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CHAPTER IV
RESULTS AND DISCUSSION
What nature delivers to us is never stale
because what nature creates has eternity in it.
Issac Bashevis Singer - (1904-1991)
Herbalism is sometimes maligned as a collection of home made
remedies to be applied in a placebo fashion to one symptom or another
provided the ailment is not too serious and provided there is a powerful
chemical wonder drug at the ready to suppress any ‘real’ symptoms.
We often forget, however, that botanical medicine provides a complete
system of healing and prevention of disease. It is the oldest and most natural
form of medicine. Its history of efficacy and safety spans centuries and covers
every country on the planet. Because herbal medicine is a holistic medicine it
offers very real and permanent solutions to real health problems. Nowhere is
the efficacy of herbalism more evident than in problems related to the nervous
system. Stress, anxiety, tension and depression are the most prevalent
occupational hazards in today’s lifestyle and these events are intimately
connected with most illness.
Botanical nervines are free from toxicity and habituation because they
are organic substances and not man-made synthetic molecules, therefore
possess a natural affinity for the human organism. They are extremely efficient
in balancing the nervous system starting from diabetics to heart attack. More
particularly these stress related events lead to ailments related to nervous
system. Thus remedies to stress related disorders assumes greater significance
in today’s lifestyle of people both men and women.
There is a great scope for the development of herbal medicine in the
area of nervous diseases and of its application in so-called ‘mental illnesses’
where pharmaceuticals seem at best to be applied for their ‘management’
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effect. Thus this is an area where the benefits of a whole food diet and holistic
lifestyle are badly neglected. Traditional or folk medicines have been widely
employed for centuries, and they remain to be one of the important sources for
the discovery of new bio-active compounds. Ayurveda, an ancient traditional
system of medicine that has been practiced in India since 200 B.C. employs a
large number of medicinal plants used in the prevention and treatment of a
wide number of nervous system related diseases. One of these includes the
plant Celastrus paniculatus Willd. (Cp), a plant known for centuries as “the
elixir of life”. According to Ayurveda, depending upon the dose regimen,
Celastrus paniculatus may be employed as a stimulant-nerve tonic, rejuvenate,
sedative, tranquilizer and diuretic. It is also used in the treatment of leprosy,
leucoderma, rheumatism, gout, paralysis and asthma. Anti-anxiety activity of
Celastrus paniculatus seeds is also documented (Patwardhan et al., 2003).
Most of the claims for this plant have not been substantiated in rigorous
scientific settings. This includes the purported property of Celastrus
paniculatus germane to this study, especially its ability to stimulate the intellect
and sharpen the memory. This forms the core rationale of the objectives and
experimental designs of the present study.
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. The past two decades have 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. Currently, several mechanisms underlying such a magnificent
phenomenon of learning and memory have been known. However, many of
them remain to be convincingly revealed. Several of central nervous system
(CNS) disorders are often associated with impairment in cognitive functions.
For example Alzheimer’s disease has a primary impact on learning and
memory and other disorders like schizophrenia, bipolar depression are
associated with secondary deficits in learning and memory functions.
Experimental paradigms of this phenomenon are therefore enlightening in both
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elucidation of the pathophysiology and development of new medication for the
treatment of learning and memory disorders.
4.1 BEHAVIOURAL STUDY – Radial arm maze experiment
The radial arm maze was introduced in 1976 to study hippocampal
dependent learning and memory (Olten and Samuelson, 1976) .The radial arm
maze has been widely used in behavioural neuroscience and behavioural
pharmacology. Thus RAM tests are useful in evaluating the effect of drugs,
stress and various other environmental factors on learning and memory
(Bhagya et al., 2008; Srikumar et al., 2006, 2007; Titus 2007)..
Reference memory and working memory are the two variables that
report the physiological status of the brain. Amongst the various functions of
the brain, one of the most interesting one is the ability to acquire new
information and store them for further retrieval. Learning is defined as ‘an
enduring change in mechanisms of behaviour that results from experience with
environmental events’. Learning is a process of acquiring new information,
while Memory refers to the persistence of a change in behaviour overtime, in a
state that can be retrieved later.
The effect of Celastrus paniculatus on normal rats on performance of
a partially baited RAM task is expressed in Table -4. Results on the correct
choice of a partially baited RAM task are illustrated in Figure – 4. Four trials
form one block and eight such trials were conducted. The data in the Table 4
clearly shows that the % of correct choice is 47.45 ± 1.91 in the first block of 4
trials and as the trials increased the % correct choice goes up to 88.33 ± 1.78,
i.e., almost 2 fold (1.86) and the increase of % correct choice is in arithmetic
progression as the number of trial increases. Another equally important
phenomenon revealed in this experiment is that active principle in the
Celastrus paniculatus has not much role to play in healthy animals which could
be observed in Cp 100, Cp 200 and Cp 400. Results of this experiment reveal
that as the trials are increased the % correct choice also increased (almost
parallel) and hence the RAM experiments could be reliably used for our actual
study that is alleviating role of Celastrus paniculatus in stress animals.
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Observations of the effect of Celastrus paniculatus to normal rats on
reference memory errors and working memory errors (correct) in partially
baited RAM task are expressed in Tables – 5 and 6 respectively. The effect of
Celastrus paniculatus to normal rats on reference memory errors and working
memory errors (correct) in partially baited RAM task are illustrated in Figures
-5 and 6.
The data in Table -5 and Figure -5 shows a significant decrease in
reference memory errors as trials progressed. In normal control animals the
reference memory error (RME) is 3.58+ 0.21 in the first block of 4 trials and as
trials increased the RME is brought down to 0.50+ 0.08 in the eighth block of
four trials. Animals administered with Cp seed oil (100 mg/kg body weight,
200 mg/kg body weight and 400 mg/kg body weight) also showed a similar
trend. But the treated animals did not show a greater decrease than control
animals. So this decrease in RME cannot be attributed to Cp oil.
The Table – 6 and Figure – 6 which give the data on working
memory error (WME) also showed a decrease in WME as trials progressed.
This trend was also observed in animals given the different doses of the drug
treatment (100 mg/kg body weight, 200 mg/kg body weight and 400 mg/kg
body weight). But this decrease in treated animals was not appreciable
compared to normal control.
