Ph. D. THESIS – M.

GOMATHI MAY 2015

REVIEW OF LITERATURE

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REVIEW OF LITERATURE

Rauwolfia serpentina is an important medicinal plant belongs to the family

Apocynaceae. This plant is native to Bangladesh, India and other humid region of the

world (Roy et al., 1994). R. serpentina contains some 50 indole alkaloids and most of

the total alkaloid content percent mainly in root back (Klyushnichenko et al., 1995).

Cultivation of Rauwolfia in India was reported by many authors (Biswas, 1956; Dutta

et al., 1963; Badhwar et al., 1955). Rauwolfia serpentina L. Benth. Ex Kurz. is an

evergreen perennial shrub which grows upto 60m of height. The family includes 50

species, distributed worldwide in the region of the Himalayas, Indian peninsula,

Burma, Indonesia and Sri Lanka and is indigenous to India, Bangladesh and other

regions of Asia (Ghani et al., 1998). It is also commonly known as Sarpagandha,

Chandrabagha, Snake root plant, Chotachand, Chandrika and Harkaya etc.(Mallick et

al., 2012).

Rauwolfia serpentina Medicinal uses

Among all the alkaloids reserpine, serpentine, yohimbine, deserpidine, ajmalicine,

ajmaline, etc are used to treat hypertension (Vonposer et al., 1990; Vakil, 1949;

(Dass, 2009; Satyavativ, 1955) breast cancer (Stanford et al., 1986), high blood

pressure, insomnia, anxiety, schizophrenia, insanity, epilepsy, hypochondria and other

disorder of the central nervous system (Bhatara et al., 1997; Kirtikar & Basu, 1993;

Dastur, 1988; Bleuler & Stooll, 1955). The root extract used to treat painful affection

of bowels, diarrohea (Tona et al., 1999), dysentery, cholera and colic (Ghani, 1998).

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For centuries, root of Rauwolfia serpentina has been used in the traditional unani and

ayurvedic medicine (Andrew and Chevallier 1996). Rauwolfia serpentina contains a

variety of compounds with antioxidant capacity and other health benefits like

treatment of diabetes, cardiovascular disease, cancer and hypertension. The

antibacterial and antifungal activities were high in petroleum ether and acetone extract

of Rauwolfia serpentina (Harisaranraj et al., 2009). Rauwolfia serpentina has long

being used in India for the treatment of snakebites, hypertension, high blood pressure

and mental illness. Different ethnic groups use this plant to treat snake, insect and

animal bite, mental illness, schizophrenia, hypertension, blood pressure,

gastrointestinal diseases, circulatory disorders, pneumonia, fever, malaria, asthma,

skin disease, scabies, eye diseases, spleen diseases, AIDS, rheumatism, body pain,

veterinary diseases etc. this plant is also being used to prepare fermented food

products (Dey and Den, 2010). It has been stated that this plant is used as antidote

against snakebite (Khyade et al., 2011). Plant derived indole inhibitors were identified

from the extracts of Rauwolfia serpentina which has the capacity to acts as aldose

reductase, a potent drug for diabetes (Pathnania et al., 2013). The methanol root

extract of this plant provides the high antioxidant compound phenol of 233mg/gm. So

this plant has significant antidiabetic and hypolipidemic activity. (Azmi and Qureshi,

2012).

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Rauwolfia serpentina

Scientific classification

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Gentianales
Family: Apocynaceae

Genus: Rauwolfia
Species: R. serpentina

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Chemical constituents

The phytochemical analysis of Rauwolfia serpentina has many medicinal values. It is

used for the diseases such as Anthelmintic, anti hypertensive, snake bite, diarrhoea,

dysentery, cholera, fever, pneumonia, malaria, body aches, eczema, burns, menstrual

disorders, scabies, skin cancers, asthma, respiratory problems, eye inflammation,

spleen diseases and feveopacity of the cornea and central epilepsy (Rai, 2004; Itoh et

al., 2005; Mohanta et al., 2006; Bhattarai et al., 2009; Dey and De, 2010; Azmi et al.,

2013; Ghani, 1998). Because of presence of high amount of alkaloids it is used for the

treatment of circulatory disorders (Fabricant and Farnsworth, 2001), treat liver,

abdomen pain, gastrointestinal disorders and to expel intestinal worms from the

childrens (Nayak et al., 2004; Anisuzzaman et al., 2007; Dey and Den, 2011).

