenylalanine has been reported

to increase the accumulation of shikonins in a cell suspension

culture of L. erythrorhizon (Mizukami et al., 1977). Fukui

et al. (1984) observed that cells of L. erythrorhizon, cultured

in M-9 medium containing activated charcoal, started

producing an orange color benzoquinone derivative, echino-

furan B instead of shikonin. The presence of an adsorbent

PVP (1.0 g/l) in cell suspension culture of A. euchroma was

found to promote cell growth and shikonin derivative

production (Zakhlenjuk et al., 1992).

Use of elicitors/inducers

Elicitation is one of the strategies employed in plant cell

culture to improve the productivity of secondary metabolites

(Namdeo, 2007). Endogenous polysaccharides were found to

induce the biosynthesis of shikonin derivatives in cell cultures

of L. erythrorhizon (Fukui et al., 1990). Ge et al. (2006)

reported the use of rare earth elements in cell cultures of

A. euchroma for enhanced cell growth and shikonin derivative

production. In order to enhance the shikonin content in cell

culture of A. euchroma, fungal elicitors were reported by

Fu & Lu (1999). They obtained 2.24 times higher production

of shikonin derivatives from cells on addition of Rhizopus

oryzae in the medium. Ning et al. (1994) observed the higher

production of shikonin derivatives in cell suspension cultures

of Onosma paniculatum on addition of fungal elicitor. There

are reports of enhanced production of shikonin derivatives in

cell culture of boraginaceous plants by methyl jasmonate.

Enhanced production of shikonin was observed from cells of

L. erythrorhizon (Gaisser & Heide, 1996) and A. euchroma

(Bychkova et al., 1993) and there was an increase in alkannin

pigment, an enantiomer of shikonin from Alkanna tinctoria

cells (Urbanek et al., 1996) in response to methyl jasmonate.

Urmantseva et al. (1999) reported that methyl jasmonate

induced shikonin biosynthesis in cells of A. euchroma by

activation of phenylalanine ammonia-lyase. The elicitation

activity of methyl jasmonate was higher than that of yeast

extract (Mizukami et al., 1992, 1993). Touno et al. (2005)

studied the role of ethylene on shikonin biosynthesis in stem

cultures of L. erythrorhizon.

Permeabilization

Permeabilization of the cell membrane enables the passage of

various enzymes or molecules into and out of the cell. Chung

et al. (2006) studied the effect of gamma-irradiation on

shikonin derivatives production from callus cultures of

L. erythrorhizon. A brief exposure of cells to ultrasound

resulted in a 60–70% increase in the yield of shikonin

derivatives in L. erythrorhizon (Lin & Wu, 2002). It has been

reported that the addition of 1–2% (w/v) Na2EDTA and 1–5%

(v/v) Triton X-100 at the end of growth phase stimulated

the accumulation of shikonin derivatives in cell suspension

cultures of A. euchroma (Zakhlenjuk et al., 1992).

Immobilization

Immobilization can be an effective tool to improve the

effectiveness of a plant cell production process (Dornenburg,

2004). Kim & Chang (1990b) showed 2.5 times higher

production of shikonin from cells of L. erythrorhizon by

immobilization using calcium alginate.

In situ product removal and two-phase culture

Use of a two-phase culture system to improve secondary

metabolites production from different plant species, its types

as well as applications have been described in a recent review

by Malik et al. (2013b). In situ extraction of shikonin in cell

suspension culture of L. erythrorhizon using n-hexadecane as

lipophilic phase was reported by Deno et al. (1987).

Shimomura et al. (1991) and Sim & Chang (1993) success-

fully employed this method in hairy roots of L. erythrorhizon

(Bruce & Daugulis, 1991). The time period for addition

of organic solvent affects the cell growth and secondary

metabolite production. The addition of n-hexadecane to the

0 100 200 300 400 500 600 700

2

4

6

8

10

12

14

Day

s

Shikonin derivatives (µg/g FW)

20 °C 25 °C 30 °C

c

a

b

a

a

a

a

b

b

b

b

bb

cc

cc

cc

Figure 10. Effect of temperature on shikonin derivatives production incell suspension cultures of A. euchroma (± SD). Different letters column(a, b, c) on the bars showing the significant difference (Tukey test,p� 0.05). Reprinted from Malik et al. (2011b) Copyright (2011)International Federation for Cell Biology, with permission from JohnWiley and Sons.

Figure 9. Cell suspension cultures of A. euchroma under (a) light and(b) dark conditions after 8 days of culture. Reprinted from Malik et al.(2011b) Copyright (2011) International Federation for Cell Biology, withpermission from John Wiley and Sons.