In the present study the higher dose 400mg/kg /day for 14 days
showed slightly better performance than 100 and 200 mg/kg doses. Incidentally
chronic Cp administration was associated with no observable side effects in
animals even with the 400 mg/kg dose regimen.
Chronic treatment with 400 mg/kg Cp alone resulted in a small degree
of enhancement. A marked degree of task enhancement was not expected since
the rats were young and presumably cognitively unimpaired.
Learning and memory is a complex phenomenon that is affected by
various factors. These factors can either enhance the performance by
facilitating the learning process or impair by inhibiting the process of learning.
Stress is one such factor, which has been found to have a prolonged effect on
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cognitive functioning and is associated with hippocampus damage. 21 days of
restraint stress in rats is known to impair performance on different types of
spatial memory tasks like T-maze, Y-maze, radial arm maze and Morris water-
maze (McEwen, 2000; Ramkumar, 2001; 2005; Srikumar, 2004, 2006, 2007;
Sunanda et al, 2000a).
In one study (Karanth et al., 1980), rats were treated with 400 mg/kg of
Cp (by oral gavage) once daily for 3 days. The animals were then given 10
trials in a raised platform shock-avoidance task. Each trial was spaced 5 min
apart. The Cp treated rats exhibited a significantly increased learning curve
compared with vehicle treated animals in the avoidance paradigm. In another
study, rats treated daily with 850 mg/kg of Cp oil for 15 days exhibited a
significant improvement in their retention times in a two-way passive
avoidance task. Cp also produced a significant decrease in the content of
norepinephrine, dopamine and serotonin, and certain of their respective
metabolites in both brain and urine (Nalini et al., 1995).
4.2 BIOCHEMICAL STUDY – AChE activity
The mechanisms underlying learning and memory include an interaction
between the various neurotransmitter systems, amongst which the central
cholinergic system is known to play a prominent role. Estimation of
acetylcholinesterase (AChE) activity provides a relatively easy and valuable
assessment of cholinergic function.
Ever since the discovery of acetylcholine (ACh) as a neurotransmitter by
Sir Henry Dale and Otto Loewi (for which they were awarded the Nobel Prize
in 1936), its function in health and dysfunction in disease has been increasingly
recognized. In the recent past, the role of ACh in learning and memory has
been demonstrated indubitably. Further, pharmacological manipulation of
cholinergic function has been found useful in the treatment of CNS disorders
like Alzheimer’s and Parkinson’s disease. Thus assessing cholinergic function
is considered as an important tool in neuroscience research.
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Acetylcholine per se has a very short half-life and direct estimation
of ACh is a little difficult in brain homogenates. There are several approaches
to evaluate cholinergic function indirectly. Estimating the expression of choline
acetyltransferase (ChAT) and acetylcholine esterase (AChE) by
immunochemical and histochemical techniques provide information on the
cholinergic function, but are tedious and time consuming. Estimation of AChE
activity provides a relatively easy and valuable assessment of cholinergic
function.
The Table - 7 shows the AChE activity in the five brain regions
namely frontal cortex, hippocampus, septum, hypothalamus and brain stem.
The Figure – 7 clearly shows that AChE activity showed a gradual
decrease in frontal cortex when treated with an increasing dose of Cp oil.
Figure -8 shows a marginal increase in activity in the hippocampus when the
animal is treated with 200 mg/kg body weight of Celastrus seed oil. Figure – 9
shows enhanced AChE activity in the septum when treated with 100 and 400
mg/kg body weight of the rats while Figure – 10 shows a decline in activity at
higher dose in the hypothalamus. Figure – 11 shows an increase in AChE
activity in the brain stem in Cp 200 and Cp 400.
At higher dose, Cp did appear to inhibit brain cholinesterase activity in
the hippocampus and hypothalamus without affecting in frontal cortex. The
results of this study are consistent with the possibility that there is a basis for
the conflict derived mainly from anecdotal reports that Cp may enhance
learning and memory in humans.
Furthermore, this plant seed oil may be more effective in individuals
who are cognitively impaired as a result of chemical or organic brain damage
as compared with normal subjects. In the least, these data may provide the
impetus for further study of the material, and isolation of its active components.
The mechanism of action by which Cp enhances learning and memory
performance in behavioural tasks is as yet unknown. At higher dose, Cp did
appear to inhibit brain cholinesterase activity in the hippocampus and
hypothalamus without affecting in frontal cortex. The results of this study are
75
consistent with the possibility that there is a basis for the conflict derived
mainly from anecdotal reports that Cp may enhance learning and memory in
humans. Furthermore, this plant seed oil may be more effective in individuals
who are cognitively impaired as a result of chemical or organic brain damage
as compared with normal subjects. In the least, these data may provide the
impetus for further study of the material, and isolation of its active components.
Studies on brains from patients suffering from Alzheimer’s disease have
shown reduced AChE activity in the hippocampus and cortex (Nakano et al.,
1986).
Intracranial induced behavioural self- stimulation which is shown to
enhance operant performance and reverse the stress deficits is associated with
an increase in AChE activity (Ramkumar et al., 2008; Shankaranarayana Rao et
al.,2008b; Yoganarasimha et al., 1998). Thus there is a tight correlation
between cholinergic function, AChE activity and cognition. Extensive evidence
supports the view that cholinergic mechanisms modulate learning and memory
formation. Learning requires combinational participation of multiple neural
systems. ACh might be a neuromodulator important for regulating the relative
balance in neural systems. Today, the evidence supporting the important role
for ACh in modulating cognitive functions includes findings from a host of
pharmacological studies showing that interfering with cholinergic function
generally impairs and augmenting cholinergic increase or decrease ACh
functions in different memory systems.
4.3 STRESS AND ANTIOXIDANT ENZYMES –
The immobilization stress induced by both acute and chronic
immobilization resulted in the decrease in the levels of Superoxide dismutase
(SOD) as in Table -8 and Figure – 12, Glutathione peroxidase (GPx) as in
Table – 9 and Figure – 13, Glutathione S- transferase (GST) as in Table –
10 and Figure – 14, Glutathione reductase (GSH) as in Table – 11 and
Figure – 15 and Catalase as in Table- 12 and Figure – 16. On treatment
with Celastrus paniculatus seed oil the levels increased significantly and the
effect was greater in chronic immobilization stress induced animals which
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received nearly 14 days of drug treatment than the acute immobilized mice.