The alkaloids of R. serpentina were reported by Siddiqui and Siddiqui (1931, 1932,

1935) and Siddiqui, (1939); Chatterjee and Bose, (1951); Popelak et al., (1953); Bose

(1954). Rauhimbin und Isorauhimbin from this plant was reported by Hofmann, 1954.

Reserpinin, alkaloid compound was reported by Schlittler et al., (1954); Agbalyam,

(1961); Karim et al., (1961). TLC of Rauwolfia alkaloids was performed by

Schlemmer and Link (1959). The alkaloid contant of the plant may vary according to

the ecological region (Wakhloo, 1963), and geographical condition (Dhar, 1965;

Farooqi and Sreeramu, 2001). The methods of quantitative determination was

reported by Habib and Court (1974). The quantitative determination of alkaloids from

tissue culture grown Rauwolfia was reported by Vollosovich et al., (1977); Stockigt et

al., (1981); Stockigt, (1995); Shimolina et al. (1984); Uesato et al. (1986).

Spectrophotometric determination of alkaloids (Singh et al., 2004) and

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characterization of oxidation products of alkaloids (Azeem et al., 2005), analyzed

with NMR (Itoh et al., 2005). Functional expression of an ajmaline pathway-specific

esterase from Rauvolfia in a novel plant-virus expression system was reported by

Ruppert et al., (2005). Quantitative determination of reserpine, ajmaline and

ajmalicine in this plant by reversed-phase HPLC (Srivastava et al., 2006).

Quantization of Reserpine, Ajmaline and Ajmalicine from the plant by HPLC (Goel et

al., 2009).

Alkaloids are huge cluster of organic molecules which contain a heterocyclic nitrogen

ring. The pure alkaloids are used as analgesic, antispasmodic and bactericidal effects

(Okwu et al., 2004). Different types of alkaloids in Rauwolfia serpentina are as

follows

Reserpine

Reserpine is a pure crystalline single alkaloid, derived from the roots of Rauwolfia

and was first isolated in 1952 (Schlitter et al., 1954). It is useful for the treatment of

hypertension, cardiovascular diseases and neurological diseases (Weiss et al., 2000;

Pullaiah, 2002). Reserpine (3, 4, 5-trimethyl benzoic acid ester of reserpic acid, an

indole derivative of 18- hydroxy yohimbine type) are used in hypersensitive reactions

and also act as natural tranquillizer (Banerjee et al., 2010). Reserpine can be used in

the antihypertensive actions by act on peripheral nervous system by binding to

catecholamine storage vesicles present in the nerve cell (Ellenhorn and Barceloux

1988; Gilman et al., 1990; Nammi et al., 2005).

Ajmaline

The compound was first isolated by Salimuzzaman Siddiqui in 1931 from the roots of

R. serpentina. He named it ajmaline, after Hakim Ajmal Khan, one of the most
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illustrious practitioners of Unani medicine in South Asia (Siddiqui et al., 1985). It

has been used in as antiarrhythmic agent, hereditary cardiac disorder (Rolf et al.,

2003). This type of compounds used in four different treatments such as sodium

channel blockade, betaadrenergic blockade, repolarization prolongation and calcium

channel blockade (Kostin et al.,1986; Brugada et al., 2000)

Ajmalicine

Ajmalicine has been used for the treatment of circulatory diseases, prevent strokes

and lowers blood pressure (Srivastava et al., 2006). Annually 3500kg of ajmalicine

was isolated from this plant. An estimated 3500 kg of Rauvolfia or Catharanthus spp.

by pharmaceutical industries for the treatment of circulatory diseases. The ajmalicine

is derived from tryptophan which is converted to tryptamine via secologanin,

strictosidine and cathenamine. Reduction of cathenamine to ajmalicine is facilitated

by enzyme NADPH and tryptophan decarboxylase (TDC). Decarboxylase might be

the key enzyme involved in the synthesis of ajmalicine in Rauwolfia (Liu et al.,

2012).