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medium on 12th day in A. euchroma culture yielded 2.49

times higher shikonin derivatives as compared to normal

culture without solvents (Fu & Dewei, 1998). In

A. hispidissima, addition of adsorbents such as liquid paraffin

and charcoal in liquid culture medium stimulated alkannin

production by ca. 3-fold (Singh et al., 2002). Zare et al.

(2010) used liquid paraffin to extract shikonin and alkannin

from cell suspension cultures of Echium italicum. There are

reports on the combined effect of in situ extraction and

immobilization, elicitation or permeabilization for better

production of shikonin derivatives (Fu & Lu, 1999; Kim &

Chang, 1990a; Lin & Wu, 2002; Zhang et al., 2013).

Organ culture

Although plant cell cultures showed great potential to produce

an array of valuable products, limited success has been

achieved at the industrial scale. This is attributed to poor

genetic stability of in vitro cell cultures and reduced

metabolite yield with scalability (Shanks & Morgan, 1999).

The culture of differentiated cells or organs viz., shoot or root

culture offer an advantage as these are more stable and do

not undergo rapid genetic variation as observed in unorgan-

ized cells.

Shoot culture

Touno et al. (2000a) reported the production of shikonin

derivatives in shoot cultures of L. erythrorhizon. Shoot

cultures showed the highest shikonin production in liquid

B5 medium at 25 �C and under dark conditions. On the basis

of microscopic studies of shoot culture, Touno et al. (2000b)

showed the presence of a red pigment on stem surface

and hairs.

Scale up studies

Efforts of scientific groups for several years enabled the

Mitsui Petrochemical Company, Japan to become the first of

its kind in the production of shikonin derivatives on the

commercial scale using plant cell culture technology. The

initial experiments on optimization of nutrient media, culture

conditions and downstream parameters increased the yield of

shikonin derivatives up to 13.6% (DW) using L. erythrorhizon

cell cultures, as compared to 1–2% (DW) in roots of intact

plants (Fujita et al., 1981b; Tabata & Fujita, 1985). Later,

shikonin derivative production has been scaled up success-

fully by Mitsui Chemicals Inc. in stirred airlift type bioreac-

tors having 200 L working volume in the growth stage and

750 L in the production stage with residence times of 9 and 14

days, respectively (Fujita, 1988a, b; Tabata & Fujita, 1985).

The cells in bioreactor produced 23% of shikonin in only 23

days as compared to only 2% in 3–7 years from wild grown

plants. Approximately, 5 kg of shikonin was produced in each

bioreactor run and out of 150 kg of its annual demand, 65 kg

was produced by Mitsui Chemicals (Renneberg, 2008). The

production of shikonin derivatives from hairy roots of L.

erythrorhizon by employing a bubble phase bioreactor was

reported by Sim & Chang (1993).

Experiments on the up-scaling of shikonin derivative

production from cell suspension cultures in A. euchroma

using 7.5 L stirred tank bioreactor were carried out

(Figure 11). Cell suspension cultures in growth medium

were transferred from 250 ml flasks to 500, 1000, 2000, 3000,

5000 flasks and finally to 7.5 L BioFlo 110 Benchtop

Fermentor (New Brunswick Scientific Company Inc.,

Edison, NJ) containing APM for large scale production of

shikonin derivatives.

Dong et al. (1993) studied some characteristics of cell

suspension and fermentation culture in A. euchroma. Process

of progressive scale-up culture in A. euchroma from 250 mL

shake flask to 9 L and 25 L airlift bioreactors has been

established by Chen et al. (1994). The use of periodically

submerged airlift bioreactor systems was investigated in a

two-stage culture system of plant cell culture and enhanced

secondary metabolite production by Ge et al. (2006). The

advances in shikonin derivative production from different

plant species under in vitro conditions are highlighted in

Table 3.

Figure 11. (a) Shikonin derivatives production from cell suspension cultures of A. euchroma in 7.5 L bioreactor and (b) Close up of the same.


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Table 3. Chronological studies on in vitro culture of shikonin bearing plant species.

Year References

1974 Shikonin derivatives obtained from callus cultures of Lithospermum erythrorhizon. Tabata et al. (1974)1978 Stable cell lines in L. erythrorhizon with improved growth and pigment production obtained using cell-

aggregate cloning technique .Mizukami et al. (1978)

1981 Effect of different basal media was studied on cell growth and shikonin derivatives production in cellsuspension culture of L. erythrorhizon.

