To Elucidate Pharmacological Profile of Bioactive Principles of some Himalayan Medicinal and Aromatic Plants A Thesis Submitted for the Degree of DOCTOR OF PHILOSOPHY in CHEMISTRY KUMAUN UNIVERSITY 2009 by SANGEETA PILKHWAL M. Pharm DEPARTMENT OF PHARMACY KUMAUN UNIVERSITY, NAINITAL UTTARAKHAND
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To Elucidate Pharmacological Profile of Bioactive Principles of some Himalayan
Medicinal and Aromatic Plants
A Thesis Submitted for the Degree of DOCTOR OF PHILOSOPHY
in
CHEMISTRY KUMAUN UNIVERSITY
2009
by
SANGEETA PILKHWAL M. Pharm
DEPARTMENT OF PHARMACY
KUMAUN UNIVERSITY, NAINITAL UTTARAKHAND
CC EE RR TT II FF II CC AA TT EE
This is to certify that the work incorporated in this thesis
entitled “To Elucidate Pharmacological Profile of Bioactive Principles
of some Himalayan Medicinal and Aromatic Plants” has been carried
out by Mrs. Sangeeta Pilkhwal, under our supervision. She has fulfilled
the requirement of prescribed period for the Degree of Doctor of
Philosophy in Chemistry of Kumaun University, Nainital.
Unless stated otherwise, the work included in this thesis is
original and has not been submitted for any other degree.
DR. KANWALJIT CHOPRA PROF. C. S. MATHELA Co-supervisor Supervisor Head, Pharmacology Division Professor & Head UIPS, Panjab University Department of Chemistry Chandigarh. Kumaun University, Nainital.
DDeeddiiccaatteedd TToo
MMyy FFaammiillyy MMeemmbbeerrss
AACCKKNNOOWWLLEEDDGGEEMMEENNTT
As I sit back to write this little piece, I think of the innumerable hands that
have steered me through this arduous journey and put me on the right path of
learning and enlightment.
At the outset, I deem it my prime duty and obligation to express my
sagacious sense of gratitude and indebtedness to my esteemed teacher and guide
CONTENTS Introduction................................................................................... Aims and objectives.................................................................................... CHAPTER 1
Review of literature.................................................................................... CHAPTER 2 Chemical screening of Valeriana wallichii collected from different regions of Kumaun Himalaya
Section A: Isolation and identification of constituents from Valeriana wallichii essential oils 2.1.a. Introduction …………….......................................................... 2.2.a. Material and methods............................................................... 2.3.a. Results..................................................................................... Section B: Isolation and analysis of valepotriates of Valeriana wallichii chemotypes 2.1.b. Introduction………………………………………………………………… 2.2.b Material and methods……………………………………………………… 2.3.b Results…………………………………………………………………………
CHAPTER 3
Psychopharmacological profile of Valeriana wallichii chemotypes
3.1. Introduction …………………………………………………………. 3.2. Material and methods ………………………………………………. 3.3. Results ………………………………………………………………………. 3.4. Discussion ...…………………………………………………………
CHAPTER 4
Screening of Valeriana wallichii chemotypes for antidepressant effect
4.1. Introduction ………………………………………………………… 4.2. Material and methods ……………………………………………… 4.3. Results ……………………………………………………………… 4.4. Discussion …………………………………………………………..
CHAPTER 5
Pharmacological and neurobiochemical evidence for antidepressant like effect of Valeriana wallichii chemotypes
5.1. Introduction ………………………………………………………… 5.2. Material and methods ……………………………………………… 5.3. Results ……………………………………………………………… 5.4. Discussion …………………………………………………………..
CHAPTER 6
Studies on analgesic activity of Valeriana wallichii chemotypes
6.1. Introduction ……………………………………………………… 6.2. Material and methods …………………………………………… 6.3. Procedure ………………………………………………………… 6.4. Results …………………………………………………………… 6.5 Discussion………………………………………………………………..
CHAPTER 7
Evaluation of in vitro antioxidant profile of Valeriana wallichii chemotypes
7.1. Introduction ……………………………………………………… 7.2. Material and methods …………………………………………… 7.3. In vitro antioxidant assays ………………………………………. 7.4. Results …………………………………………………………… 7.5. Discussion ………………………………………………………..
CHAPTER 8
Modulation of antioxidant defense system in mice brain by Valeriana wallichii chemotypes
8.1. Introduction ………………………………………………………. 8.2. Material and methods ……………………………………………. 8.3. Results ……………………………………………………………. 8.4. Discussion …………………………………………………………
Compound VW-IC was another major component. The IR absorption band at
3507 indicates the compound VW-IC possesses an alcoholic group (Fig. 2.14.a). The 1H-NMR spectral data δ 0.82 (3H, s), δ 1.02 (3H, s) and δ 0.79 (3H, d) showed –CH3,
(CH3)2C-OH and >C-CH3, respectively (Fig. 2.15.a). The mass spectrum showed
molecular ion peak (m/z) 222 corresponding to C15H26O which is for a tricyclic
sesquiterpene alcohol (Fig 2.16.a). The carbon attached to –OH showed up at δ 72.8
ppm. Comparison of the data with literature report ((Nishiya et al., 1995; Sati, 2002;
Tewari, 2002; Sammal, 2005) confirms the compound VW-IC as patchouli alcohol.