Serpentine

Serpentine, a type II topoisomerase inhibitor, exhibits antipsychotic properties.

(Dassonneville et al., 1999, Costa-Campos et al., 2004) The enzyme peroxidase

(PER) is responsible for oxidation of ajmalicine to serpentine by catalyzing bisindole

alkaloid localized in the vacuole (O’Connor and Maresh, 2006).

Rescinnamine

Rescinnamine, a purified ester alkaloid of alseroxylon fraction in species of Rauwolfia

this was first identified in 1950’s used for the treatment of hypertension as an

antihypertensive agent. Rescinnamine inhibits angiotensin converting enzyme,

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peptidyl dipeptidase that catalyzes the conversion of angiotensin I to the

vasoconstrictor substance, angiotensin II which stimulates aldosterone secretion by

the adrenal cortex. First it inhibits the Angiotensin Converting Enzyme (ACE) and

then blocks the conversion of angiotensin I to angiotensin. Inhibition of ACE results

in decreased plasma angiotensin II. As angiotensin II is a vasoconstrictor used for

lowering blood pressure and decreased vasopressor activity and aldosterone secretion

(Kolh et al., 1954).

Deserpidine

Deserpidine is an ester alkaloid isolated from Rauwolfia. It is used mainly for its

antipsychotic and antihypertensive properties. It can able to reduce high blood

pressure by controlling nerve impulses along various nerve pathways (Varchi et al.,

2005).

Yohimbine

Yohimbine, is used as a selective alpha-adrenergic antagonist or alpha blocker in the

blood vessels for the treatment of erectile dysfunction It dilates blood vessels and

increases blood flow in the penis, which helps in improving erectile function (Morales

et al., 2000; Andersson et al., 1993; Goldberg and Robertson, 1983).

Phenols

Phenols are toxic for the various pests and pathogens (Singh and Sawhney, 1988).

Phenolic compounds in R. serpentina has antidiabetic and hypolipidemic properties

(Qureshi and Udani 2009). It also used as anti-microbial agent.

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Saponins

Saponins are glycoside of both triterpenes and sterols and have been detected in over

70 families of plants. The high content of saponins in Rauvolfia serpentina helps to

stop the bleeding in treating wounds (Basu and Rastogi, 1967).

Indole alkaloid reserpine

Rauwolfia serpentina is rich source of indole alkaloid of medicinal values which are

used in the treatment of circulatory disorders (Tyler et al., 1981). Root of Rauwolfia

serpentina is mainly used in mild hypertension in combination with a diuretic agent

AHFSDI. It is also used as tonic states of asthenia, snake & insect bite &for

constipation, liver diseases flatulence, insomnia & rheumatism (Fransworth, 1995). It

is well accepted that the pharmacological effects of rauwolfia serpentine are due to its

alkaloids, especially the reserpine, rescinnamine group (Rand and Jurevices, 1977).

Reserpine is an effective indole alkaloid first isolated from Rauwolfia serpentina used

as an antihypertensive (Anonymous, 2003). Due to its poor seed germination rate, it

cannot be easily increases the number of plants, because of its high medicinal use, R.

serpentina is becoming extinct and is now listed as an endangered species by the

International Union for Conservation of Nature and Natural Resources (IUCN) (Jain

et al., 2003; Singh et al., 2009; Sushila et al., 2013).

The roots contain 50 alkaloids including the therapeutically important reserpine,

deserpidine and yohimbime. Insulin binds to insulin receptors that is present on

different cells of the body and mediates the absorption of glucose in to the cells.