The decrease in the levels from normal was drastic in chronic immobilization
than acute and increase in levels of these antioxidant enzymes in the treated
group was significant.
In the present study the TBARS (Lipid peroxide) level was significantly
increased (as in Table – 13 and Figure – 17) during stress condition and was
found to be decreased on treatment with oil, this effect also being dose
dependent. The oil caused a decrease in TBARS level in enzymatic assay in
immobilization stress induced animals.
The tissue protein level was found to be decreased in case of
immobilization stress induced mice brain.( Table-14 and Figure – 18). The
degree of decrease was directly proportional to the exposure of animals to
stressed condition. Hence the decrease was profound in the chronic
immobilization than acute immobilization. The treatment pre (acute and
chronic) and co treatment (chronic) to immobilized animals significantly
increased the protein level. In contrary to this the protein value was found to
be increased in the serum of animals belonging to acute and chronic
immobilization stress induced groups and it was more pronounced in chronic
stress induced animals on treatment with the Jyothismathi oil . The reversal of
the concentration was identified and it was dose dependent. Increase in
dosage decreased the protein concentration (Table- 15 and Figure – 19). The
protein levels in animals treated with the oil alone was near normal.
Significant decrease was noted in chronic stress induced animals treated with
400 mg of the drug.
The serum marker enzymes such as Serum Glutamate Oxaloacetate
Transferase (SGOT), Serum Glutamate Pyruvate Transferase (SGPT), Serum
Acid Phosphatase (ACP) and Serum Alkaline Phosphatase (ALP) indicates
the fluctuation from the normalcy during stress conditions. The results are
shown in Table– 16, Table- 17, Table- 18 and Table -19 respectively. Acute
and chronic immobilization resulted in the increase in these enzymes and
treatment with the oil brought the levels considerably. Again the decrease in
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the activity of these enzymes are dose dependent and the activity is much
pronounced in the immobilization stress induced group. Figure-20, Figure-
21, Figure- 22 and Figure- 23 show the graphical representation of the
same.
A DETAILED ANALYSIS OF THE TABLES AND FIGURES –
4.3.1 SUPEROXIDE DISMUTASE(Cu-Zn SOD) –Table-8 & Figure -
12 The finding on the effect of acute and chronic stress on the SOD
activity and the recovery role of Celastrus paniculatus (Cp) in
different doses on these two stress conditions are given in Table – 8.
The percentage of SOD reduction due to acute and chronic stress is
23 and 28.2 respectively. Cp 200 dose could recover 31% of the
reduction due to acute stress and Cp 400 could recover 62.2 % of the
reduction due the same stress. Cp 200 dose could recover 33.94% of
the reduction due to chronic stress and Cp 400 could recover 82.48
% of the reduction due the same stress. The findings indicate that i)
Immobilization stresses considerably reduce the SOD level; ii) Cp
definitely plays a curative role in mitigating the stress effect; iii) the
maximum recovery is 82.48% due to Cp 400 in Chronic stress; iv)
however the Cp is not able to contribute much in the healthy animal
(Control. = 230.4 and Cp to healthy animal =233.2).
4.3.2 GLUTATHIONE PEROXIDASE (GPx ) – Table-9 & Figure-13
The finding on the effect of acute and chronic stress on the Glutathione
peroxidase activity and the recovery role of Celastrus paniculatus(Cp) in
different doses on these two stress conditions are given in Table – 9. The
percentage of GPx reduction due to acute and chronic stress is 13.63 and
22.72 respectively. Cp 200 dose could recover 27.77 % of the reduction
due to acute stress and Cp 400 could recover 44.44 % of the reduction due
the same stress. Cp 200 dose could recover 16.66 % of the reduction due to
chronic stress and Cp 400 could recover 56.66 % of the reduction due the
same stress. The findings indicate that i) Immobilization stresses
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considerably reduce the GPx level; ii) Cp definitely plays a curative role in
mitigating the stress effect; iii) the maximum recovery is 56.66 % due to
Cp 400 in chronic stress; iv) however the Cp is not able to contribute much
in the healthy animal (Control. = 13.2 and Cp to healthy animal =13.1).
4.3.3 GLUTATHIONE-S-TRANSFERASE(GST)–Table-10&
Figure- 14
The finding on the effect of Acute and Chronic stress on the Glutathione
transferase activity and the recovery role of Celastrus paniculatus (Cp) in
different doses on these two stress conditions are given in Table – 10.
The percentage of GST reduction due to acute and chronic stress is 9.54
and 14.38 respectively. Cp 200 dose could recover 19.40 % of the
reduction due to acute stress and Cp 400 could recover 76.11 % of the
reduction due the same stress. Cp 200 dose could recover 23.76 % of the
reduction due to chronic stress and Cp 400 could recover 77.22 % of the
reduction due the same stress. The findings indicate that i) Immobilization
stresses considerably reduce the GST level; ii) Cp definitely plays a
curative role in mitigating the stress effect; iii) the maximum recovery is
77.22 % due to Cp 400 in chronic stress iv) however the Cp is not able to
contribute much in the healthy animal (Control. = 70.2 and Cp to healthy
animal =71.4).
4.3.4 GLUTATHIONE REDUCTASE (GSH)- Table-11 & Figure- 15
The finding on the effect of acute and chronic stress on the Glutathione
reductase activity and the recovery role of Celastrus paniculatus (Cp) in
different doses on these two stress conditions are given in Table – 11.
The percentage of Glutathione reductase reduction due to acute and
chronic stress is 4.93 and 6.79 respectively. Cp 200 dose could not bring
about any recovery under acute stress and Cp 400 could recover 12.5 % of
the reduction due the same stress. Cp 200 dose could recover 18.18 % of
the reduction due to chronic stress and Cp 400 could recover 63.63 % of
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the reduction due the same stress. The findings indicate that i)
Immobilization stresses reduce the Glutathione reductase level; ii) Cp
definitely plays a curative role in mitigating the stress effect except Cp
200 in acute stress iii) the maximum recovery is 63.63 % due to Cp 400
in chronic stress iv) however the Cp is not able to contribute much in the
healthy animal (Control. = 16.2 and Cp to healthy animal =16).
