ISSN 0974-3618 (Print) www.rjptonline.org
0974-360X (Online)
RESEARCH ARTICLE
Evaluation of
Anti Depressant and MAO Inhibitory Activity of Rhodiola rhodantha rhizome
methanolic extract
Anantha Lakshmi J*,
Satyavthi D.
1Sri Venkateshwara College of Pharmacy,
Madhapur, Hyderabad, Telangana State
2Sree Datta Institute of Pharmacy,
Sheriguda, Ibrahimpatnam, Ranga Reddy Dist.
*Corresponding Author E-mail: anu_manas0108@yahoo.com
The objective of the present study was
to evaluate the antidepressant activity of methanolic extract for the rhizomes
of Rhodiola rhodantha. Depression
is the most common
feature and it
may range from a
very mild condition to
even severe depression which
is called as
psychotic depression. In the traditional systems of medicine many plants
and formulations have been used to treat depression for thousands of years. The
present study was undertaken to assess the antidepressant effect of crude
methanolic extract of Rhodiola rhodantha at doses 100mg/kg, 200mg/kg
and 400mg/kg using parameters forced induced swimming test (FST), tail
suspension test (TST). Significant dose dependent decline in immobility time
was observed in all the three doses in FST and TST and the extract showed
dose-dependent relation of immobility reduction in mice. In order to understand
the probable mechanism of antidepressant-like activity in FST and TST, the
levels of 5-hydroxy indole acetic acid in urine was taken as basis for
biochemical assessment of anti depressant activity.
The antidepressant activity of extract
was comparable to that of standard drug imipramine. The results of the present
study indicate the potential for use of RRMS as an adjuvant in the treatment of
depression.
KEYWORDS: Forced swim test, Tail suspension test,
Depression, Rhodiola rhodantha, imipramine
INTRODUCTION:
Depressive
disorder is a prevalent psychiatric disorder, which affects 21% of the
world population. Mental depression is one of the common chronic illnesses that
affect the mood, thought, physical health and behavior of an individual often
associated with suicidal tendency. In spite of the availability of various
classes of drugs, psychological disorders continue to be a major medical
problem. The drugs used to treat this disorder has a success rate of about 60%
and in addition, most therapies require
several weeks of treatment and
produces severe toxic and side effects (1,2).
People from
different regions of the world have used herbal medicines to alleviate
affective disorders for many years. In addition, the search for novel
pharmacotherapy from medicinal plants for psychiatric illnesses has progressed
significantly in the past decade.
An
increasing number of herbal products have been introduced into psychiatric
practice, among the most commonly used herbal medications for the prevention or
treatment of depression. Various plants are being used in complementary and
alternative medicines for management of stress and mood disorders, because of
their minimum toxicity (3-5).
Rhodiola
rhodantha (A. Gray) H. Jacobsen is a dicot plant belongs to the
Crassulaceae family. It is known locally as red pod stonecrop. The plant is a
perennial herb growing to 0.4 m (1ft4in). All members of this genus are
reported to have edible properties. However
no regular cultivation is practiced. It flowers from July to August and
the seeds ripen from Aug to September(6,7). Rhodiola species reported to strengthen the nervous system, fight
depression, enhance immunity, elevate the capacity for exercise, enhance
memory, aid weight reduction, increase sexual function and improve energy
levels (8).
EXPERIMENTAL:
Selection,
collection and authentification of plant
The authenticated Rhodiola rhodantha were
kindly provided by Dr. Madhavan Chetty, Botanist, Tirupathi Andhra Pradesh,
India. All the plant materials were further identified and authentified by Dr.
Madhavan Chetty, Department of Botany, Sri Venkateswara University, Tirupathi,
Andhra Pradesh. The specimens have been preserved in air tight containers.
Preparation
of Extract
A wide range of solvents with increasing
polarity were chosen and maceration technique was employed for extraction
process. In a 250ml round bottomed flask, weighed quantity of powdered drug
were macerated with the respective solvent methanol (i.e. 50gm in 100ml) and
kept with occasional shaking for a period of 72 hrs. After the maceration
process, the active ingredients present in the supernatant solvent were
collected in petridishes and concentrated under reduced pressure. Specific emphasis was given to
determination of phytomedicines.
Preliminary phytochemical screening:
The plant extract was subjected to
preliminary phytochemical screening. The extracts of Rhodiola rhizomes were treated with dilute hydrochloric acid and
filtered. The filtrate was treated with various alkaloid agents. The presence
of alkaloids was evaluated using Mayer’s test, Dragendroff’s test, Hager’s and
Wagner’s test. Along with this other tests such as Fehlings, Benedict’s test, Barfoed’s test were
conducted to evaluate the presence of reducing and non reducing sugars. The
extract was evaluated for the presence of glycosides. The extracts were treated
with million’s reagent, biuret’s test to detect the presence of proteins.