Docking studies of insulin receptor with alkaloids of Rauwolfia serpentina revealed

that few of the alkaloid present in Rauwolfia serpentina may be potential activators of
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Insulin receptor (Jayasree et al., 2012). Reserpine is an indole alkaloids chemically it

is (3β, 16β, 17β, 18β, 20β)-11,17-dimethoxy-18 (3,4 ,5-trimethoxy benzoyl)-oxyl)

yohimban -16 carboxylic acid methyl ester or 3,4,5-trimethoxybenzol methyl

reserpate used in lowering blood pressure. Alkaloid content has been studied

(Vollosovich et al., 1976; Roja et al., 1885; Mathur et al., 1987; Ruyter et al., 1976;

Roja & Heble, 1996; Patil and Jayanthi, 1997). Alkaloids were identified by TLC

(Roja et al., 1996). The isolation of reserpine from dry roots of Rauwolfia sepentina

marked a revolution in the anti hypertensive and sedative drug therapy (Mulla et al.,

1952).

Reserpine content were analysed by HPLC using methanol extracts and was reported

that the reserpine content varies with different geographical location (Hareesh et al.,

2010). Reserpine content was measured in the different parts of Rauwolfia serpentina

plant including leaf, stem, flower and root. 90% of total reserpine content was

produced from root, whereas stem and root contains 10% (Panwar et al., 2011). Seven

new indole alkaloids were isolated from dried roots of Rauwolfia serpentina, namely

methylajmaline, methylisoajmaline, 3-hydroxysarpagine, yohimbinic acid,

isorauhimbinic acid and 7-epiloganin and glomeratose A. The structures of these

compounds were determined by spectroscopic and chemical means (Itoh et al., 2005).

Antibacterial activity

The aqueous and methanol extracts of Rauwolfia vomitoria show antimicrobial

activity against Klebsiella, Pneumonia, Staphylococcus aureus, Enterobacter,

Pseudomonas aeruginosa and Escherichia coli (Olajumoke et al., 2012).

Propionibacterium acnes and Staphylococcus epidermidis have been recognized as

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pus-forming bacteria triggering an inflammation in acne. In a study antimicrobial

activities of some Indian medicinal plants were against these bacteria were evaluated.

Among others the ethanolic extracts of Rauwolfia serpentina roots had greatest

antimicrobial effect and produced strong inhibition zone against Propionibacterium

acnes. Phytochemical screening of Rauwolfia serpentina revealed the presence of

alkaloid could be responsible for activity (Harisaranraj et al., 2010). Antibacterial

activity of Rauwolfia serpentina plant extract against Escherichia coli,

Staphylococcus aureus, Salmonella typhi and Klebsiella pneumonia by disc diffusion

method showed the alcoholic extract was found effective against Staphylococcus

aureus only. The antibacterial activity is due to presence of alkaloids which

confirmed by gas liquid chromatography and positive alkaloid test (Upadhyay et al.,

2009).

Micropropagation

The Propagation of Rauwolfia serpentina by seeds was poor because the seed of the

plant has a cinamic acid derivative which affects the germination percentages (Mitra,

1976; Alamgir & Ahamed, 2005; Nair, 1955). In vitro propagation of Rauwolfia

serpentina has been reported by many researchers as (Ahmad et al., 2002; Sarkar et

al., 1996, Roy et al., 1994; Mathur et al., 1993; Sudha & Seeni 1996; Butenka 1964;

Mitra & Kaul 1964; Vollosovich & Butenka 1970; Kukreja et al., 1989; Roy et al.,

1994; Iiahi & Akram 1987; Mukhopadhyaya et al., 1991; Gupta et al., 1950;

Iiahi,1993, Sarkar et al., 1996; Iiahi et al., 2007; Kataria & Shekhawat 2005; Pandey

et al., 2007; Baksha et al., 2007; Goel et al., 2009; Pant & Joshi 2008; Salma et al.,

2008; Bhatt et al., 2008; Singh et al., 2009; Harisaranraj, 2010 and Rani et al., 2013).

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Callus formation was studied by Perveen & Iiahi (1978). Vollosovich et al., (1979)

have optimized the composition of macro salts in the culture media, root callus

regeneration (Akram & Iiahi, 1985; Akram & Iiahi 1986).

Production of reserpine and optimization in cell culture (Yamamoto and Yamada,

1986), in vitro for alkaloids production (Sen & Datta,1990), addition of Copper in the

callus culture improve the reserpine production (Nurcahyani et al., 2008) and

different hormone concentration & combination in enhancing callus induction was

studied by (Salma et al., 2008).

