CHAPTER 11
REVIEW
OFLITERATURE
The Human brain is the most complex organ in any living thing. Encased in a bony
armour (the skull) and guarded by a series of chemical barriers, the brain has long
defied analysis. The convoluted cerebral cortex crowns the brain which at maturityweighs 3 pounds and numbers 50 to 100 billion nerve cells. These nerve cells are
highly specialized units called neurons. The neurons hold the key to the brain's subtle
and efficient system of communication. Each neuron has a long, white-sheathed tail
called an axon which transmits nerve impulses. The brain develops nerve cells at a
rate of about 360 million a day during the foetal development stage, this process halts
at birth. From then on, we live with what we have and less every day as the brain cells
die at a regular rate. However, proper nutrition, a stress free life and various other
environmental conditions impact the way our brain cells live. To protect these
complex cells, the brain is protected by protein sheets. These protein deposits protect
the brain from injury but an excess of protein deposits can damage the brain.
Therefore it is essential for the process to be regulated. Stress is a major cause of
damage to nerve cells.
Memory is an often misunderstood term. Everyone has a memory, some
have a better power of recall than others, but that is because those individuals have
trained one part of the brain to fixnction better that others. The only significant
difference is stress. Stress causes short term memory loss due to a lack of focus. This
is what is referred to as memory loss. Unfortunately, most of us must deal with stress
as a part of our daily existence.
Ayurveda is a truly holistic healing system that has been practiced in India
for more that 5,000 years. Ayurveda is a Sanskrit term meaning "science of life". Ayu
means "life" or "daily living"; veda mean "knowing". Ayurveda encompasses both the
spiritual and physical aspects of healing. A key concept in the Ayurvedic system of
healing is that of restoring the body's balance. The use of herbs, both as medicine and
15
as food, is one of the ways of restoring balance. Many of tlie herbs that have
traditionally been used in Ayurveda are being "rediscovered" by Westerners and are
being studied by Western and Indian scientists, with promising results.
In traditional practices of Ayurvedic and Chinese medicine, numerous
plants have been used to treat cognitive disorders, including neurodegenerative
diseases such as Alzheimer's disease (AD). An ethno pharmacological approach has
provided leads to identifying potential new drugs from plant sources, including those
for cognitive disorders. Many drugs currently available in Western medicine were
originally isolated from plants or are derived from templates of compounds isolated
from plants.
The nature provides a new opportunity to regain one's full mental
capacity. A number of herbs traditionally employed in the Indian System of medicine
'Ayurveda', have yielded positive results. Some of the herbs used as memory
enhancers are Centella asiatica, Bacopa monnieri ,Celastrus paniculatus, Sida
cordifolia, Valeriana officinalis, Withania somnifera, Vitex negundo, and Acorus
calamus .
Some of the Ayurvedic plants are reputed to be memory enhancers
(Medhya) and antiaging drugs (Vayahsthapana). Shankhapushpi (leaf) is one of the
prime medhya plants of Ayurveda; it may be useful for neural regeneration and
synaptic plasticity. Nordostachys Jatamansi (Jataamansi rhizome) appears to be an
excellent candidate for a potential inhibitor of AChE. Terminalia chebula (Haritaki
fruit) is highly prized in Ayurveda for antiaging. It's extract has displayed several
activities. Withania somnifera (Ashwagandha root) is another important antiaging
plant (Sandra et al.,2006). Aswagandha, chemically rich with its varied content of
active compounds, such as withanolidess, sitoindosides and many usefiil alkaloids,
and used for centuries to treat a wide range of diseases, constitutes a promising
candidate as a multi-purpose medicinal agent. However, more clinical trials need to be
carried out to support its therapeutic use (Mohammad et al. 2009). For example,
although the high potential of withanolide A for neuronal regeneration is well-known,
it would be dangerous to simply imply that this compound could be an excellent antidementia drug.
16
Withania somnifera, commonly known as Ashwagandha, is an important medicinal
plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years.
(The Wealth of India, CSIR: 1976). In Ayurveda, it is also used as a general energyenhancing tonic known as Medharasayana, which means 'that which promotes
learning and a good memory' and in geriatric problems (Nadkami, 1976; Williamson,
2002).
Ayurvedic medicine is the indigenous medicine of India. Although its
principles and prescriptions originate in Hindu scriptures (Veda) which predate
Alexander the Great (327BC), Ayurveda is still the main source of healing for India's
population. Balaraman (1973) cited ancient Ayurvedic prescriptions in which
Jyothismati was alleged to stimulate 'Medha' (intellect) and 'Smrithi' (memory).
The drugs promoting medha (intellect) are termed as medhya drugs.
Ayurvedic System of Medicine has mentioned several naturally occurring medicinal
plants under the category 'Medhya'. By virtue of inducing mental upliftment as major
influence several medicinal plants mentioned as 'Rasayan Drugs' in Ayurveda are
primarily claimed as 'medhya'.
Learning, memory, and cognitive disordersThis area of research is important for three reasons: a) there is a paucity of modern
drugs/agents facilitating acquisition, retention, and retrieval of information and
knowledge; b) with the increasing number of elderly people in the world population,
the need for drugs to treat cognitive disorders, such as senile dementia and
Alzheimer's disease, have acquired special urgency and c) Ayurveda claims that
several plants, the so-called "medhya" plants, possess such activities. The past two
decades liave seen tremendous advances in the area of brain physiology, learning,
memory, and various brain disorders, and a host of mechanisms at molecular level
have been delineated. Synapses-the junctions of nerve cells representing the basic
interactive unit of neuronal circuits - constitute the fundamental systemic relationship
within the brain. Understanding how this interactive multitude of neuronal circuitry is
established initially, and refined continuously throughout life, is fundamental to
understanding the molecular basis of learning and memory. At present, an impressive
17
array of chemical entities affecting synapse formation, neuronal differentiation,
neurotransmission, nerve growth and repair, and several other functions are
recognized.
Approximately 50 neurotransmitters belonging to diverse chemical groups have been
identified in the brain. Acetylcholine, the first neurotransmitter to be characterized,
has a ver>' significant presence in the brain; recently, Winkler et al., 1995 determined
that acetylcholine is essential for learning and memory.
Acetylcholine has been a special target for investigations for almost two
decades because its deficit, among other factors, has been held responsible for senile
dementia and other degenerative cognitive disorders, including Alzheimer's disease.
Approximately 5% of the neurons in the hippocampus (the part of the brain central to
learning, memory, and emotions) disappears with each decade after 50 years of age, and
the brain and the brain tries to compensate for this by further growth of the neurites
(neuron axon and dendrites) , which are vital for normal functioning of the brain. Thus,
nerve growth factor has an important role to play. The knowledge about
neurotransmitters, enzymes, growth factors relevant to memory and learning, and
cognitive disorders is already being used for the discovery and development of suitable
therapeutic agents. Major emphasis has been on acetylcholine. Because the number of
acetylcholine receptors declines with advancing age, inhibitors of acetylcholine esterase
(AChE), which terminates the action of acetylcholine, have been special targets for
development. Some anticholinesterase (anti-ChE) alkaloids isolated from plants have
been investigated for their potential in the treatment of AD, and are now in clinical use.