Additionally all the plant extracts were evaluated for the presence of sterols,
steroids, mucilage and terpens.
Acute toxicity studies:
Standardization
of the therapeutic dose and confirmation of any acute toxicity was assessed by
conducting acute toxicity studies using OECD guidelines. The acute toxicity was
determined by using female albino mice (20-30g) those maintained under standard
husbandry conditions. The animals were fasted 3 hrs prior to the experiment, up
and down procedure (OECD guideline no. 425) of CPCSEA was adopted for toxicity
studies. Animals were administered with single dose of formulation and observed
for its mortality during 48 hours study period (short term) toxicity. Based on
short term profile of drug, the dose of the next animals was determined as per
as OECD guideline 425. The treated animals were carefully observed individually
for the toxicity signs and mortality. The animals were observed for mortality
for 24 hr after drug administration. The LD50 of the test extract
was calculated using AOT 425 software provided by Environmental protection
agency, USA.
In acute toxicity studies all the plant extracts were evaluated
for parameters such as Alertness, Aggressiveness, Pile Increased Motor
Activity, Tremors, Convulsions, Respiration and Number of Deaths (Mortality).
Anti depressant activity
The
antidepressant effect of selected plant extracts were evaluated using two
behavioral models the forced swim test (FST) and tail suspension test (TST). In
addition MAO inhibitory studies were conducted for the methanolic extract of rhodiola
root.
Drug treatment and schedule
The animals were
divided into five groups consisting of 6 mice per group. Group I received
vehicle (control), group II received the
standard imipramine (20 mg/kg,p.o.) group III, IV, V received methanolic
extracts of Rhodiola Rhizomest
Methanolic extract,
(100mg/kg,p.o), (200mg/Kg, P.O) and (400mg/Kg, P.O) respectively.
Each extracts and the reference compound were injected 30 min before
administration of imipramine(9).
Forced
swim test (FST)
Behavior despair was proposed as a model to
test for antidepressant activity by, Mice
were forced to swim individually in a glass jar (25 × 12 × 25 cm3sub)
containing fresh water of 15 cm height and maintained at a temperature of 25°C (± 3°C). After an
initial 2 min period of vigorous activity, each animal assumed a typical
immobile posture. A mouse was considered to be immobile when it remained
floating in the water without struggling, making only minimum movements of its
limbs necessary to keep its head above water. The total duration of immobility
was recorded during the next 4 min of a total 6 min test. The changes in
immobility duration were studied after administering drugs in separate groups
of animals. A decrease in the duration of immobility is indicative of an
antidepressant effect.
Tail
suspension test (TST)
The total duration of immobility induced by
tail suspension was means measurement according to the method described by
Steru et al. Mice were suspended on the edge of a table 50 cm above the floor
by the adhesive tape placed approximately 1 cm from the tip of the tail.
Immobility time was recorded during a 6 min period. Animal was considered to be
immobile when it did not show any movement of body and hanged passively. The
immobility time was calculated.
MAO
Inhibitory activity
The animals were randomly divided into five
groups each containing six rats. They were marked with unique identifications
and they were arranged in metabolic cages suitable for the collection of urine
(10).
Rats of group A served as control and were
treated orally with 2% gum acacia suspension. Rats of groups B were treated
respectively with moclobemide 100mg/kg orally suspended in 2% gum acacia. Rats
of group C, D, E were administered with methanolic extracts of Rhodiola Root extract at a doses (100mg/kg,p.o), (200mg/Kg,P.O) and
(400mg/Kg,P.O) respectively in 2%gum acacia suspension. The treatment was given
for 3 days in all groups and 24 hr urine
samples were collected daily for 3 days before, during and after for 4 days of the treatment. The volume of urine collected
during 24 hrs was noted and the aliquots of urine sample were analysed for
5-HIAA as described below.
Preparation of stock solution of 5-HIAA: A
stock solution was prepared by dissolving 50 mg of 5-HIAA in 100 ml of
distilled water in a volumetric flask, which represents 0.5mg/ml of solution.
From the above stock solution further dilutions were made to represent
50,100,200,400,600 and 800mcg/10ml of the solution.
1,1-Nitroso-2-napthol
(0.1%
solution) in ethanol, Nitrous acid
reagent (5ml of 2N sulphuric acid, 0.2ml of 2.5% of sodium nitrate), 2,4-Di
nitro phenyl hydrazine in 2N HCl and Phosphate
buffer (0.5N) PH>7.0
were used as reagents. Diethyl
ether (reagent grade) was washed with dilute solution of ferrous sulphate to
remove peroxides.