4.3.5 CATALASE (CAT) – Table- 12 & Figure- 16
The finding on the effect of acute and chronic stress on the catalase
activity and the recovery role of Celastrus paniculatus (Cp) in different
doses on these two stress conditions are given in Table – 12. The
percentage of CAT reduction due to acute and chronic stress is 25.45 and
47.27 respectively. Cp 200 dose could recover 21.42 % of the reduction
due to acute stress and Cp 400 could recover 57.14 % of the reduction due
the same stress. Cp 200 dose could recover 26.92 % of the reduction due
to chronic stress and Cp 400 could recover 69.25 % of the reduction due
the same stress. The findings indicate that i) Immobilization stresses
reduce the CAT level; ii) Cp definitely plays a curative role in mitigating
the stress effect; iii) the maximum recovery is 69.25 % due to Cp 400 in
chronic stress iv) however the Cp is not able to contribute much in the
healthy animal (Control. = 0.55 and Cp to healthy animal =0.54).
HIGHLIGHTS : The general trend seen in the behaviour of these
antioxidant enzymes is very similar. There is a decrease in the levels of
these enzymes under stressed conditions, the decrease being more
pronounced in chronic stress. On treatment with Jyothismati oil under the
same restrained stressed conditions there was lesser reduction in these
antioxidant enzymes. This effect was clearly dose dependent with 400
mg/kg bodyweight of the oil being more effective than 200 mg/kg
bodyweight.
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4.3.6 LIPID PEROXIDES (TBARS ) – Table- 13 & Figure- 17
The finding on the effect of acute and chronic stress on the lipid
peroxide activity and the recovery role of Celastrus paniculatus (Cp) in
different doses on these two stress conditions are given in Table –13.The
percentage of TBARS increase due to acute and chronic stress is 37.5 and
50 respectively. Cp 200 dose could bring down 50 % of the increase due
to acute stress and Cp 400 could bring down 33.3 % of the increase due
the same stress. Cp 200 dose could bring down 100 % of the increase due
to chronic stress and Cp 400 could bring down 56.25 % of the increase
due the same stress. The findings indicate that i) Immobilization stresses
increase the TBARS level; ii) Cp definitely plays a curative role in
mitigating the stress effect; iii) the maximum recovery is 100 % due to
Cp 200 in chronic stress iv) however the Cp is not able to contribute
much in the healthy animal (Control. = 3.2 and Cp to healthy animal
=3.1).
HIGHLIGHTS : Due to the damaging effect of stress the release of
harmful lipid peroxides in the brain tissue is increased, this effect being
more in chronic stress. But on treatment with Celastrus paniculatus seed
oil the level of lipid peroxides is reduced even under stressed conditions.
4.3.7 BRAIN TISSUE PROTEIN – Table- 14 & Figure – 18
The finding on the effect of acute and chronic stress on the brain tissue
protein and the recovery role of Celastrus paniculatus (Cp) in different
doses on these two stress conditions are given in Table – 14.The
percentage of brain tissue protein decrease due to acute and chronic stress
is 18.33 and 48.26 respectively. Cp 200 dose could recover 16.26 % of
the decrease due to acute stress and Cp 400 could recover 52.03 % of the
reduction due the same stress. Cp 200 dose could recover 46.54 % of the
decrease due to chronic stress and Cp 400 could recover 87.02 % of the
decrease due the same stress. The findings indicate that i) Immobilization
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stresses decrease brain tissue protein level; ii) Cp definitely plays a
curative role in mitigating the stress effect; iii) the maximum recovery is
87.02 % due to Cp 400 in chronic stress iv) however the Cp is not able
to contribute much in the healthy animal (Control. = 201.2 and Cp to
healthy animal =199.2).
HIGHLIGHTS: There is a fall in the level of brain protein under
conditions of acute and chronic stress which could be attributed to tissue
damage. The treated animals show lesser reduction in tissue protein which
suggests lesser tissue damage and hence the positive influence of
Jyothismati oil. This reduction of damage is clearly dose dependent.
4.3.8 SERUM PROTEIN- Table – 15 & Figure – 19
The finding on the effect of acute and chronic stress on the serum protein
and the recovery role of Celastrus paniculatus (Cp) in different doses on
these two stress conditions are given in Table – 15. The percentage of
serum protein increase due to acute and chronic stress is 11.36 and 4.5
respectively. Cp 200 dose could recover 40 % of the increase due to acute
stress and Cp 400 could recover 60 % of the increase due the same stress.
Cp 200 dose could recover 50 % of the increase due to chronic stress and
Cp 400 could recover again 50 % of the increase due the same stress. The
findings indicate that i) Immobilization stresses increase serum protein
level; ii) Cp definitely plays a curative role in mitigating the stress effect
though the effect is marginal iii) the maximum recovery is 60 % due to
Cp 400 in acute stress iv) there was no difference between the Cp 200
and Cp 400 treatments v) however the Cp is not able to contribute much
in the healthy animal (Control. = 4.4 and Cp to healthy animal =4.43).
HIGHLIGHTS : It is clear from the above figures that under stress there
is an increase in the level of serum protein. It is also to be specifically
noted that acute stress has more damaging effect than chronic stress which
is seen by the higher level of serum protein in Group II animals. On
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treatment with Jyothismati oil the increase in the level of serum protein in
stressed animals is reduced which again suggests less tissue damage. There
is no difference in the chronic stress groups (Group VII & Group VIII)
treated with 200 mg/kg body weight and 400 mg/kg body weight of
Jyothismati oil.