Galantamine, isolated from several plants including Lycoris radiata Herb, which was
used in Traditional Chinese Medicine (TCM), is licensed in the United Kingdom for the
treatment of mild to moderate AD. Various other plant species have shown
pharmacological activities relevant to the treatment of cognitive disorders, indicating
potential for therapeutic use in disorders such as AD.
Plants and their constituents with pharmacological activities that may be
relevant for the treatment of cognitive disorders, including enhancement of cholinergic
function in the central nervous system (CNS), anti-inflammatory and antioxidant
activities (Melanie-Jayne Howes and Peter Houghton,2003.) Alzheimer's disease (AD)
18
is a progressive, neurodegenerative disease that primarily affects the elderly population,
and is estimated to account for 50-60% of dementia cases in persons over 65 years of
age (Francis et al., 1999). The main symptoms associated with AD involve cognitive
dysfunction, primarily memory loss (Desgranges et al.,1998; Forstl et al., 1995;
Grafman et al., 1990 and Grosse et al., 1991). Other features associated with the later
stages of AD include language deficits, depression, behavioural problems including
agitation, mood disturbances and psychosis (Kumar et al., 1998; Mc Guffey, 1997 and
Wragg&Jeste, 1989).
The pathological features that have been identified in the central nervous
system (CNS) in AD are senile plaques and neurofibrillary tangles, oxidative and
inflammatory
processes
and
neurotransmitter
disturbances.
A
consistent
neuropathological occurrence associated with memory loss is a cholinergic deficit,
which has been correlated with the severity of AD ( Bierer et al.,1995; CoUerton,
1986; Giacobini,1990; Perry et al., 1978; Perry, 1986; Plotkin and Jarvik, 1986 and
Read, 1987). Thus, attempts to restore cholinergic function have been a rational target
for drugs used to treat the symptoms of AD. Approaches to enhance cholinergic
function in AD have included stimulation of cholinergic receptors or prolonging the
availability of acetylcholine (ACh) released into the neuronal synaptic cleft by
inhibiting ACh hydrolysis by acetylcholinesterase (AChE); the latter may be achieved
through the use of AChE inhibitors.
Implicated in the pathology of many diseases, including neurodegenerative
diseases such as AD, are free radical reactions, which are reported to initiate cell
injury. Consequently, the use of antioxidants has been explored in an attempt to slow
AD progression and neuronal degeneration. In traditional practices of medicine, plants
have been used to enhance cognitive function and to alleviate other symptoms
associated with AD. An ethno pharmacological approach may be useful in providing
leads to identify plants and potential new drugs that are relevant for the treatment of
cognitive disorders, including AD. The pharmacological basis of some plants and their
active constituents that have been used in traditional Ayurvedic medicine and TCM
for their reputed cognitive-enhancing effects. The reputed effects for some traditional
19
TABLE - 3: A list of plants used in Ayurvedic medical practice to treat mental
disorders.
SI.
Botanical Nomenclature
No.
Acorus calamus
j2. \Alternanthera
sessilij
|3^ \Argyreia speciosa
k._ ^spara£us
racemosus
5. \Bacopa monniera
T
jAyurvedic
Family
iNomenclature
• '
iVacha
.Araceae
JMatsyakshi
lAmarnanthaceae
jvidhara
iconvolvulaceae
jShatavari
jLiliaceae
iBrahmi
iScrophulariaceaej
6. Celastrus
JyothismatijCelastraceae
paniculatus
7. \Centella asiatica
;8^ \Clerodendrum
infortunatum
9. ]ciitoria
ternatea
10. Convolulus PjjJricau[[s^
11. {Curcujigo orchioides
12. Ib/qscorea bulbifera
ilS.^hydra
fluctuans
14. ]Glycyrrhiza glabara
15. \Jasminum sambac^
16. Mucuna pruriens
17. 'Nardostachys
jatamansi
1 8 . [Piper longum
_
il9. ^Terminalia chebula
So. ^Tinospora cordifolia
2 1 . \Valeriana wallichii
|22^ iWtex negundo
23. |l'V/t/7an/a somnifera
1
jMandukparni
iUmbelliferae
fshant
[Verbanaceae
JAparajita
iLeguminosae
jshankhpushpi [Convoluvalaceae
iKrishna musli iAmaryllidaceae
iDioscoreacae
fvarahi kand
jCompositae
iHilmochika
p/ashtimadhu
Leguminosae
jOleaceae
iBela
iKewanch
ILeguminosae
jJatamansi
ivalerianceae
jPiperaceae
'Pippali
Icombretaceae
IHaritaki
jMenispermacea
Amrita
iValerianaceae
Tagar
Verbenaceae
jNirgundi
Ashwagandha [Solanaceae
herbal drugs may not only be relevant in managing the cognitive decline that can be
associated with general ageing but may also be relevant in the treatment of specific
cognitive disorders such as AD. Thus, plants reputed to have antiageing or 'memoryenhancing' effects could also be considered for potential efficacy in disorders now
recognised to be associated with cognitive dysfunction, including conditions that
feature dementia. Plants have shown favourable effects in relation to cognitive
disorders, including anticholinesterase (anti-ChE), anti-inflammatory and antioxidant
activities, or other relevant pharmacological activities indicating the potential for
clinical use.
In Ayurveda, rasayana or science of rejuvenation is a unique concept. The
main objective of rasayana therapy is the medical management with the advancement
of age. The principal physiological effect of rasayana is to improve and revitalize the
physiological and endocrine functions of the body, to deaccelerate the aging process
and to make an individual more responsive and resistant to disease, i.e., to improve
body function by strengthening the immune system. The rasayanas are interpreted to
act through a psycho-endocrinological-immune axis. There are various classifications
of rasayanas, i.e. according to mode of administration (Carak) or site of action
(Susruta) etc. Medhya rasayana is used for rejuvenation of brain and mental health and
to promote intellect and memory. The names of plants used for treatment of mental
disorder and based on a contemporary authentic text are listed in the Table - 3
Stress is manifested as irritability, nervousness, sleeplessness etc. If the
process is not checked the person gets additional symptoms like palpitation, raised
blood pressure etc. As these changes continue he becomes a victim of one of the
psychosomatic disorders such as hypertension, ulcerative colitis, ischaemic heart
disease, peptic ulcer, diabetes mellitus, bronchial asthma, migraine, rheumatoid
arthritis etc. Large number of synthetic compounds are being practiced to minimize
the stress and its induced complications but none of them are devoid of their ill effects
in the long run. A much clear concept of health, physical as well as mental, has been
described in Ayurveda. They have also mentioned a large number of herbal
preparations which reduce anxiety and stress and are harmless too!
