what nature delivers to us is never stale because what...

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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

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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


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


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



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


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



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


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


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


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


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.

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