Fujita et al. (1981b)

1981 A new medium developed for the production of shikonin derivatives from cell suspension culture ofL. erythrorhizon.

Fujita et al. (1981a)

1981 Production of shikonin derivatives in callus cultures of Echium lycopsis. Inouye et al. (1981);Fukui et al. (1983)

1983 Analysis of shikonin derivatives in cell suspension cultures of L. erythrorhizon. Fujita et al. (1983)1983 Commercial production of shikonin derivatives achieved from cell culture of L. erythrorhizon. Fujita (1988a)1987 Tissue culture of Arnebia euchroma initiated.1987 Factors affecting the production of shikonin derivatives in callus and cell suspension culture studied

in L. erythrorhizon.Hara et al. (1987)

1989 Reversible effect of light on shikonin biosynthesis in L. erythrorhizon. Heide et al. (1989)1991 Two phase culture system for the extraction of shikonin derivatives from hairy roots of L. erythrorhizon. Shimomura et al. (1991);

Sim & Chang (1993)1993 PFP-resistant cell lines selected in A. euchroma. Zakhlenjuk et al. (1993)1996 Cell lines of A. euchroma selected during different stages of culture by in vitro screening method. Sokha et al. (1996)1996 Important enzymes involved in regulation of shikonin biosynthesis were identified. Gaisser & Heide (1996);

Lange et al. (1998)1997 Effects of elicitor methyl jasmonate were investigated on shikonin biosynthesis in cell culture of

L. erythrorhizon.Yazaki et al. (1997b)

1997 Effect of various auxins and cytokinins on organogenesis and embryogenesis in selected line ofL. erythrorhizon.

Yu et al. (1997)

1999 Synergistic effect of in situ extraction and elicitation on shikonin derivatives production in cellsuspension cultures of A. euchroma.

Fu & Lu (1999)

1999 Isolation of naphthoquinones and pyrrolizidine alkaloids from callus and cell suspension culturesof A. euchroma.

Pietrosiuk et al. (1999)

1999 Arnebins showing antibacterial and antifungal activity were found in cell culture of A. hispidissima. Jain et al. (1999)1999 Studies were carried out to modify the biosynthetic pathway for enhanced production of shikonin

derivatives.Sommer et al. (1999)

2000 Cell suspension culture of L. erythrorhizon were found to produce different groups of compounds. Yamamoto et al. (2000)2001 Regulatory mechanism of light was studied on formation of shikonin derivatives in L. erythrorhizon. Yazaki et al. (2001)2001 Direct regeneration from leaves of A. euchroma. Ji & Wang (2001)2001 High shikonin content in the selected p-fluorophenylalanine resistant callus lines in A. euchroma. Bulgakov et al. (2001)2002 Production of alkannin from hairy roots of A. hispidissima. Singh et al. (2002)2002 Enhancement in production of shikonin derivatives in cell suspension culture of L. erythrorhizon by using

low energy ultrasound.Lin & Wu (2002)

2002 Different shikonin derivatives were observed in cell suspension cultures of L. erythrorhizon. Yamamoto et al. (2002)2005 Direct regeneration from cotyledons and hypocotyls in A. euchroma. Jiang et al. (2005)2005 Shoot regeneration and somatic embryogenesis from leaf derived callus in A. euchroma. Manjkhola et al. (2005)2008 Role of pH on shikonin derivatives production in cell suspension culture. Malik et al. (2008)2008 Presence of acetylshikonin and b-acetoxyisovaleryl shikonin in cell suspension cultures of A. euchroma. Sharma et al. (2008)2009 Shikonin formation in cell suspension culture of Onosma paniculatum was reported to be regulated by

Nitric oxide.Wu et al. (2009)

2010 In vitro induction of hairy root culture and shikonin production in A. hispidissima. Chaudhury & Pal (2010)2010 Direct plant regeneration, micropropagation and shikonin induction in A. hispidissima. Pal & Chaudhury (2010)2010 The importance of pre-culture time and concentration of TDZ for direct regeneration from in vitro leaves

of A. euchroma.Malik et al. (2010b)

2010 Pathway for shikonins biosynthesis was identified. Singh et al. (2010)2010 A two-phase liquid culture system using liquid paraffin was established to elicit shikonin and alkannin

derivatives in E. italicum.Zare et al. (2010)

2011 Different physico-chemical factors affecting the shikonin derivatives production in cell suspensioncultures of A. euchroma were studied.

Malik et al. (2011b)

2011 Micropropagation and establishment of callus and cell suspension culture of A. hispidissima foroptimization of alkannin production.