20
Stress is considered to be a risk factor of several diseases. The effect of
acute and chronic immobilization stress on brain acetylcholinesterase (AChE) enzyme
activity and cognitive function in mice was investigated by Das, A. et ah, in 2000.
Mice were immobilized by strapping for 150 min. One group of mice were only
immobilized once (acute stress) while in another group mice were immobilized (150
min) daily for 5 consecutive days (chronic stress). Specific AChE enzyme activity
ipimolmin" mg~') was estimated by a spectrophotometric method in the whole brain of
mice subjected to acute and chronic stress. In the acute stress group, AChE activity
was found to be significantly decreased in comparison to the control group. Chronic
stress did not cause any significant change in AChE activity .The study shows that
acute immobilization stress may enhance cognitive function in mice which may be
attributed to a decrease in AChE activity leading to an increase in cholinergic activity
in the brain.
Liu et al., in 1996 reported that immobilization stress of male Sprague-Dawley
rats induces oxidative damage to lipid, protein, and DNA in the brain. Significant
increases in lipid peroxidation were found in the cerebral cortex, cerebellum,
hippocampus, and midbrain compared to the unstressed controls. Significant increases
in levels of protein oxidation were also found in the cortex, hypothalamus, striatum,
and medulla oblongata. Oxidative nuclear DNA damage increased after stress in all
brain regions. Depletion of glutathione showed some stimulation to oxidative damage
in the unstressed control and stressed animals. Further studies of the mitochondrial and
cytosol fractions of cerebral cortex demonstrated that mitochondria showed a
significantly greater increase in lipid peroxidation and protein oxidation than cytosol.
Data fi"oin plasma and liver showed oxidative damage similar to that of the brain.
These findings by Liu et al., 1996 provide evidence to support the idea that stress
produces oxidants, and that the oxidative damage in stress could contribute to the
degenerative diseases of aging, including brain dysfunction. A number of Indian
medicinal plants have been used for thousands of years in the traditional system of
medicine (Ayurveda). Amongst these are plants
for
the management
of
neurodegenerative diseases such as Parkinson's, Alzheimer's, loss of memory.
21
degeneration of nerves and other neuronal disorders by the Ayurvedic practitioners.
Though the etiology of neurodegenerative diseases remains enigmatic, there is
evidence, which indicates that defective energy metabolism, excitotoxicity and
oxidative damage may be crucial factors (Beal, 1995).
The part of the Ayurvedic system that an approach to prevention and
treatment of degenerative diseases is known as Rasayana, and plants used for this
purpose are classed as rejuvenators. This group of plants generally possesses strong
antioxidant activity, but only a few have been investigated in detail. The relative
antioxidant capacity for the water infusions of the following plants was observed in
the following order: Evolvulus alsinoides > Cyanodon dactylon > Sida cordifolia. The
results of water infusions of the plants on lipid peroxidation were as follows:
E.alsinoides > S.cordifolia > C.dactylon (Auddy et al. 2003).
The active principles of Withania somnifera have been tested for
antioxidant activity by observing the levels of the major free-radical scavenging
enzymes, superoxide dismutase, catalase and glutathione peroxidase, in the rat brain
frontal cortex and striatum. The increase in these enzymes after treatment with
withanolides represent enhanced antioxidant activity and a corresponding protective
effect on neuronal tissue, suggesting that the antioxidant effect of W. somnifera in the
brain may be responsible for its diverse pharmacological properties (Bhattacharya et
al.l997). Similarly, oral administration of W. somnifera extracts prevented an increase
in lipid peroxidation in mice and rabbits (Dhuley, 1998).
Celastrus paniculatus Willd. (Jyothismati), commonly called the climbing
staff tree or the intellect tree, is an important medicinal plant belonging to the family
Celastraceae. It is a large woody unarmed climbing shrub reaching up to a height of
10 m and is distributed throughout India upto an altitude of 1200m mainly in
deciduous forests. The species is vulnerable in the Western Ghats of South India.
(Rajasekaran & Ganesan, 2002). Root bark extract shows antimalarial activity
(Rastogi and Mehrotra, 1998). Seeds are good at stimulating intellect (Prajapathi et
al.,2003). Among the Gonds tribe of Uttar Pradesh, India the powdered root is
22
considered useful for the treatment of cancerous tumours (Parotta, 2001).The seed oil
is bitter, thermogenic, intellect promoting and is useful for treating abdominal
disorders, beriberi and sores (Warrier et al.,1994).Chemical constituents are revealed
by phytochemical analysis were sesquiterpene alkaloids like celapagine, celapanigine
and celapanine (CSIR, 1992).
According to Ayurveda, leaves are emmenagogue whereas seeds are acrid,
bitter, hot, appetizer, laxative, emetic, aphrodisiac, powerful brain tonic, cause
burning sensation. Oil enriches blood and cures abdominal complaints. According on
Unani system of medicine, seeds are bitter, expectorant, brain and liver tonic, cure
joint-pains, paralysis and weakness. Oil stomachic, tonic, good for cough and asthma;
used in leprosy, cures headache and leucoderma. (Pankaj Oudhia, 2001).
Seed oiliness more than 50%. The oil is used for the lamp oil or for soap
making in Yunnan. Many pharmacological studies deal with its effects on the central
nervous system, and the tranquilizing property of the alkaloidal fractions or the oil.
Folk names frequently alert researchers to the therapeutic potential of
plants. Hence, two of the given names for C. paniculatus may be thought suggestive.
These are the evocative 'Intellect Tree' (Chopra, 1956) and the romantic ' Jyothismati'.
Jyothismati is an ancient Sanskrit word which means possessor of the light' (Dymock
et al., 1890). Jyothismati is pungent and bitter in taste pungent in the post digestive
effect and hot in potency. It alleviates kapha and vata doshas. It possesses tiksna sharp, sara - laxative and sthira - stable attributes. It stimulates intellect and promotes
the memory with its special potency - prabhava. It is a rejuvenative, nervine, appetizer
and aphrodisiac in properties and is used in the diseases like anorexia, erysipelas,
wounds, minor memory disturbances and anxiety neurosis.