4.3.9 SERUM GLUTAMATE OXALOACETATE TRANSFERASE –
(SGOT) – Table- 16 & Figure- 20
The finding on the effect of acute and chronic stress on the SGOT and the
recovery role of Celastrus paniculatus (Cp) in different doses on these two
stress conditions are given in Table – 16. The percentage of SGOT increase
due to acute and chronic stress is 86.65 and 119 respectively. Cp 200 dose
could recover 26.24 % of the increase due to acute stress and Cp 400 could
recover 43.83 % of the increase due the same stress. Cp 200 dose could
recover 51.52 % of the increase due to chronic stress and Cp 400 could
recover 88.02 % of the increase due the same stress. The findings indicate
that i) Immobilization stresses increase SGOT level; ii) Cp definitely plays
a curative role in mitigating the stress effect; iii) the maximum recovery is
88.02 % due to Cp 400 in chronic stress iv) however the Cp is not able to
contribute much in the healthy animal (Control. = 44.2 and Cp to healthy
animal = 45.7).
4.3.10 SERUM GLUTAMATE PYRUVATE TRANSFERASE –
(SGPT)- Table – 17 & Figure - 21
The finding on the effect of acute and chronic stress on the SGPT and the
recovery role of Celastrus paniculatus (Cp) in different doses on these two
stress conditions are given in Table – 17. The percentage of SGPT increase
due to acute and chronic stress is 19.6 and 37.8 respectively. Cp 200 dose
could recover 27.55 % of the increase due to acute stress and Cp 400 could
recover 52.04 % of the increase due the same stress. Cp 200 dose could
recover 69.31 % of the increase due to chronic stress and Cp 400 could
recover 86.24 % of the increase due the same stress. The findings indicate
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that i) Immobilization stresses increase SGPT level; ii) Cp definitely plays
a curative role in mitigating the stress effect; iii) the maximum recovery is
86.24 % due to Cp 400 in chronic stress iv) however the Cp is not able to
contribute much in the healthy animal (Control. = 50.0 and Cp to healthy
animal = 52.1).
4.3.11 SERUM ACID PHOSPHATASE (ACP) – Table – 18 & Figure
- 22
The finding on the effect of acute and chronic stress on the ACP and the
recovery role of Celastrus paniculatus (Cp) in different doses on these two
stress conditions are given in Table – 18. The percentage of ACP increase
due to acute and chronic stress is 82.05 and 101.99 respectively. Cp 200
dose could recover 9.3 % of the increase due to acute stress and Cp 400
could recover 31.98 % of the increase due the same stress. Cp 200 dose
could recover 34.52 % of the increase due to chronic stress and Cp 400
could recover 58.63 % of the increase due the same stress. The findings
indicate that i) Immobilization stresses increase ACP level ii) Cp definitely
plays a curative role in mitigating the stress effect iii) the maximum
recovery is 58.63 % due to Cp 400 in chronic stress iv) however the Cp is
not able to contribute much in the healthy animal (Control. = 30.1 and Cp
to healthy animal =31.6).
4.3.12 SERUM ALKALINE PHOSPHATASE (ALP)-Table-19
&Figure -23
The finding on the effect of acute and chronic stress on the ALP and the
recovery role of Celastrus paniculatus (Cp) in different doses on these two
stress conditions are given in Table – 19. The percentage of ALP increase
due to acute and chronic stress is 61.05 and 91.34 respectively. Cp 200
dose could recover 17.32 % of the increase due to acute stress and Cp 400
could recover 41.73 % of the increase due the same stress. Cp 200 dose
could recover 53.15 % of the increase due to chronic stress and Cp 400
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could recover 76.31 % of the increase due the same stress. The findings
indicate that i) Immobilization stresses increase ALP level; ii) Cp definitely
plays a curative role in mitigating the stress effect; iii) the maximum
recovery is 76.31 % due to Cp 400 in chronic stress iv) however the Cp is
not able to contribute much in the healthy animal (Control. = 20.8 and Cp
to healthy animal =21.6).
HIGHLIGHTS: The above mentioned four serum marker enzymes show a
similar trend with an increase in their levels with stress. On treatment with
Jyothismati oil the levels of these enzymes is brought down even under
stressed conditions.The 400 mg/kg body weight oil treatment was far
superior than 200 mg/kg body weight.
The above observations lead to inferences such as
Chronic stress is more harmful than acute stress.
JO oil undoubtedly relieves stress.
400 mg/kg body weight fares better in alleviating the stress
effect in both the types of stress.
4.4 Histological analysis were carried out to study the extent of damage
caused by immobilization stress to the brain tissue. The brain sections reveal
the changes that occurred in the brain with stress and Cp oil treatment. The
results are seen in Plate -8 to Plate- 16. It is seen that stress causes tissue
degeneration as shown in Plate – 9 and Plate – 10. On treatment with JO
under stress conditions the extent of damage is drastically minimised as seen in
Plate – 12 to Plate – 15. Plate – 16 reveals a comparative view of the brain
sections of all the eight animals. Plate – 11 clearly shows that normal animals
on treatment with JO show no difference in the brain tissue compared to
normal animals. Therefore the histological studies act as a confirmation for the
results obtained in the biochemical tests.
Stress is defined as a disruption of homeostasis (Rivier and Rivest,1991), and
stimuli that challenge homeostasis are designated as stressors. Stressors can be
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divided into 3 general categories (Pacak et al, 1998;Van de Kar and Blair,
1999; Tilbrook et al, 2000): 1) physical (for example, restraint, foot shock, and
exercise); 2) psychosocial, including isolation, anxiety, fear, or mental
frustration; and 3) metabolic, including upright tilt, heat exposure,
hypoglycemia, and haemorrhage. Stress has been further subdivided based on
duration: acute (single, intermittent, and time-limited exposures) and chronic
(intermittent-and-prolonged or continuous exposures). Stressors used in
research are often of a mixed type. For example, immobilization stress is a
mixture of physical and psychological stressors, restricting movement and
isolating the individual from its group (Pacak and Palkovits, 2001). In adult
mice, IMO stress induced a sharp fall in both serum marker enzymes and tissue
antioxidant levels relative to unstressed controls. It is likely, therefore, that
IMO stress, as an acute stressor, does not decrease the levels of the marker and
antioxidant enzymes only to a lesser extent, which agrees with data from other
laboratories (Charpenet et al, 1981; Mann and Orr, 1990; Srivastava et al, 1993;
Orr et al, 1994). This contrasts with chronic stress where there is evidence for
decreases in the marker enzyme concentration and increase in antioxidant
enzymes (Veldhuis, 1997). Stress induced by immobilization (restraint stress)
is particularly effective because it combines physical stress (i.e., increased
muscular work) and emotional stress (i.e., enhanced flight reaction). Here, we
used this well established model of stress to investigate for the first time the
effects of stress induced by immobilization at a definite number of
immobilization sessions (i.e.,1(acute) and 7 (chronic) immobilization sessions).