Monamine Oxidase Inhibitory Activity
The biogenic amine 5-HT is
oxidized by MAO to 5-hydroxy indole acetic acid (5-HIAA) as a major metabolite
which is excreted in urine (Udenfriend et al., 1955). Hence the reduction in
the urinary 5-HIAA level from normal after treatment with the test compound was
taken as a criteria for it’s MAO inhibitory action (11).
In the present work, the MAO inhibitory
activity was studied by biochemical method where the urinary excretion of
metabolite of 5-HT was determined in urine samples of rats and it was compared
with that of moclobemide, a known MAO inhibitor which was used as a reference
substance.
As per the method reported by Udenfriend
et.al the 5-HIAA in solutions and urine samples was determined by measuring the
optical density at 540nm on treating it with nitroso napthol reagent followed
by nitrous acid reagent, after removal of the interfering keto acids by
treating with 2,4dinitrophenyl hydrazine and extracting with chloroform.
Method: To prepare 10ml of standard
solution in a 50 ml glass stoppered bottle, 10ml of 2,4dinitrophenyl hydrazine
reagent was added. After 30min 25ml of chloroform was added, shaken for 10min
and then centrifuged. After phase separation organic layer was removed and
replaced with 25ml of chloroform and the extraction was repeated. After
centrifuging, 15ml of aqueous layer was taken into 50ml stoppered bottle to
this 8g of sodium chloride and 25ml of ether were added. These bottles were
shaken for 5min on a shaker and centrifuged. After centrifugation and phase
separation 20ml ether was taken into 50ml glass stoppered bottle containing 4ml
of phosphate buffer at pH 7.0. It was shaken for 5min, centrifuged and ether
was removed by aspiration. Later 3ml of buffer layer was taken into 15ml
stoppered tubes and to this 1.5 ml of nitroso napthol reagent and 1.5ml of
nitrous acid reagent were added and mixed well and were kept at 37°c for 5min. Then 5ml of
ethyl acetate were added and after phase separation the ethyl acetate layer was
aspirated. The above step was repeated with another 5ml of ethyl acetate and
the absorbancy of aqueous solution was measured at 540nm against reagent blank.
Statistical
analysis:
In this investigation, the
results were expressed as mean ± standard deviation (n = 6). The data were
analyzed statistically by one way analysis of variance (1ANOVA) compared. while
p<0.05was considered statistically significant in all cases. The
software package graph pad prism version 6.0 was used for analysis of data.
RESULTS
AND DISCUSSION:
The phyto chemical constituent evaluation
of Rhodiola rodantha rhizomes extract
showed the presence of alkaloids, glycosides, flavonides, tannic acid, tannins
and pseudo tannins. The presence of alkaloids, glycosides and tannins which was
attributed to be responsible for psycho neuro pharmacological activity.
In the acute toxicity studies no toxic
symptoms or mortality were observed in any animals, which lived up to 14 days after
the administration of methanolic extract
at single dose level of 5000 mg/kg body weight. A starting dose of 2000mg/kg
body weight /p.o of methanolic extracts were administered to 3 female rats,
observed for 14 days. When the experiments were repeated again with the same
dose level, no changes were observed from the first set of experiment. The
extracts at lower does not changed alteration in Alertness, Aggressiveness,
Pile erection, Muscle relaxant and
Hypnosis effect and a significant differences were observed at higher doses.
The signs of tremors, convulsions, respiratory depression and death were not
observed at low and higher doses. The above studies indicate that the extract
of rhodiola is safe and the selected doses are below the lethal dose.
Forced
Swim Test (behavioural despair test):
In this
study, we evaluated the antidepressant effects of methanolic extract of R. rhodantha using forced swimming test and
tail suspension test. Both the paradigms are widely accepted behavioral models
for assessing antidepressant activity. For this purpose duration of immobility
and swimming times of the three doses of methanolic extract from R. rhodantha
in comparison with negative control and imipramine (positive control) were
studied. In this test when animal forced to swim in a restricted area initially
has vigorous activity and then showed an immobile posture and restricted
movement which is indicative of lowered mood was presumed to have
antidepressant effects.
A
significant (P<0.01) decrease in the duration of immobility was seen with
the standard drug imipramine in all the tested doses. All the extracts in doses
of 200mg/kg and 300mg/kg produced a greater decrease in the duration of
immobility as compared to the standard drug imipramine. Results indicated that
in lower dose of the extract (100 mg/kg) immobility times (Figure-2) did not
changed compared to the control group. However, at higher concentrations of
extract duration of immobility (200 and 400mg/kg) significantly increased and
decreased immobility respectively.
Exact
mechanisms underlying the antidepressant action cannot be concluded at the
moment due to the presence of large number of phyto chemicals. However the
antidepressant activity may be attributed to the presence of tannic acid,
gallic acid, polyphenols in the extract. Tannic acid has been shown to be a non
selective inhibitor of monoamine oxidase, thereby increasing the levels of mono
aminergic neurotransmitters in the brain. Another possible mechanism of action
is the attenuation of oxidative stress produced during depression, by the
polyphenols and tannic acid.