The seeds and seed - oil have great medicinal value. Internally, Jyothismati
is used in vast range of diseases. It is one of the best nervine and its seed oil is given
along with ghee (prepared from cow's milk) to stimulate intellect and to promote
memory. The leave's juice, daily 50 ml. relieves the opium addiction. The seed oil
tackles the digestive problems of anorexia and flatulence. Being hot and sharp in
23
properties, Jyothismati is rewarding in cough and bronchial asthma to alleviate kapha
dosa. It also is a stimulant to kidneys, hence useful in dysuria to augment the urinary
output. In males, it works well as an aphrodisiac and in females it is salutary to
regulate the menstrual cycle, in arthritis, the seeds are given along with sunthi and
ajamoda in equal quantity, with honey. Jyothismati works well as anti pruritic in skin
diseases. It is also used as a tranquilliser in anxiety neurosis. It is diaphoretic and
benevolent in fever. Jyothismati stimulates the heart, improves circulation and
alleviates the edema. The leaves, cooked as vegetables, are recommended in
dysmenorrhea to alleviate the pain (Rajkumar et al., 2007). Recent preclinical studies
of the seed extract of Celastrus paniculatus on male rats showed an improvement in
learning and memory in both the shuttle-box and step-through paradigms. The study
also demonstrates that the cognitive- enhancing properties of extract of Celastrus
paniculatus seed could attributed to its antioxidant effect.
'Jyothismati' {Celastrus paniculatus) - Powerful nervine and brain tonic which
stimulates the intellect and sharpens the memory. Celastrus paniculatus is an antistress agent and improves motor learning process. In children with mental deficiency,
this gives amazing results of improvement (Alchemy for sound health - Nurax herbal
solutions; Copyright 2004 Rich International India Pvt Ltd).
Gaitonde et al (1957) found tranquilizing effects when they gave high doses of
Jyothismati to rats. Joglekar and Balwani (1967) reported that a polyester of
Jyothismati demonstrated a mild tranquilizing effect and potentiated morphine
analgesia in high dose rate studies. Extensive clinical work was conducted by Hakim
(1965). He used a formula comprised mainly of Jyothismati against a wide variety of
mental dysfunction. He reported good results against depression and excellent results
against hysteria (78.9%) success. There were no side effects.
As previously reported Singh et al (1974) studied an alcoholic extract
(including seeds) in a variety of tests. They concluded the extract possessed "several
important pharmacological properties and the low toxicity indicate the chemical
isolation and characterization of active principle(s)".
24
Jyothismati is frequently referred to as a therapeutic with low toxicity Hakim
(1965) reported the usual doses of Jyothismati is between 300 - 500 mg a day. He
further reported that some Ayurvedic literature prescribed up to 2 grams a day. In
addition he drew attention to animal studies in which unspecified 'high doses' were
non-toxic. All the evidence on hand supports the claim Jyothismati has low toxicity
hence giving in an exceptional margin of safety.
The adaptogenic, antibacterial, and possible antiviral activity and activity
against Beriberi's polyneuritus suggests it may be of help to HIV sufferers. Activity
against polyneuritis and anticonvulsant activity suggest Jyotishmati may also offer
some relief to multiple sclerosis victims. Its range of physiological activity and low
toxicity suggests Jyothismati may find use as an ideal fortification for those over 40.
Is it possible for a herb to exhibit effectiveness against a range of
disorders? Several lines of evidence are persuasive. Apart from ancient, enduring and
widespread traditional claims for Jyothismati, there is scientific confirmation for
earning, memory and antibacterial activity. Moreover, there is scientific support from
Singh et al's (1974) observation that the plant extract possesses 'several important
pharmacological properties. Furthermore, it is well established that natural brain
messengers (neuro hormones) frequently mediate various physiological functions. For
example, acetycholine is known to be integral to memory, all body movement, and
implicated with pain control. Hence one would expect a compound that demonstrated
acetylcholine activity to be active over several areas. Finally a complex natural oil
such as Jyothismati possesses many chemical compounds, these may mediate various
physiological activities.
The effect of aqueous extract of Celastrus paniculatus (Cp) (100, 200 and 300
mg/kg for 21 days once a day) on ICV streptozotocin (STZ) induced cognitive
impairment and oxidative stress in rats. Male Wistar rats were injected ICV STZ (3
mg/kg) bilaterally on days 1 and 3. The cognitive behaviour was assessed using
passive avoidance and elevated plus maze paradigms on days 13, 14 and 21. The rats
were sacrificed on the 21st day for the estimation of oxidative stress parameters
25
(malondialdehyde [MDA], glutathione, superoxide dismutase [SOD] and catalase) in
the whole brain upon completion of the behavioural task. The rats treated with Cp
showed a dose dependent increase in the cognitive behaviour in both the paradigms.
Significant lower level of MDA, and higher level of glutathione was observed in only
200 and 300 mg/kg Cp treated rats. These findings indicate that aqueous extract of Cp
was effective in preventing the cognitive deficits as well as the oxidative stress caused
by ICV STZ in rats( Veerendra Kumar & Gupta ,2003).
Ayurvedic prescriptions specified the use of Jyothismati against mental
illness. Prakasa and Sreeramulu (1985) report Jyothismati is used in Andhra Pradesh
to treat unspecified mental disorders. Dandiya and Chopra (1966) cite Desai (1927) as
authority for the use of Jyothismati against mental illness.
Many pharmacological studies deal with the effects of Celastrus paniculatus on
the central nervous system and the tranquilizing property of the alkaloidal fractions of
the oil (Joglekar and Belwani, 1967). The oil obtained from the seeds of Celastrus
produced sedation in the rats in a dose of Ig/kg intramuscularly. On intraperitoneal
administration, the same dose of oil produced sedation but the effect was not so
marked. The oil showed anticonvulsant activity in rats. The oil (as emulsion in
between 80 and water), at a dose of 50-100 mg/kg, produced a gradual fall in the
blood pressure of cats. At a dose of 20mg/kg, the oil produced a fall in cardiac output,
brachycardia and a marked increase in pulse pressure on isolated heart lung
preparation. (Gaitonde et.al.,1957). The extract showed marked CNS depressing
activity (230 + 5 mg/kg body weight, intra peritoneal) without loss of the righting
reflex even in larger doses than above ( Singh et. all974).
Studies have been done
on the tranquilisingfiractionof the Celastrus seed oil. (Seth et al.,1963).
The effect of Celastrus paniculatus on the memory process has also been evaluated
(Karanth et.al.l980). Clinical observations on the use of the oil in psychiatric practice
were promising (Hakim, 1964).
The products of Celastrus paniculatus used clinically are considered safe, with
low toxicity. (Singh et al. 1974).
C. paniculatus (Celastraceae) seeds and seed oil have been used in Ayurvedic
medicine for "stimulating intellect and sharpening the memory" (Nadkarni,1976 and
26
Warrier et al.,1995). Most of the studies undertaken to establish any pharmacological
basis for the reputed effects have focussed on the seeds and seed oil. When
administered orally to rats, the seed oil decreased levels of noradrenalin, dopamine
and 5-hydroxytryptamine (5-HT) in the brain which was correlated with an
improvement in learning and memory processes; in addition, the oil was not shown to
be neurotoxic. (Nalini et al. 1995)
An antioxidant effect in the CNS, observed with an aqueous seed extract, may
also explain the reputed benefits on memory, since this extract enhanced cognition in
vivo (Kumar and Gupta, 2002a).