The protein value was found to be increased in the serum of animals belonging
to acute and chronic immobilization stress induced groups and it was more
pronounced in chronic stress induced animals on treatment with the Jyothismati
oil the reversal of the concentration was identified and it was dose dependent ,
increase in dosage decreased the protein concentration. The protein levels in
animals treated with the oil alone was near normal. Significant decrease was
noted in chronic stress induced animals treated with 400mg of the drug. This
increase in the total serum protein content might be attributed by the draining
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of degenerated tissue protein in the circulatory fluid. In contrary to this the
tissue protein level was found to be decreased in case of the immobilization
stress induced mice brain. The degree of decrease was directly proportional to
the exposure of animals to stressed condition. Hence the decrease was profound
in the chronic immobilization than acute immobilization. The treatment pre
(acute& chronic) and co treatment (chronic) to immobilized animals
significantly increased the protein level which is an indication of lesser tissue
degeneration or necrosis.
SOD, CAT, and GR are known to be inactivated in vitro by H2O2, O2-, and
.OH, respectively. SOD and CAT are major antioxidant defense components
that primarily catalyze the conversion of superoxide radical O2- to H2O2 (SOD)
and decomposition of H2O2 to H2O (CAT). H2O2 is normally detoxified in cells
by either CAT and or GPx (Glutathione peroxidase). GPx catalyzes the
reduction of H2O2 by reduced glutathione (GSH). GSH is readily oxidised to
glutathione disulfide (GSSG) by the GPx reaction. GSSG can be reduced by
NADPH- dependant reaction catalysed by glutathione reductase. A decrease in
SOD, CAT and GPx activity with the stress induction probably results in
accumulation of O2- and H2O2 which react with metal ions to promote
additional radical generation, with the release of the particularly reactive
hydroxyl radical.
Hydroxyl radicals reacts with lipids, DNA and proteins, caused a loss of cell
integrity, enzyme function and genomic stability. The immobilization stress
induced by both acute and chronic immobilization resulted in the decrease of
the enzymes levels of SOD (Cu-Zn SOD), CAT, GPx, GST and GSH . On
treatment with the Celastrus paniculatus seed oil the levels increased
significantly and the effect was greater in chronic immobilization stress
induced animals which received nearly 14 days of drug treatment than the acute
immobilized mice. The decrease in the levels from normal was drastic in
chronic immobilization than acute and increase in levels of these antioxidant
enzymes in the treated group was significant.
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The free radicals generated as a result of immobilization stress induction
propagate a chain reaction, leading to lipid peroxidation in cellular membranes,
destruction of Ca2+ homeostasis that induces neuronal cell injury and finally
results in cell death.
Emel Sahina and Saadet Gümülü, 2007, determined the effects of
immobilization stress on antioxidant status, protein oxidation and lipid
peroxidation in brain, liver, kidney, heart and stomach of rats. Sixteen male
Wistar rats (3 months old) were divided into control (C) and immobilization
stress group (IS). IS rats were immobilized for 180 mins/day for 15 days.
Copper, zinc-superoxide dismutase activities were increased in brain, liver and
kidney, but decreased in the heart and stomach after immobilization.
In the present study the TBARS level was significantly increased during
condition and was found to be decreased on treatment with oil, this effect was
also dose dependent. The oil caused a decrease in TBARS level in enzymatic
assay in immobilization stress induced animals. The enzymatic NADPH-
dependant LPO is catalysed by the NADPH-cytochrome P450 reductase and
propagated by Cytochrome P450 with generation of free radicals i.e O2 - and
ROS. The oil of Celastrus paniculatus might have inhibited the activity of
NADPH-dependant LPO could be associated with its free radical scavenging
ability. Elevations in the levels of products of free radicals like TBARS in
brain of acute and chronic stress induced group again support the low
antioxidant enzyme activity that elevate the lipid peroxidation while TBARS is
the product of lipid peroxidation. Another possibility for such elevation in
TBARS may be due to ischemia- reperfusion phenomenon (Freeman et al.,
1982 ; Jewet et al.,1989) or due to high rate of catecholamine secretion that
generate free radicals either through auto oxidation or through metal ion or
superoxide-catalyzed oxidation (Dilard et al., 1978).
Immobilization -induced oxidative stress in mice brain has been established
here by noting the low activities of SOD, CAT, GST- important antioxidant
enzymes, which is consistent with the observation of others (Venkateswaran
and Pari, 2003). The decrease in antioxidant enzyme activities due to
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immobilization might be due to their use against the free radicals destruction
and or their inhibition by free radical species (Debnath and Mandal, 2000). It is
well established that SOD activity is inhibited by hydrogen peroxide that
reduced Cu2+ to Cu1
+ in SOD .The reduced Cu1+ can act as promoter of
hydroxyl by Haber-Weis reaction (Marklund and Marklund, 1974). Low-
antioxidant enzyme activities further facilitate the increased susceptibility to
lipid peroxidation (Ji, 1999).
The reduction of hydrogen peroxide is catalyzed by CAT that protects the
tissues from highly reactive hydroxyl radicals (Chance et al, 1952). Reduction
of hydrogen peroxide and hydro peroxides to non-toxic products are catalyzed
by GST and peroxidase.