In vitro propagation of Rauwolfia serpentina using liquid medium was reported by

(Goel et al., 2009). Effect of growth regulators on direct root induction from leaf

explants. The separation & identification micro quantities of alkaloids were

performed by (Habib and Court, 1974). Methods for the quantitative determination of

the sum of alkaloid in tissue culture of Rauwolfia serpentina were reported by

(Vollosovich et al., 1977). The indole alkaloid patterns of cell suspension & tissue

culture have been investigated (Stockigt et al., 1981; Stockigt, 1984).

Propagation of Rauwolfia serpentina through tissue culture has previously been

described but most of them have focused on micropropagation by means of

stimulating axillary shoot growth (Mathur et al., 1977; Roy et al., 1994).

Use of higher concentration of growth hormone leads to multiple shoot formation

from nodal segment (Verma et al., 2002; Selvakumar et al., 2001; Sudha and Seeni,

1996).

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Auxin helps to enhance shoot bud initiation in nodal explants of many plant species,

and cytokinin for the auxiliary bud initiation and multiplication (Hamdy and Hattori,

2006; He et al., 2005; Baskaran and Jayabalan, 2005; Gupta et al., 2001). The

elongation and proliferation of shoots can be enhanced by addition of auxin at low

concentration (Hu and Wang, 1983).

Auxins induces root in micropropagation along with the MS medium in many

medicinal plants (Ahamed et al., 2005; Mederos-Molina, 2004; Huda et al., 2003).

MS medium along with different concentration of IAA and IBA in combination or

separately can induce root intitation during micropropagation (Ahamed et al., 2005;

Vesperinas, 1998)

The unorganized growth of plant cells by growth hormones (auxins and cytokinins)

are known as callus (Shah et al., 2003). The concentrations of the plant hormones

vary from explants of plant to plant (Charriere et al., 1999). 2,4-D is a auxin used for

inducing callus (Bhaskaran and Smith, 1990; Chaudhury and Qu, 2000). Cytokinins in

use of low concentration helps in the regeneration of callus (Alpeter and Posselty,

2000; Chaudhury and Qu, 2000; Bai and Qu, 2001; Bradely et al., 2001). The

cytokinins and auxins in the medium avoid somoclonal variation and efficiently

produce true to type plantlets (Edson et al., 1996).

Archana et al., 2014 has standardized the protocol for micropropagation through in

vitro culture of Rauwolfia serpentina. MS media in combination with 0.5 mg L-

1Indole Acetic Acid + 0.5 mg L Nephthalene acetic acid was identified has better

induction of callus. Axillary shoot growth was obtained when MS media combined

with 0.5 mg L-1 Indole Acetic Acid + 0.5 mg L-1 Benzyl Amino Purine. Better shoot
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elongation was found on MS media combined with 3.0 mg L-1 IAA plus 3.0 mg L-

1BAP. High root induction was obtained from the MS medium supplemented with

Indole Butyric Acid (3.0 mg L-1).

High frequency regeneration from various explant of Rauwolfia serpentina has been

reported (Sehrawat et al., 2002 ; Ahamad et al., 2002). Shoot tips and nodal explants

cultured on MS medium with different concentration of BA & NAA has better

regeneration (Singh & Guru, 2007; Kataria & Sekhawat, 2005). Nodal segments are

significant source for micropropagation and plants grown from them are

comparatively more resistant to genetic variation (Pierik, 1991).

Various parts of the Rauwolfia serpentina plant were used as explant and inoculated

in Murashige and Skoog medium containing 2, 4-D and 6-BAP. Maximum callus

inductions of 93.65% were obtained in leaf and stem explants. The callus was

inoculated in shooting medium containing BAP and NAA and maximum shoots were

obtained. The rooting was induced in invitro regenerated shoots in MS medium

containing IBA and NAA and 100% rooting was obtained (Panwar et al., 2011).

Callus was induced from root explants of Rauwolfia serpentina using 2mg/l BAP and

0.8mg/l NAA and later induced to bud formation which further developed into shoots.