The effects of aqueous extracts of Celastrus paniculatus (Cp) seeds were
shown to have antioxidant properties in rats. In a study the free radical scavenging
capacity of three aqueous extracts (WSEs) obtained from Cp seeds: a room
temperature extract (WF); a hot water extract (HF); an acid extract (AF) were
investigated. All the WSEs exhibited a dose-dependent free radical scavenging
capacity for l,l-diphenyl-2-picryl-hydrazyl radical (DPPH) and also for superoxidegenerated assays (in vitro assays). Enriched forebrain primary neuronal cell (FBNC)
cultures were used to evaluate the neuroprotective effects of the three Cp-WSE
extracts on HiOi-induced toxicity. FBNC were pre-treated with the Cp-WSE and then
with H2O2 to evaluate the protection afforded against H202-induced toxicity. All the
WSEs significantly attenuated H2O2 induced neuronal death, and AF was the most
effective in protecting the neuronal cells against oxidative injury caused by H2O2. In
10 day FBNC, cellular superoxide dismutase activity was not affected by the WSEs or
H2O2, but catalase activity was decreased and levels of malondialdehyde were
increased by H2O2 treatment. When the neuronal cells were treated with WSEs prior
to H2O2 exposure, catalase activity was increased and levels of malondialdehyde were
decreased significantly. The data presented suggest that Cp seed WSEs protected
neuronal cells in part by their free radical scavenging properties, by reducing lipid
peroxidation, and also by their ability to induce the antioxidant enzyme catalase. The
results indicate that WSEs might exert neuroprotective effects against increased
27
oxidative stress resulting from free radical damage that is associated with a number of
neurodegenerative diseases. (Godkar et al.,2003).
I'he oil of Celastrus paniculatus was fractionated into polar and semipolar
compounds and these fractions were injected to rats for one month. Serum
transaminases (SGOT and SGPT), alkaline phosphatase, calcium, creatinine, uric acid
and blood urea of treated rats were estimated. These oil fractions were not found to be
harmfial in the long run (Bidwai, et.al.,1990/ Celastrus has been traditionally used in
products that enhance memory. Researchers in India found that the alcohol extract of
Cpaniculatus
possesses tranquilizing, sedative, central muscle relaxant, anti-
inflammatory, anti-pyretic, anti-ulcerogenic, anti-emetic, analgesic and adaptogenic
properties. Other researchers reported the seed oil extract oi C.paniculatus obtained
from Amsar Private Limited, to have memory enhancing effect besides anti-anxiety
and lipid lowering properties in albino rats of either sex. Celastrus paniculatus
decreased transfer latency in elevated plus maze in the rats receiving 60 mg/kg and
decreased the secondary avoidance blockade in conditioned avoidance response.
(Ayurvedic medicine from Amsar Pvt. Ltd. Av Memvita - Antistress, memory
enhancing agent)
FUTURE HORIZONS:
The most salubrious of all therapeutic are those which prevent attack by diseases.
Hence adaptogens and the closely related class of compounds known as
immunostimulants, are of great interest to medical science and healers. Adaptogens
are compounds that strengthen the body against all forms of stress. Stress is known to
provoke a wide range of dysftmction's including a weakening of the immunosystem,
thus opening the way for opportunist infections. Vohora (1985) reports Unani-Tibb
medicine considers .Tyothismati to be an adaptogen. Singh et al., 1974 found activity
indicative of adaptogenic properties, when they conducted a scientific evaluation of
the whole plant extract.
28
CITATIONS IN MATERIA MEDICA ON CELASTRUSPANICULATUS
Indian Materia Medica (Nadkarni, 1927)... "It is also used in the form of a
pomatum made by mixing one part of the oil in 8 parts of butter for application to the
head. It is known as Magzsudhi (brain clearer) and is believed to promote intelligence.
Indian Medicinal Plants (Kirtikar and Basu, 1918)..." The seeds are supposed to
have the property of stimulating the intellect and sharpening the memory..."
Indigenous Drugs of India (Chopra, 1958) "The oil is reputed to be a nerve
stimulant and is considered as a brain tonic.
Pharmacographia Indica (Dymock et al., 1890)..." Light possessing is an
allusion to their supposed property of stimulating the intellectual powers and
sharpening the memory", and the" oil is used in the Courts and Colleges of India by a
great many pundits to increase the intelligence of their pupils".
^^Celastrus paniculatus
has been highly recommended by Ayurvedic
physicians as a good nerve tonic."Vegetable Drugs of India ,Sanyal and Ghose (1934).
Scientific confirmation of Jyothismati's ability to enhance learning and
memory, was provided by Karanth and fellow scientists at the Pharmacology
Department of Kasturba Medical College. (Karanth et al.,1980, 1981).
Animals provide reliable models for learning and memory, because there is no
chance of a psychological bias. Hence rats were used and the assessment method
devised by Plotnikoff (1966). Rat controls were given a placebo, or vasopression, a
natural hormone known to enhance the learning process in animals and humans, but
possessing serious side effects that limit continual clinical use; other rats were given
an oral dose of 1 millilitre of 5% emulsion of Jyothismati for 3 days and 7 days. Rats
were place in a shuttle box with a metal grill under foot, at the other end of the shuttle
box was a raised platform. A buzzer was sounded for 15 seconds. During the last 5
29
seconds a mild electric current was passed through the metal grill. The time required
for the rats to jump up to the raised, platform was used to assess learning and memory
over a number of trials. In the first trial the time to jump was about 28 seconds for rats
given a placebo and rats given vasopressin or Jyothismati. But on the third trial, rats
given a placebo took about 26 seconds to jump, whereas the rats treated with
Jyothismati and vasopressin, took about 15 seconds, This difference between the two
groups persisted to the 10th and final trial. These scientists concluded that Jyothismati,
significantly hastened the learning process in rats after 3 days administration,
fiirthermore, they found it also brought about a highly significant improvement in the
memory process.
Hakim (1951) reported to the British Medical Journal under the heading.
'Indian Remedies for Poor Memories'. He wrote that he possessed clinical records
covering over 100 years of continual family medical practice. He fiirther wrote that
Jyothismati had been used with very favourable results in a large number of cases
such as juvenile feeble mindedness, impairment of memory and deterioration of
general mental function (dementia).So far neuroscience has presented medicine with
no answers for treatment of Alzheimer's disease. For example. Perry (1987) wrote in
the authoritative psychopharmacology. The Third Generation of Progress: 'The critical
need for a drug to relieve the cognitive, especially memory impairment in the early
stages (of Alzheimer's Disease) and to control the broad range of dementing
symptoms in later stages can hardly be over emphasized.
In light of this 'critical need' the Plant Medicine Research Institute is presently
investigating fiarther studies with Jyothismati, mindful that even small amounts of
relief are gains against this distressing disease.