Natural antioxidant enzymes manufactured in the body provide an important
defense against free radicals. Glutathione peroxidase, glutathione reductase,
catalase, lipid peroxidase, superoxide dismutase are the most important
antioxidant enzymes. The enzyme superoxide dismutase converts two
superoxide radicals into one hydrogen peroxide and one oxygen. To eliminate
hydrogen peroxide before the Fenton Reaction can create a hydroxyl radical,
organisms use catalase and/or glutathione peroxidase. The brain, which is very
vulnerable to free radical damage (due to high metabolic rate, high unsaturated
fat in neurons, and the fact that neurons are post-mitotic) has seven times more
glutathione peroxidase activity than catalase activity
• The Superoxide dismutase (SOD) molecule in the cytoplasm contains
copper & zinc atoms (Cu/Zn−SOD), whereas the SOD in mitochondria
contains manganese (Mn−SOD). Superoxide dismutase without
glutathione peroxidase or catalase (CAT) to remove hydrogen peroxide
is of little value.
• The glutathione system (glutathione, glutathione peroxidase and
glutathione reductase) is a key defense against hydrogen peroxide and other
peroxides. There are four forms of glutathione peroxidase (GPx) enzymes:
(1) cystolic Glutathione Peroxidase (cGPx), ubiquitously distributed),
(2) Phospholipid Hydroperoxidase Glutathione Peroxidase (PHGPx), in
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plasma membranes to reduce hydroperoxides of complex lipids), (3) plasma
Glutathione Peroxidase (pGPx), in blood plasma) and (4) Gastro-Intestinal
Glutathione Peroxidase (GIGPx), in the liver and GI tract only.
• O2- + O2-+ 2H+ -----------------------------> H2O2 + O2
• Hydrogen peroxide should be scavenged as it is harmful to the system.
Super oxide dismutase (SOD) removes the H2O2.
• Glutathione is a metabolic intermediate and it is a tripeptide made up of
three amino acids – glycine, glutamic acid and cysteine
Glutathione Peroxidase • 2GSH + H2O2 -----------------------------> G-S-S-G + H20
Reduced form Oxidised form Glutathione Reductase
• G-S-S-G + NADPH + H+ -----------------------------> 2GSH + NADP
• Electrophilic xenobiotics ( R ) is carcinogenic
Glutathione S-transferase • R + GSH ---------------------------------------> R-S-G (not harmful and
excreted by kidneys) • Lipid peroxidation –
Polyunsaturated fatty acids release a number of free radicals during lipid
peroxidation.All these free radicals combine with LDL to form oxidised LDL.
This oxidised LDL form plaques leading to atherosclerosis. Lipid
peroxidases scavenge all the free radicals formed during lipid peroxidation.
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Catalase • 2 H2O2 ----------------------------------- 2H2O + O2
4.5 ANTI-FUNGAL PROPERTY OF CELASTRUS PANICULATUS:
It is seen from the above experiment that the extracts of Celastrus paniculatus
has antifungal property. The aqueous and methanolic extracts have remarkable
antifungal property followed by petroleum ether and chloroform. It is seen in
that seed extract of Cp in various solvents like chloroform, petroleum ether,
methanol and water had different effect on the growth of the three fungi –
Aspergillus, Penicillium and Trichoderma considered here. Figure – 24 and
Plate – 17 show that aqueous extract of Cp seed shows maximum inhibition of
the growth of Aspergillus followed by methanol and petroleum ether extract.
Chloroform extract did not show significant inhibition. The same trend is seen
with the growth of Penicillium and Trichoderma as shown in Figure – 25 and
Figure – 26 respectively. Plate – 18 and Plate – 19 confirm the same. Table –
20 gives the growth in cms of Aspergillus, Penicillium and Trichoderma in the
solvents like chloroform, petroleum ether, methanol and water.
Flowers of Celastrus paniculatus were extracted in absolute methanol. Extracts
were tested for their oral analgesic and anti/inflammatory potentials. Results
showed that C. paniculatus had both analgesic and anti/inflammatory activities
(Ahmad et al., 1990).
4.6 IN VITRO PROPAGATION
4.6.1 Multiple shoot formation from shoot tip explants:
Shoot tip culture is an accepted technique in micro propagation because
shoot tips are less prone to pathogens. In this method, the response of axillary
or terminal buds is controlled by hormones to produce multiple shoots and a
large number of plantlets can be produced.
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Shoot tip explants of C. paniculatus were inoculated on MS medium
supplemented with different concentrations (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg/l)
of BAP + 0.5 mg/l IAA + 0.5 mg/l GA3 for multiple shoot induction. The shoot
buds were initiated after 5 days of inoculation from the shoot apex in all the
concentrations.(Plate-20). A maximum number of 25 shoots were observed in
3.0 mg/l BAP + 0.5 mg/l IAA + 0.5 mg/l GA3. The least number of shoots (2
shoots / explant) were observed at 6.0 mg/l BAP + 0.5 mg/l IAA + 0.5 mg/l
GA3. The shoots attained a length of 3-3.5 cm within 15 days of inoculation
and it was used for rooting process (Table -21).
In the present study, a maximum of 25 shoots/ explant was obtained at
3.0 mg/l BAP + 0.5 mg/l IAA + 0.5 mg/l GA3. A similar finding was observed
on Mentha piperita L. which produced 44 shoot / explant (Kiran Ganti et al.,
2004). In the present study, BAP + IAA were proved to be the best hormones
for multiple shoot formation from shoot tip explants of C. paniculatus. Jyoti
Sardana et al. (1999) demonstrated that BAP (8.0 mg/l) and IAA (3.0 mg/l) are
needed for multiple shoot formation from shoot tip explants of Trachyspermum
ammi.
Similar results were documented by various scientists. Conchou et al.
(1991) reported that multiple shoots showed regeneration from the shoot tips of
Arnica montana on MS and B5 media supplemented with BAP (1.0 mg/l) and
NAA (0.1 mg/l). Multiple shoot from the shoot tip explants of Piper nigrum
inoculated with 1.5 mg/l BAP on MS medium was achieved by Philip et al.
(1992). Micropropagation of Chlorophytum borivillianum from shoot tip
explants were induced on MS medium supplemented with 22.2 μΜ BAP
(Purohit et al.,1994). Sanju and Elisabet Claveria (1995) produced multiple
shoots from the shoot tips of Pistacia vera on MS medium containing 5 μM
BAP and 0.5 μM IBA. Incorporation of 0.5 mg/l GA3 along with BAP in the
culture medium resulted in marked increase in the number of axillary branches
and multiple shoot formation in Ocimum sps. (Sitakanta Pattnaik et al.,1996).