OH
H
H VW-IC (Patchouli alcohol)
Fig. 2.14.a. Infra Red spectrum of compound VW-IC
Fig. 2.15.a. 1H-NMR spectrum of compound of compound VW-IC
Fig. 2.16.a. Mass spectrum of compound VW-IC
2.4.a. DISCUSSION
The percentage yield of the essential oil was found to be 0.60% (v/w) in case
of plant VW-II. Gas chromatogram revealed 28 peaks out of which 20 compounds
have been identified. The major constituents were separated by column
chromatography followed by their purification. The minor constituents were identified
with the help of GC-MS. Patchouli alcohol (VW-IIC), an isolated compound was
found to be major constituent. 8-Acetoxyl patchouli alcohol (VW-IID) constituted
10.45 % of the oil. The percentage yield of the essential oil of plant VW-I was found
to be 0.16% (v/w). The GC-MS analysis showed the presence of 30 compounds, out
of which 26 compounds constituting 92.41% of the total oil have been identified. An
interesting feature in the mother liquor was the presence of oxygenated
sesquiterpenoid like maaliol (36.82%) followed by β-gurjunene (21.28%), guaiol
(8.60%), α-santalene (5.42%), bicyclogermacrene (2.87%). The major component was
found to be maaliol. The results showed that the composition of the essential oil
isolated from roots and rhizomes of V. wallichii varied depending on the origin. The
plant VW-I was found to be chemotype maaliol of V. wallichii and VW-II was found
to be patchouli alcohol chemotype.
Section B
Isolation and Analysis of Valepotriates of Valeriana wallichii Chemotypes
2.1. b. INTRODUCTION
Same as in section 2.1.a. of chapter 2.
2.2.b. MATERIAL AND METHODS
2.2.1.b Plant material
Roots and rhizomes of Valeriana wallichii (Maaliol type), Valeriana wallichii
(Patchouli alcohol type) were collected from regions as mentioned in section 2.2.1.a.
2.2.2.b. Extraction
After collection, the plant materials were immediately stored at cool and dry
place. The plant material (1kg) were dried in shady place and subjected to
dichloromethane (CH2Cl2) extraction. The extract was concentrated to dryness with
rotary evaporator at 35°C to yield a brown dry mass (2 g). So the yield being 20% for
both the chemotypes. Extract from plant VW-II (Patchouli alcohol type) was coded as
VVDR03 and that from VW-I (Maaliol type) was coded as VVW02. Extraction of the
plant material for HPLC screening was carried out by homogenizing 10 g of dried
material of each Valeriana plant with 50 ml of dichloromethane for 3 days. The
homogenate was filtered and the residue was washed with 5 ml portion of
dichloromethane. Evaporation of the solvent was done below 35°C by rotary
evaporator, and the residue was again dissolved in dichloromethne (3 ml).
2.2.3.b HPLC Analysis
Thermoelectron Quaternary HPLC system with an injection valve system and
a 20µl sample loop and stainless steel column (Thermoelectron C-18) 25 cm x 4.6 mm
i.d was used. The column outlet was connected to UV detector (UV-1000) and RI-
detector (RI-150). The detection wavelengths were 254 nm for diene valepotriates and
210 nm for monene valepotriates.
2.2.4.b. Solvents
Dichloromethane and acetonitrile used were of HPLC grade (Qualigens). The
water used for solvent system was deionised and the HPLC system was isocratic with
binary solvent system, acetonitrile: water (70:30).
2.2.5.b. Analytical separation of valepotriates
Diene valepotriates show absorption maximum at 254 nm, due to their two
conjugated double bonds, and can be detected with high sensitivity at this wavelength.
Monene valepotriates were detected at 210 nm. Acetonitrile: water (70:30) was used
as eluent with 1ml/ min flow rate (Fig. 2.1.b, 2.2.b, 2.3.b & 2.4.b).
Fractionation
The residue (oily mass) was fractionated on column chromatography silica gel
(60-120 mesh) in Et2O: n-hexane (8% to 30%). Repeated column chromatography of
the column fractions followed by purification afforded three compounds (VW-I≠01,
VW-I≠02 and VW-I≠03) from Valeriana wallichii (Maaliol type) and three
compounds (VW-II≠04, VW-II≠05 and VW-II≠06) from Valeriana wallichii
The compound VW-I≠01 was obtained as a light brown viscous liquid. 13C-
NMR showed the presence of twenty-three carbons. Valtrate type skeleton was
confirmed by the presence of one epoxide group at the C-8 carbon, one methyl
protons at δ 4.74 adjacent to an oxygen function and one methine carbon δ 83.0
having an oxygen function with an acetate residue at C-7. 13C-NMR (Fig. 2.5.b) and 1H-NMR (Fig. 2.6.b) signals confirmed two isovaltrate groups at C-1 and C-11. The
structure of the compound VW-I≠01 was therefore characterized as didrovaltrate.