Jyothismati is widely used against bacterial and viral induced diseases in traditional
medicine. These include skin diseases, stomach orders, syphilis and sores. In addition
it is used to treat leprosy, pneumonia in children and wounds. Vohora (1985) reported
that Jyothismati is considered to be anti-bacterial and anti-viral in Unan-Tibb
30
medicine. Jyothismati holds much promise in the area, particularly so when one
considers its antibacterial and possible anti-viral activity.
The effects of an indigenous drug, Celastrus oil, extracted from the seeds
of Celastrus paniculatus on learning and memory in a two compartment passive
\avoidance task was studied in albino rats. The effects on the contents of
norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the brain and on the
levels of their metabolites both in the brain and urine were also assessed. Significant
improvement was observed in the retention ability of the drug treated rats compared
with the saline administered controls. The contents of NE, DA and 5-HT and their
metabolites in the brain were significantly decreased in the drug treated group. The
urinary metabolite levels were also significantly decreased except for total 3-methoxy4-hydroxyphenyl glycol. These data indicate that Celastrus oil causes an overall
decrease in the turnover of all the three central monoamines and implicate the
involvement of these aminergic systems in the learning and memory process.
(Nalini,et al., 1995).
The Celastrus oil, extracted from seeds of Celastrus paniculatus tested at 2 dose
levels (1 and 1,5 g/kg), exhibited significant anxiolytic activity and did not produce
tolerance. The non-sedative nature and reversal of buspirone induced behaviour (in
open field exploration) point to the serotonergic mechanism underlying the anxiolysis,
inspiring further research( Ramamoorthy Rajkumar et al., 2006).
BR-16A is a herbal (non allopathic) medication used in India to enhance
cognition. In the first experiment conducted by Jerry Joseph et al., in 1994, 28 Wistar
rats received either BR-16A (200 mg/kg/day) or vehicle alone for 3 weeks. During the
third week, the rats were tested for learning in the Hebb Williams complex maze. BR16A treated rats showed significantly better learning than did controls. Experiment 2
was conducted identically except that during the second week all of 32 rats
additionally received six once-daily electroconvulsive shocks (ECS). An advantage
for learning was again demonstrated for the BR-16A group. It is concluded that BR16A facilitates learning, and that this effect extends to a protection against ECSinduced anterograde amnesia.BR-16A (Mentat: The Himalaya Drug Company) is a
31
herbal medication derived from Ayurveda, a system of healing in India's rich culture
in traditional medicine. Important ingredients of BR-16A suggested to improve
memory
function
Mandookaparni
include
(Centella
the
following:
asiatica),
Jal-brahmi
Ashwagandha
{Bacopa
{Withania
monnieri),
somniferd).
Shankapushpi (Evolvulus alsinoides), Jatamansi {Nardostachys jatamansi). Vach
(Acorus calamus) Malkangni (Celasirus paniculatus) & Sonth {Zingiber officinale).
In normal volunteers, BR-16A was found to improve memory parameters and
decrease anxiety parameter after 12 weeks of administration in the dose of 350 mg
twice daily (Agrawal et al., 1990a, 1990b). Verma and Kulkarni (1991) studied mice
in an elevated plus maze. Transfer latency, the time taken by the animal to leave the
open arm and enter the closed arm, was used as a measure of learning. Pre-treatment
with BR-16A (50-100 mg/kg) was found to produce a dose-dependant reversal of
scopolamine-induced prolongation of the transfer latency.
Kulkarni and Verma (1992a) studied mice using an avoidance paradigm. The
apparatus consisted of a grid through which an electric shock could be delivered with
an elevated, shock-free zone in the center of the grid. Latency to reach the shock-free
zone was recorded as were the number of descents onto the grid over a period of 15
min. These two variables operationalised the efficiency of recall of the electric shock
offered by the grid during training. In separate experiments, scopolamine, single ECS
and,6 once daily ECS were found to induce amnesia as evidenced by increases in
latency and increases in the number of descents. Pre-treatment with BR-16A (50 to
500 mg/kg) was found to result in dose-dependant attenuations of these indices of
amnesia.
Andrade et al., (1994) used a reward-oriented paradigm to study the baseline
learning behaviour of rats in a T maze and in a complex maze. Number of trials to
satisfactory learning and number of wrong arm entries were recorded as the learning
variables in the T maze experiment, whereas time taken by the animal to reach the
reward chamber was recorded as the learning variable in the complex maze
experiment. After establishing baselines, rats were treated with BR-16A (200
mg/kg/day) or vehicle for 3 weeks. During the second week of treatment, all rats
32
received 6 once daily ECS. Learning assessments were resumed during the tliird week
of treatment and were compared with baseUne scores. In both experiments, BR-16A
(200 mg/kg/day) was observed to attenuate ECS-induced learning deficits.
The mechanism of action of BR-16A is unclear. Cholinergic facilitation may be
involved, as the drug reverses scopolamine-induced memory deficits (Verma and
Kulkami 1991; Kulkarni and Verma 1992). BR-16A may also influence opioid
neurotransmission: Kulkarni and Verma found the drug to prevent the development of
tolerance and dependence to morphine in mice. Preliminary work indicates no effects
on adrenergic or dopaminergic neurotransmission.
TISSUE CULTURE:
DIRECT ORGANOGENESIS:
Pioneering work in the field of direct organogenesis has been reported in
legumes (Favre, 1977; Bajaj and Dhanju, 1979; Horseh et al., 1987) and the trend has
extended to ornamental plants i.e. direct adventitious shoot organogenesis from the leaf of
African violet-a popular omamental plant (Start and Cummig, 1976). Similar innovative
studies pertaining to direct organogenesis has been achieved from fragmented leaves and
shoots apices of Vigna and intemodal segments of grape vine (Rajasekaran and MuUins,
1931) yet another plant of commercial value.
Adventitious shoots were formed at a high concentration of BAP, Kn and Zn
(Huetteman and Preece, 1993). BAP was ideally suitable for direct organogenesis in a
number of plants like Vitis species (Gray and Meredith, 1992, Stamp et al., 1990 and
James et al., 1990), mango stem (Chong et al.,1994), Sesamum leaf (Manjusharma and
Pareek, 1998). Augustine and D' Souza (1997) observed direct organogenesis in leaf
explants of Curcutigo orchioides on MS medium without cytokinin. Similar results
were observed in the leaf explants of grapevine (Tang and Mullins, 1990);
Liquidambar styraciflua (Brand and Lineberger, 1991) and Enicostemma axiiiare
(Sudherson, 1998), whilst on the other hand stem explants produced maximum
number of adventitious shoots in Onobrychis viciifolia cultured on MS medium
33
supplemented with BA (20 uM) in combination with NAA (0.5 uM) (Mural ozgen et
al., 1998) and carnation (Nugent et al., 1991; Van Altvorst et al, 1995). In
contradiction to both observations, direct shoot formation was observed from
intemodal explants (Shanthi and Anne Xavier, 2002) in Ruta graveolens on MS
medium containing BAP (13.32 uM) and NAA (5.37 uM).