4.6.2 Multiple shoot formation from node explants:
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In many plant species, potential axillary bud remains inactive due to
apical dominance. These dormant axillary buds can be activated either by
causing an injury or destroying the apical bud. This principle is used in single
node culture, wherein the apical dominance is controlled by cytokinin. This
causes the axillary bud to develop which in usual cases when subcultured in
similar medium proliferates into multiple shoots.
Node explants of C. paniculatus were inoculated on MS medium
supplemented with different concentrations (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg/l)
of BAP + 0.5 mg/l IAA + 2.0 mg/l GA3 for multiple shoot induction. Shoot
buds were initiated from the node, 5 days of inoculation in all the
concentrations (Plate-21). A maximum number of 15 shoots were observed in
4.0 mg/l BAP + 0.5 mg/l IAA + 2.0 mg/l GA3. The shoots attained a length of
2– 3 cm in length after 25 days of inoculation and it was ready for rooting
process (Table-22).
In the present study, the combination of cytokinin and auxin was found
to be effective in multiple shoot formation from node explant of C. paniculatus.
Similar findings were reported in Pelargonium graveolens (Satyakala et al.,
1995) and Plumbago indica (Smitachetia and Handique, 2000)
The increasing concentrations of BAP were found to be the best for
multiple shoot induction in C. paniculatus. However, studies on the same plant
showed an average of 5 shoots/ explant in MS medium supplemented with 1.5
mg/l BAP and 0.1 mg/l NAA (Gerald Martin et al., 2006).
4.6.3 Indirect organogenesis from leaf explant:
a). Callus induction from leaf explant :
The leaf explants of C. paniculatus were inoculated on MS medium
supplemented with different concentrations (0.5, 1.0, 2.0, and 3.0 mg/l) of
2,4 - D for callus induction. Initially swelling of the leaf explants were
observed from the cut end after 3 days of inoculation. Callus initiation was
noticed after 7 days of inoculation (Plate –22). The entire surface of the
explants was covered with proliferated callus (Plate – 22b). A maximum of
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89% callusing was observed at 2.5 mg/l 2,4 - D. The calli were hard, compact
and dark green in colour. The percentage of response decreased with the
decreasing concentrations of 2,4 - D. The results are presented in Table – 24.
In the preset study, a maximum of 89% of callusing was observed at 2.5
mg/l 2,4 - D. Our results are in accordance with report of John Britto et al.
(2002) in Anisomeles indica. The node explants showed green, nodular
organogenic calli.
b). Regeneration of shoots from leaf callus:
The well developed organogenic calli were transferred to regeneration
medium containing different concentrations (1.0 – 5.0 mg/l) of BAP. The leaf
calli showed shoot bud initiation after 10 days of inoculation. A maximum of
87% calli showed regeneration at 4.0 mg/l BAP and produced 7 shoots / callus
(Plate – 22c). The length of the shoot was measured to be 2 – 5 cm. The results
are presented in Table –25.
In the present study, BAP was found to be more effective in the
regeneration of shoots. Our results are in conformity with a report of
Arulmozhi and Ramanujam, (1997) in Solanum trilobatum , Sreelekha and
Ramanujam (1997) in Solanum nigrum. In our study a single cytokinin was
sufficient for the regeneration of shoots.
4.6.4 Indirect organogenesis form internode explant:
a). Callus induction from internode explant :
Internodal explant of C. paniculatus were inoculated on MS medium
supplemented with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/l)
of 2,4-D. Internode explant showed callus initiation after 7 days of inoculation
(Plate –23 a). Later on, the entire surface of the explants was fully covered by
the proliferated callus (Plate – 23b). The calli were hard, compact and dark
green in colour. A maximum of 87% callusing was observed at 3.0 mg/l 2,4-D.
The callus formation showed increase with increasing concentrations of 2,4 –D
up to 3.0 mg/l. The results are presented in Table –23.
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In the present study, the maximum callus formation (87%) was observed
at 3.0 mg/l 2,4-D. Our results are in accordance with a report of Srinath Rao et
al. (2005). They reported that 2,4 – D was proved to be better than NAA and
Kn for callus proliferation in various explants of Vigna radiata. The
combination of BAP and IAA was found to be the best hormone in Solanum
nigrum (Jawahar et al. (2004). Arulmozhi and Ramanujam, (1997) reported
that organogenic green calli was produced in Solanum trilobatum on MS
medium supplemented with BAP and NAA.
b) Regeneration of shoots from internode callus :
The well developed green organogenic calli of C. paniculatus obtained from
the internodes were transferred to MS medium containing different
concentrations (1.0 – 5.0 mg/l) of BAP for regeneration of shoots. The inter
node calli showed shoot bud initiation after 10 days of inoculation. The shoots
were dark green and healthy in appearance. A maximum of 96% calli showed
regeneration at 4.0 mg/l BAP and produced 7 shoots / callus (Plate – 23c). The
length of regenerated shoot was measured to be 2 – 5 cm. The results are
presented in Table –25. The well developed elongated shoots were excised
from shoot clumps and transferred to rooting medium.
In the present study BAP was more effective in regeneration of shoots.
Similar findings were observed in Datura metal (Arockiasamy et al., 1999).
4.6.5. Rooting of regenerated shoots from direct and indirect
explants of Celastrus paniculatus.
The shoots were excised from the shoot clumps of direct and indirect
organogenesis and inoculated on MS medium containing different
concentrations (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/l) of IBA for rooting. The root
initiation was noticed within 5 days of inoculation. A maximum of 73% of
rooting was observed at 4.0 mg/l IBA. They were pale white, long with an
average of 15 roots / shoot (Plate – 20d &21d). The results are tabulated in
Table –26.
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Our findings are in accordance with the findings of Siddique et al.
(2009) which report that the rhizome cutting of Picrorhiza kurrooa responded
significantly to various growth regulators. In their study, 1000 ppm of IBA
showed maximum rooting percentage (81.3) and was found to be most
effective. Shanthi and Anne Xavier (2003) recorded that the combination of
auxins (NAA, IAA and IBA) were essential for rooting in Enicostemma
littorae.