Shekhawat & Shekhawat(2011)

2011 Shikonin production potential of E. italicum callus. Zare et al. (2011)2011 LeERF-1, a novel AP2/ERF family gene within the B3 subcluster was down-regulated by different

light conditions in L. erythrorhizon.Zhang et al. (2011)

2012 Enhanced production of shikonin derivatives in hairy roots of L. canescens. Sykłowska-Baranek et al.(2012)

2013 In vitro propagation and genetic fidelity of micropropagated plants was assessed for A. hispidissima. Phulwaria et al. (2013)2013 The effects of fungal elicitor and macroporous adsorption resin were studied on shikonin accumulation in

hairy roots of A. euchroma.Zhang et al. (2013)

2013 Shikonin production from cell suspension culture of Arnebia sp. and it’s up-scaling through bioreactor. Gupta et al. (2013)


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Genetic transformation and transformed plants

A low yield of secondary compounds from plant tissues under

in vitro conditions is a major bottleneck in their production at

the commercial level (Charlwood & Pletsch, 2002). Through

genetic engineering, it is possible to manipulate the regulatory

steps of the biosynthetic pathway in order to increase the yield

of required compounds (Verpoorte et al., 1999). There are

reports on the transformation of shikonins bearing plants with

Agrobacterium rhizogenes to enhance the secondary metab-

olite content (Fukui et al., 1998; Shimomura et al., 1991;

Yazaki et al., 1998). Yazaki et al. (1998) obtained transfor-

mants in L. erythrorhizon using A. rhizogenes strain 15 834

with stable and higher production of shikonin derivatives.

Shimomura et al. (1991) transformed the shoot cultures of L.

erythrorhizon with A. rhizogenes strain 15 834 and found a red

pigment after 2–3 weeks culturing on agar gelled and liquid

root culture media. Similarly in L. canescens, hairy roots were

shown to produce ca. a 10% higher content of acetylshikonin

and isobutrylshikonin as compared to natural roots (Pietrosiuk

et al., 2006). Hairy root transformation for other genera of

the Boraginaceae family has also been reported. In another

report by Singh et al. (2002), alkannin was obtained on a

half-strength agar solidified MS medium from hairy roots

of A. hispidissima induced with A. rhizogenes strain 15 834.

Sommer et al. (1999) modified the biosynthetic pathway

to 4HB in hairy root cultures of L. erythrorhizon by

introducing bacterial gene ubiC but it did not show any

significant increase in yield of shikonin derivatives in

transformed plants. Boehm et al. (2000) made an attempt to

manipulate the biosynthetic pathway of secondary metabol-

ism by the introduction of the bacterial gene ubiA in hairy

root cultures of L. erythrorhizon. The transformed hairy roots

showed high ubiA activities, however, ubiA overexpression

was not found sufficient to increase the shikonin content. In A.

euchroma, callus cultures were transformed with

pCAMBIA1302 containing HMGR cDNA (unpublished

data). The cell suspension cultures, raised from transformed

callus, did not show significant alteration in the shikonin

derivative content as compared to the control. Despite high

activity of HMGR, the shikonin content remained unchanged.

A high activity of HMGR in L. erythrorhizon hairy root

cultures was reported by transformation with A. rhizogenes

containing HMGR and ubiC genes under the control of

(octopine synthase)3 mannopine synthase promoter (Kohle

et al., 2002). In spite of high activity using the soluble

cytosolic domain of HMGR in cultures, the shikonin content

remained unchanged. Based on this, it was suggested that the

overexpression of CPL or HMGR was insufficient to increase

shikonin formation. Therefore, it seems that either simultan-

eous overexpression or down regulation of different genes

may influence the biosynthesis of shikonin derivatives.

Conclusions and future prospects

Considering the consumer driven market demand for natural

products as well as commercial and societal importance of

and shikonin derivatives, noticeable efforts on in vitro

production R&D activities are evident from the recent

literature. Biotechnological interventions not only help in

the conservation of biodiversity of natural habitats by

reducing the demand for raw materials from the wild, but

will also lay a foundation towards development of alternative

strategies to fulfill industrial demand. Production of second-

ary compounds using various biotechnological means could

offer an important strategy, but so far have provided with only

limited commercial success. More detailed studies are

required to understand the complex biosynthetic pathways

in order to obtain the desired product. The technology could

be utilized for studies related to metabolic engineering in

shikonins bearing plants to suppress or overexpress the genes/

enzymes involved in the pathway for enhanced production of

secondary compounds.

Acknowledgements

The authors are grateful to the Council of Scientific and

Industrial Research (CSIR), India for providing financial

support. SM would also like to acknowledge the grant from

Centre of the Region Hana for Biotechnological and

Agricultural Research, Czech Republic (CZ.1.05/2.1.00/

01.0007 (SPP 813103061/15)).

Declaration of interest

All the authors have read the manuscript and there are no

conflicts of interest.

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biotechnological approaches to the production of shikonins: a critical review with recent updates

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