An efficient adventitious shoots of Guazuma crinita was reported by
Maruyama et al., (1997) on MS medium containing Zn (10 u.M), while Son and Hall,
(1990) achieved it with Zn (22 uM) in Poputus albs x P. grandidentata hybrids. On
the other hand adventitious shoots were produced in BAP (4.41 uM) in Curcutigo
orchioides by Augustine and D'Souza (1997) which contradicted to the above two
reports.
Regeneration of plantlets through micro propagation has been established
(HoUings, 1965) and further strengthened by Murashige (1974). Various explants
such as shoot tip of Aristolochia indica (Manjula et al., 1997), axillary node of
Coriandrum sativum (Kataeva and Popowich, 1993), young shoot base of
Chlorophytum borivitianum (Purohit et al., 1994), cotyledonary node of Solarium
viarum (Tejavathi and Bhuvana, 1996) and axillary bud of Psoralen corylfolia
(Saxena et al., 1998) have been manipulated to bring forth regeneration.
Multiple shoots were achieved using shoot tip of Withania somnifera (Sen and
Sharma, 1991), Naregamia alata (Benny Daniel et al., 1999), Holarrhenapubescens
(Sumana et al., 1999), Cunila galioides (Fracaro and Echeverrigaray, 2001),
Solarium surattense (Pawar et al., 2002), Wedelia calendulacea (Emmanuel et al.,
2000) and Vitex negundo (Thiruvengadam and Jayabalan,2000). Multiple shoot
proliferation have been reported using axillary buds of Adhatoda beddomei (Sudha
and Seeni, 1994), Ocimum amehcanum and O. sanctum (Pattnaik and Chand, 1996),
Cleistanthus collinus (Quraishi and Mishra,1998), Uraria picta (Ajithanand et al.,
1998), Withania somnifera (Handique and Pranjal Bora, 1999) and Lilium nepalense
(Wawroschetal.,2001).
34
Nodal explants have been extensively used for micropropagation in
Angelonia salicahefolia (Datta and Datta, 1984), Asparagus racemosus (Dipak
Kumar and Sumitrasen, 1985), Eclipta alba (Franca et al., 1995), Gymnema elegans
(Komalavalli and Rao, 1997), Mondia whitei (McCartan and Van Staden, 1998),
Leucas lavandulifolia (Das et al., 1999), Bacopa monnieri (Tiwari et al., 2000),
Wedelia chinensis (Puspa-Bhuyan et al.,2000) .Anisomeles indica (John Britto et al.,
2001) and Plumbago zeylanica (Rout et al., 2001).
Growth regulators such as BAP, NAA, AdS, BA, lAA and Kn have been
used to regenerate plantlets, either individually or in combination with other
regulators. Among these growth regulators, BAP is the most frequently used (Sen
and Sharma, 1991; Begum et al., 2000; Fracaro and Echeverrigaray, 2001) for
micropropagation of Ocimum sanctum (2 mg/1), Withania somnifera (2 mg/1) and
Cuniia galioides (8.8 uM). Besides BA (1 mg/1) and AdS (5.44 uM) were the other
growth regulators successfully employed individually for regeneration of Eucommia
ulmoides (Li-Jingchen et al., 1995) and Gloriosa superba (Sivakumar and
Krishnamurthy, 2000).
In vitro regeneration of multiple shoots on MS medium supplemented with
BAP (1 mg/1) in combination with NAA (0.5 mg/1) was observed in Centella
asiatica (Hossain et al., 2000). Similar combination has been used by Krishna and
Seeni (1994), Boro et al., (1998) and Anitha & PuUaiah (2002) for Woodcordia
fruticosa, Alternanthera sessilis and Sterculia foetida respectively. Like wise BAP
has been used to induce shoot proliferation with other regulators such as AdS (25
mg/1) in Plumbago indica (Smitachetia and Handique, 2000), Kn (0.5 mg/1) in
Canavalia virosa (Kathiravan and Ignacimuthu, 1999) and lAA (2 mg/1) in
Boerhaavia diffusa (Phukan et al., 1999).
MS medium supplemented with BA (0.5 mg/1) and lAA (0.01 mg/1) had been
used to induce multiple shoot production in Psoralen corylifolia (Saxena, et al.,
1998) and Tridax procumbens (Sahoo and Chand, 1998). Sudha and Seeni (1996)
have reported micropropagation in Rauvolfia micrantha on MS medium with BA
35
(13.2 uM) in combination with NAA (2.68 uM). Amaranthus hypochondriacus has
been successfully regenerated (Pramod Kumar and Dube, 1997) using combination
of Kn (0.5 mg/1) and lAA (0.5 mg/1).
Clonal propagation studies on tree species on MS medium include that of
Emblica officinalis (Bhanu Verma and Kant, 1996), Ficus religiosa (Nagaraju et al.,
1998) and Sterculia foetida (Anitha and PuUaiah, 2002).
INDIRECT ORGANOGENESIS
Totipotency is the base for regeneration and propagation of selected genotypes
through tissue culture (Haberlandt,1902) and likewise studies pertaining to
organogenesis were carried out in vitro using callus cultures (Hickes, 1980; Gosal and
Bajaj, 1979; Singh et al., 1982). Tobacco was the classical plant species used for
illustrating de novo organogenesis (Skoog and Miller, 1958). Explant tissues generally
show distinct planes of cell division, various specializations of cells and organization
into specialized structures such as the vascular systems. Callus formation from explant
tissue involves the development of progressively more random planes of ceil division,
less frequent specialization of cells and loss of organized structures (Thorpe, 1980;
Wagley et al., 1987).
BA was found to be more effective than Kn in the proliferation and
development of apple shoots (Lundergan and Janick, 1980). However, in leaf and
intemodal explants of Datura metel, the maximum shoot proliferation was
observed at BAP (2.0 mg/1) (Muthukumar and Arockiasamy, 1998; Arockiasamy et
al.,
1999).
Auxin
and
cytokinin
was
beneficial
for
inducing
regeneration in Frageria x Ananassa hybrids (Liu and Sanford, 1988) and
Fagopyrum esculentum (Nescoric et al., 1987). Regeneration potential of different
portions of leaf explants has been studied in Rosmarinus officinalis. (Mishra, 1987)
and Piper colubrinum (Kelkar et al., 1996). In both cases shoot buds were induced and
elongated on half strength MS medium containing NAA (2.0 mg/1).
Shoot differentiation was obtained from green compact nodular calli derived from
different explants of Azadirachta indica (Abubacker and Alagumanian, 1999) in
36
Plumbago zeylanica, plant differentiation was achieved from leaf and stem explants
(Rout et al, 1999) on MS basal medium supplemented with BAP (4.44 micromole),
lAA (1.42 micromole) and sucrose (3%). Prem and Xu, (1999) studied shoot bud
regeneration from primary callus of root and leaf on MS medium supplemented with BAP
(3.0 mg/1), coconut milk (5%) and sucrose (60 gm/1).