The plantlet formed were transferred to soil which initially watered with half strength

Knop’s solution till they became autotrophic and were noticed to grow well in open

field condition (Akram and Ilahi, 1986). High frequency of 96.43% callus was

induced when nodal segments from invitro raised shoots were cultured on MS

medium supplemented with 0.5mg/l BA and 2.0mg/l NAA (Salma et al., 2008).

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Varying concentration of BAP and combination of BAP with IBA produced multiple

shoots. Maximum multiple shoots (85.6%) were obtained when MS medium

supplemented with 5.0mg/l BAP and 0.5mg/l IBA along with 2.5% sucrose and

0.85% agar. The developed shoots were excised and implanted on MS medium with

varying concentration of IBA. Maximum rooting of 76.6% was obtained and 74% of

regenerated plantlets survived in open field condition (Vandana et al., 2003).

Richa et al., (2008) has been reported that callus was obtained from leaf and stem

explants, whereas direct regeneration observed when apical and nodal explants were

used. Combination of IBA (0.125 mg/l) + BAP (1.0 mg/l) produced better results for

both callus induction and direct regeneration.

The callus of Rauwolfia serpentina consists of cell colonies with different

fluorescence (yellow-green and blue-white) under 365nm UV- light. HPLC analysis

showed that the yellow-green fluorescent strains produced more reserpine, whereas

the blue-white strains produced more 3, 4, 5-trimethoxy benzoic acid. Combination of

10µM NAA and 10µM BA enhanced the production of reserpine in the yellow-green

fluorescent cell strains (Osamu, 1987).

Stress induced by alteration of medium composition (hormones) for several

generations’ results in changes the in level of production of alkaloids. Such changes

were noted in a study that Rauwolfia serpentina cells that have been cultured and

maintained on modified Linsmaier-skoog medium for over 13 years produced more

ajmaline (0.005-0.012g /dw) than reserpine (0-0.003g dw) (Osamu, 1986).

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Growth hormones play vital role in somatic embryogenesis. Leaf explants were

cultured in the MS medium containing different combinations of growth hormones.

Callus induction was highest in 2.5 mg/L BAP + 2.0 mg/L IAA and 1.0 mg/L BAP +

0.5 mg/L IAA, shoot regeneration was highest 75% in 2.5 mg/L BAP + 0.4 mg/L

IAA, root regeneration containing the combination of BAP (2.5 mg/L) + IAA (0.5

mg/L) + NAA (0.5 mg/L). Survival percentage of plants after hardening was 67%

(Prabhat et al., 2009).

Rauwolfia serpentina inoculated on MS medium supplemented with NAA induced

callus under 24 hour light while 2, 4-D induced callus under 16 hour light (Ihsan and

Akram, 1987). When the leaf and stem explants of Rauwolfia serpentina inoculated

on modified Murashige and Skoog (MS) medium with 2.5mg/l 2, 4-D maximum

regeneration of shoots from callus (90%) was observed in MS medium supplemented

with 0.2mg/l NAA and 1.5mg/l BA. Direct regeneration (96%) was recorded best in

MS medium supplemented with BAP 2.5mg/l. higher induction of root (100%) was

observed in MS medium supplemented with NAA 0.5mg/l (Subhadra et al., 2012).

Biosynthesis of Strictosidine in Rauwolfia serpentina

Rauwolfia serpentina produces many terpene indole alkaloids such as ajmaline,

yohimbine and ajmalicine. The biosynthetic pathway for ajmaline in Rauwolfia

serpentina is one of the best characterized terpene indole alkaloid pathways. Over the

last 10 years, remarkable progress has been made in identifying the enzymes

responsible for ajmaline biosynthesis. Much of this progress has been detailed in a

recent extensive review (Ruppert et al., 2005).

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Like all other terpene indole alkaloids ajmaline, an antiarrhythmic drug with potent

sodium channel blocking properties is derived from strictosidine (Rolf et al., 2003).

Strictosidine is deglycosylated by a b-glucosidase, convert it to a immediate

hemiacetal intermediate (Gerasimenko et al., 2002). This reacts with the secondary

amine of the strictosidine to form 4, 21-dehydrocorynantheine aldehyde (Scheme 3).