TAIL
SUSPENSION TEST:
In this test animals were treated with three doses
of RRME (100, 200 and 300 mg/kg) showed significant decrease in their
immobility times. Similarly, animals treated with imipramine (20 mg/kg) showed
a significant decrease in the immobility time. Tail suspension test produced a marked reduction in
immobility time at doses of 200 and 400 mg/kg in the rat with a profile
comparable to that observed for the classical antidepressant drug imipramine.
The plant extracts showed dose proportional relationship with time of
immovability. In this test, the immovable time decreased significantly with
increase in the dose level of methanolic extract (200mg/kg & 400mg/kg) of
rhodiola root.
Average mean values of 3 such
determinations for rhodiola extract are given in Table-2. A linear relationship
between the amount of 5-HIAA was found in the range of 66 to 273 mcg. A
positive correlation between concentration of 5-HIAA and the concentration of
rhodiola extract was observed. The amount of 5-HIAA in the unknown sample was
directly read from the standard graph. The 5-HIAA levels in urine samples were
analysed in the same manner as described earlier and the amount of 5-HIAA
excreted during 24hrs was calculated.
The percentage changes of 5-HIAA levels
calculated from average of the values obtained on the 3 days prior to drug treatment
was also shown in the Table-3. The significance of rhodiola extract on urinary
5-HIAA levels compared to pre treatment levels was tested. All the drugs
produced significant change. The percentage off change in 5-HIAA levels
observed with (500mg/kg) rhodiola was equivalent to that of moclobemide
(50mg/kg) indicating that they produced similar effect. Also the percentage
change of 5-HIAA levels observed with rhodiola (400mg/kg) was almost equal to
that of Moclobemide (100mg/kg) indicating that they produced similar effect.
CONCLUSION:
In conclusion Rhodiola rodantha
root methanolic extract
has shown significant antidepressant activity greater than imipramine and it
has the potential to be used as an antidepressant.
ACKNOWLEDGMENT:
We are grateful to the
management of Sri Venkateswara College of Pharmacy for providing the facilities
to carry my research work.
REFERENCES:
1.
Borsini F, Meli
A, Is the forced swimming test a suitable model for revealing antidepressant
activity, Psychopharmacology, 94; 1988: 147-160.
2.
Dhingra D,
Sharma A, Antidepressant-like activity of Glycyrrhiza glabra, Neuropsychopharmacol. Biol.
Psychiatry. 30; 2006: 449.
3.
Dunham N.M,
Miya T.S. A note on simple apparatus for detecting Neurological deficit in rats
and mice, J. Am. Pharm, 46; 1957: 208-9.
4.
Guo Y, Kong L,
Wang Y, Huang Z, Antidepressant evaluation of polysaccharides from a Chinese
herbal medicine Banxia-houpu decoction, Phytother.Res.18; 2004:204-207.
5.
Van Diermen D,
Marston A,
Bravo J,
Reist M,
Carrupt PA,
Hostettmann K.Monoamine oxidase
inhibition by Rhodiola rosea L. roots. J Ethnopharmacol.2009; 122(2):397-401.
doi: 10.1016/j.jep.2009.01.007. Epub 2009 Jan 9.
6.
C. B.
Nemeroff, “The burden of severe depression: a review of diagnostic challenges
and treatment alternatives,” Journal of Psychiatric Research, 41(3-4); 2006:
189–209.
7.
Haque AM, Hashimoto M, Katakura M, Tanabe Y, Hara
Y, Shido O. Long-term administration of green tea catechins improves spatial
cognition learning ability in rats. J Nutr 2006; 136:1043-7.
8.
Joshi H,
Parle M. Brahmi rasayana improves learning and memory in mice. eCAM
2006;3(1):79-85.
9.
Calabrese
C, Gregory WL, Leo M, Kraemer D, Bone k, Oken B. Effects of a standardized Bacopa monnieri Extract or cognitive
performance, anxiety and depression in elderly: a randomized double blind
placebo controlled trial. J Altern complement Med.2008; 14: 707-3.
10. Tang JL, Zeng YS, Chen QG, Qin YJ, Chen SJ,
Zhong ZQ. Effects of valerian on the level of 5- hydroxyl tryptamine, cell
proliferation and neurons in cerebral hippocampus o rats with depression
induced by chronic mild stress. Zhan Xi Yi Jie He Xue Bao 2008; 6: 283-8.
11.
Hou WC, Lin RD, Chen CT, Lee MH. Monoamine oxidase
B (MAO-B) inhibition by active principles from Uncaria rhynchophylla. J
Ethnopharmacol 2005; 100(1-2):216-20.