Plant regeneration from axillary nodes has been reported in Gymnema sylvestre on
MS medium supplemented with BA (1 mg/1), Kn (0.5 mg/1) and NAA (0.1 mg/1)
(Komalavalli and Rao, 2000). Maximum shoot bud differentiation (100%) occurred,
when leaf explants with their adaxial surface and stem explants were placed horizontally in
the MS medium containing BAP or Kn (2.0 micromole) and gelrite (0.2%) in Bacopa
monnieri (Shrivastava and Ranjani, 1999).
A protocol for in vifro shoot regeneration in leaf explants of Neotegelia crunenta
has been developed (Carneiro et al., 1999). A method was developed for in vitro
multiple shoot production from cotyledonary node and shoot tip explants of Macrotyloma
uniflorum on MS medium containing BAP (1-5 mg/1), AdS (1.0 mg/1), 2ip (0.5 mg/1) and
GA3 (0.5 mg/1) (Mohamed et al., 1999). Cotyledon explants derived callus in
Corchorus capsularis on medium containing NAA (2.7 M) and BAP (4.4 iM) (Saha
et al., 1999); cotyledon and hypocotyl explants of Aegle marmelos on MS basal
medium supplemented with different
auxins in combination with cytokinin
(Arumugam et al., 1997); leaf and shoot explants in Sempervivum tectorum (Dobo et
al., 1994) and high frequency regeneration of plantlets from different explants and
callus culture of Paspalum scrobicufatum in MS medium containing BAP (3 mg/1)
(Arockiasamy et al., 2001) have been reported.
Shoot differentiation has been reported from callus culture of
Datura innoxia in MS medium containing BAP (Engvild,1973) and in vitro
propagation for Datura insignis using nodal explants supplemented with BA alone or
in combination with 2,4-D or lAA were studied. It was inferred that BA (1.0 mg/1)
resulted in maximum shoot multiplication and elongation. Rooting was achieved on
medium free of growth regulator (Santos et al., 1990). Several workers have described
the formation of tropane alkaloids in the callus cultures of Datura plants but their
alkaloid content was much lower than that of the mother plants (Chan and Staba,
1965; Staba and Jindra, 1968).
Sen and Sharma (1991) cultured shoot tips of Withania somnifera and
obtained maximum number of (multiple) shoots with BA (4.4 uM) from germinating
seeds.
Baburaj and Thamizhchelvan (1991) reported regeneration of Solarium
surattense from leaf callus on MS medium fortified with NAA and BAP. Regenerated
plantlets were rooted using lAA. In Solarium paludosum multiple shoots were raised from
organogenic callus cultures using root, hypocotyl and cotyledonary explants (Badaoui et
al., 1996). Magioli et al., (1998) used Solarium melongena, leaf hypocotyl, node,
cotyledon, epicotyl and leaf with thidiazuron (0.2 micro mole) resulting in shoot bud
induction with success rate of 75 -100% and rooting with lAA (0.6 micromole).
Baburaj and Gunasekaran (1995) reported shoot bud differentiation from leaf
explants of Withania somnifera cultured on MS medium containing different
combinations of auxin and cytokinin, Jaiswal and Pratap Narayan (1985) reported
regeneration from callus of stem segments of adult plants of Ficus religiosa cultured on
MS medium with 2,4-D (1.0 mg/1) and shoot regeneration in BAP (2.0 mg/1) and
rooting in NAA (1.0 mg/1).
Rekha et al. (2005) reported seed germination as low as 11.5%.
Propagation through tissue culture is a viable alternative in this species because it
could be used as a strategy for conservation (Nair and Seeni 2001; Arya et al.
2002).
Arya et al. (2002) reported multiplication in a medium containing NAA and
that was fiirther enriched with additives such as adenine sulphate, arginine, acid and
citric acid. Cultures retained for more than six weeks showed necrosis of shoot tips
caused by nutritional deficiencies.(Nair and Seeni, 2001), and this can be overcome
by reducing the subculturing cycle duration (Patnaik and Debata, 1996) to 4 weeks.
38
Arya et al .(2002)- A protocol was developed using nodal shoot segments as
explants for mass/clonal propagation of Celastrus paniculatus—an important
threatened medicinal plant. Four to five shoots differentiated from nodal region
within 15-20 days on Murashige and Skoog's (MS; 1962) medium containing 1.5
mg 1 1 6-benzylamino purine (BAP) + 0.1 mg 1 1 naphthaleneacetic acid (NAA) +
additives (50 mg 1 1 ascorbic acid and 25 mg 1 1 each of adenine sulfate, arginine
and citric acid) at 28°C temperature and 36 mol m 2 s Iphoton flux density, 10
h/day photoperiod. In vitro produced shoots were further multiplied by subculturing
on modified Murashige and Skoog's (MMS) medium containing 0.8 mg / 1 BAP +
0.1 mg / 1 NAA + additives. Five to seven shoots regenerated from each node of
subcultured shoots; on subsequent sub-culturing rates of multiplication were
increased to 10-15 folds. The mother explant could also be repeatedly transferred
onto fresh medium to yield fresh crops of shoots. After 10-12 culture cycles the
cultures exhibited hyper hydration and callusing. These could be checked by
lowering the BAP concentrations to 0.25 mg / 1 and that of NAA to 0.01 mg / 1.
The clonally-produced shoots were treated with a solution containing 100 mg 1 1
each of indole-3-butyric acid (IBA) and -naphthoxy acetic acid (NOA). The auxinpulsed shoots were subsequently treated with 10.0 mg / 1 solution of chlorogenic
acid for 3 minutes. The pulsed shoots were kept in glass bottles containing soilrite
in greenhouse at 28 °C. Of these 83% rooted ex vitro. The ex vitro rooted plantlets
were hardened and transferred to polybags /earthen pots containing black soil, fine
sand, farm yard manure and soilrite in the ratio of 4:2:1 :l(v/v). To date 7500 shoots
have been produced from 10 explants originating from a single plant. Two
thousand plantlets have been hardened and acclimatized. It is suggested that within
a period of 6 months 3000 plants can be produced from single explant of C.
paniculatus by this protocol. The protocol developed can be useful for cloning and
consei-vation of germplasm of C.paniculatus .Gerald Martin et al. 2006 reported
that the nodal explants of Celastrus paniculatus exhibited bud break within 7 days
in MS growth regulator-free initiation medium and elongation of axillary buds
were achieved in 2 weeks without any callus formation at the proximal end.
Maximum shoot induction was achieved in MS medium supplemented with 1.5
mg/1 BA and 0.1 mg/1 NAA.
40