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Dehydrocorynantheine aldehyde then undergoes allylic isomerization and enolization

to produce either the enol or keto forms of dehydrogeissoschizine. The enol form of

dehydrogeissoschizine undergoes 1, 4 conjugate addition to produce the

heteroyohimbine cathenamine (Kan-Fan and Husson, 1998). Cathenamine and

dehydrogeissoschizine comprise of experimental in equilibrium in vitro condition (El-

sayed et al., 2004; Heinstein et al., 1979). The glycosidase, strictosidine-b-

glucosidase, has been cloned from Rauwolfia serpentina (Gerasimenko et al., 2002).

Strictosidine is also deglycosylated by non specific bacterial glucosidases (Shen et al.,

1998). A crystal structure of strictosidine-b-glucosidase enzyme from R. serpentina

was identified by Dewick et al., (2002). Eight enzymes involved in biosynthesis of

ajmaline after strictosidine deglycosylation. The sarpagan type alkaloid, polyneuridine

aldehyde, is a known early intermediate of the ajmaline pathway. Experiments

suggest 4, 21-dehydrogeissoschizine may be a precursor for polyneuridine aldehyde

(Stockigt, 1986).

A membrane protein of R. serpentina extract transforms strictosidine into sarpagan

type alkaloids. The enzyme activity depends on NADPH and the sarpagan bridge

enzyme may be a cytochrome P450 enzyme (Ruppert et al., 2005; Stockigt, 1995).

Polyneuridine aldehyde esterase hydrolyzes the polyneuridine aldehyde methyl ester

and an acid which decarboxylates to form epi-vellosamine. Cell cultures of Rauwolfia

and sequencing of protein fragments enabled a clone of polyneuridine aldehyde

esterase isolated from a Rauwolfia cDNA library. This enzyme has been over

expressed in E. coli and subjected to detailed mechanistic studies (Pfitzner and

Stockigt, 1983; Mattern-Dogru et al., 2002, Dogru et al., 2000). In the ajmaline
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pathway, vinorine synthase converts sarpagan alkaloid epi vellosamine to the ajmalan

alkaloid vinorine (Pfitzner and Stockigt, 1983).

Vinorine synthase has also been purified from Rauwolfia cell culture, subjected to

protein sequencing and cloned from a cDNA library (Gerasimenko et al., 2004; Bayer

et al., 2004). The enzyme, which appears to be an acetyl transferase homologue, has

been heterologously expressed in E. coli. Vinorine hydroxylase converts vinorine to

vomilene (Falkenhagen et al., 1995). Vinorine hydroxylase is a P450 enzyme it needs

an NADPH dependent reductase. Seven full length cytochrome P450 clones were

isolated from a Rauwolfia cDNA library by homology cloning and then

heterologously expressed in combination with a reductase.

Two reduction steps follow the formation of vomilenene they are 1, 2-

dihydrovomilenene and 1, 2-dihydrovomilenene reductase, then reduces this product

to acetylnorajmaline. An acetylesterase then hydrolyzes the acetyl linkage of

acetylnorajmaline to yield norajmaline. This esterase was purified from R. serpentina

cell suspension cultures, partial amino acid sequences were obtained and a full length

clone was isolated from a cDNA library. Expression of the gene in tobacco leaves

successfully yielded protein with the expected enzymatic activity. In the final step of

ajmaline biosynthesis, an N-methyl transferase introduces a methyl group at the

indole nitrogen of norajmaline. Although this enzymatic activity has been detected in

crude cell extracts, the enzyme has not been further characterized. Five of the

enzymes, strictosidine synthase, strictosidine glucosidase, polyneuridine aldehyde

esterase, vinorine synthase and 17-O-acetyl-ajmalanesterase have been cloned.

Putative clones for vinorine hydroxylase, vomilenine reductase, and 1,2-

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dihydrovomilenene reductase have been isolated. N-Methyl-transferase activity and

sarpagan bridge enzyme activities have only been detected in crude cell extracts

(Connor and Maresh, 2006).

The biosynthetic pathway of ajmaline and indole alkaloid needs an enzyme

strictosidine synthase (STR1) which has been identified from the Indian medicinal

plant Rauvolfia serpentina. strictosidine synthase helps to initiate all the biosynthetic

pathways of monoterpenoid indole alkaloid family. The crystal structures of

strictosidine synthase is an complex substance made of tryptamine and secologanin

(Ma et al., 2006).

Expression of enzymatically active stictosidine synthase from Rauwolfia serpentina in

E.coli was reported by (Kutchan, 1989). The alkaloid content varies from 1.4-3%

depending on location, seation & soil condition (Farooqi & Sreeramu 2001).

Deserpidine which differs from reserpine only by the absence of a methoxy group at

c-11 was synthesized from reserpine (Varchi et al., 2005). The structure of Rauwolfia

serpentina stictosidine synthase is a novel – six bladed β-propeller fold in plant

protein was reported by Ma et al., (2006). Quantitative determination of reserpine,

ajimaline & ajimalicine this plant by reversed phase HPLC was reported by Sivastava

et al., (2006) and Goel et al., (2009).

The gene involved in strictosidine synthase has been identified from genomic libraries

prepared from Rauvolfia serpentina (India) and from Rauvolfia mannii (West Africa).

The gene strictosidine synthase has no introns and 100% nucleotide sequence

homology of 1180 bp, transcription of the R. serpentina gene was start at 81

nucleotides upstream from the AUG (26 nucleotides downstream from the TATA
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box). Transient expression assays in Nicotiana plumbaginifolia protoplasts of the R.

serpentina str1 5'-noncoding region fused to the beta-glucuronidase reporter gene

revealed promoter activity. R. serpentina has sequences specifically identified by Gel

retardation assays. The root and other parts of the R. serpentina plant has strictosidine

synthase poly(A)+ RNA (Bracher and Kutchan, 1992).

The cDNA sequence of Strictosidine glucosidase comparison with Catharanthus

roseus and raucaffricine glucosidase from Rauwolfia serpentina, primers were

designed and the cDNA encoding Strictosidine glucosidase was cloned from R.

serpentina cell suspension cultures and active enzyme was expressed in Escherichia

coli and purified its homogeneity (Gerasimenko et al., 2002).

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Enzymatic conversion of strictosidine and its Nβ-methyl derivative by

heterologously expressed strictosidine glucosidase from R. serpentine cell
suspension cultures.

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Structure of different types of alkaloids in Rauwolfia serpentina

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Molecular biological analysis

From the cultured plants the DNA homology degree and the number of repeated

sequences determined by Solovyan et al., (1986). The cDNA clone for strictosidine

synthase from R. serpentina and DNA sequence determination and it was expressed in

E. coli (Kutchan et al., 1988). Homogeneous strictosidine synthase from cell

suspension cultures (Hampp and Zenk, 1988). PCR reaction comparison of the gene

for strictosidine synthase from ten Rauwolfia species including R. serpentina was

reported by Bracher and Kutchan, (1992); Bracher and Kutchan, (1992); Gerasimenko

et al., (2002). Interspecies Polymorphism and variability of ribosomal RNA genes

was identified in Rauwolfia sepentina (Andreev et al., 2005). Genetic diversity of

Rauwolfia sepentina from six localities of Andhra Pradesh (India) performed by

RAPD analysis (Padmalatha and Prasad, 2007).

Conservation status

Rauwolfia serpentina is threatened with extinction in India due to indiscriminate

collection and over exploitation of natural resources for commercial purposes to meet

the requirement of the pharmaceutical industry, coupled with limited cultivation

(Nayar and Sastry, 1987, 1988, 1990; Mamagain et al., 1998; Singh et al., 2010).

Collection and conservation of this plant from south Karnataka and Western Ghats of

India were reported by Sethi and Kazim (1983). Ansari (1993) has stated that genetic

erosion has affected the species greatly and populations left in India have very poor

alkaloid content. It was found to be endangered in Southern Western Ghats of India

(Nayar, 1996). It has been categorized as globally endangered (Jadhav et al., 2001).

Raj and Sukumaran (2008) have reported this species as endangered and threatened in
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Kanyakumari district, India. The plant was described as critically endangered in the

Northeast India (Mao et al., 2009).

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