Finally, identity was confirmed by comparison of its spectral data with those reported
in literature (Tang et al., 2002; Thies et al., 1981, Sammal, 2005).
carbons and 6 quaternary carbons (Fig 2.9.b). The signals at δ 6.69 and δ 92.5
respectively were assigned to H-1 and C-1. The chemical shifts of two olefinic
methine carbons at δ 108.3 and 140.9 respectively were assigned to C-3, C-6, C-4 and
C-5. The chemical shift of one methine carbon (δ 83.0), one quaternary carbon (δ
64.1) and one methylene carbon (δ 43.0) were assigned C-7, C-8 and C-9
respectively. The quaternary carbon at δ 64.1 (C-8) and the methine carbon at δ 47.8
(C-10) were characteristic for an epoxide ring at C-8 and C-10. 1H and 13C-NMR data
of compound showed the presence of acetate residue at C-7 and a β-me-isovaltrate
group at C-11. Therefore, compound VW-II#06 was identified as 1-homoisovaltrate.
Finally, the identity was confirmed by comparison of spectral data (Fig. 2.9.b) with
that reported in literature (Tang et al., 2002; Thies et al., 1981; Sammal, 2005).
O
O
O
H
O
CH3
O
CH3
O
O
CH3
VW-II≠06 (1-Homoisovaltrate)
Fig. 2.9.b 13C-NMR spectrum of VW-II≠06
CHAPTER-3
Psychopharmacological Profile of Valeriana wallichii chemotypes
3.1. INTRODUCTION
Although a multitude of pharmaceutical agents are available for the treatment
of mood disorders, anxiety, insomnia and epilepsy; many patients have difficulty
tolerating the side effects, do not respond adequately, or eventually lose their
response. Many therapeutic herbs and nutrients have far fewer side effects and may
provide an alternative treatment or can be used to enhance the effect of prescription
medications.
Epilepsy is one of the most common disorders of the central nervous system,
worldwide. Though, the advent of newer techniques in neurobiology has provided
some insight into the pathophysiology of epilepsies, many aspects of this phenomenon
still remain unknown. It is, therefore not surprising that the currently used
antiepileptic drugs fail to provide satisfactory seizure control for nearly 15 % to 20 %
per cent of patients with epilepsy. For such patients, the combinations of antiepileptic
drugs are often prescribed in attempts to improve seizure control. However, toxicities
associated with these drugs further compromise the quality of life, while drug-drug
interactions may complicate management. Thus there is an ever lasting need for
search of newer molecules for treating seizures.
Valeriana wallichii DC. (Syn: V. jatamansi Jones) is a plant that belongs to
family “Valerianaceae” commomly called as Indian valerian. It is a small perennial
herb of 15-45 cm height with rootstock, thick branching stem, sharply pointed leaves,
white or pink flowers in clusters and hairy fruit. It is indigenous to the temperate
Himalayas and found in Kashmir, Nepal, Bhutan, Burma and Afghanistan. The plant
is widely known for its use in anxiety, insomnia, epilepsy, failing reflexes, hysteria,
neurosis and sciatica (Nadkarni, 1976). It is also considered useful as a potent
tranquilizer, emmenagogue (Nadkarni, 1976), diuretic (Said, 1970) and
hepatoprotective (Awan, 1990). Its active principles are valepotriates (Becker and
Chavadej, 1985) (like dihydrovaltrate) (Bounthanh et al., 1981), linarin-isovalerianate
(Thies, 1968), sesquiterpenoids (Ron et al., 2000), 6- methylapigenin and hesperidin
(Marder et al., 2003). Valerian preparations yield isovaleric acid, a substance
analogous to valproic acid and likely to possess anticonvulsant properties (Eadie,
2004). It is well known that the content and composition of active compounds in
herbal medicines are strongly influenced by many factors, such as genotype, climate,
harvest and preparation process. For example, V. wallichii roots from different
sources were found to contain between 0.09 and 1.30% v/w of essential oil (Kapoor,
1990). In the present work the effect of variation of active compounds between V.
wallichii growing under different environmental conditions has been correlated with
their psychopharmacological profile
3.2. MATERIAL AND METHODS
3.2.1. Plant material and its extraction
Plants were collected and identified as mentioned in section 2.2.1.a. of chapter
2 (section A). The oil was extracted by steam distillation as described in section
2.2.2.a. of chapter 2 (section B). The oil obtained from VW-II (Patchouli chemotype)
was coded as V-PA and that obtained from VW-I (Maaliol chemotype) was coded as
V-MA. Similarly the extracts were obtained by the method as mentioned in section
2.2.2.b. and were coded as VVW02 (maaliol chemotype) and VVDR03 (patchouli
alcohol chemotype).
3.2.2. Animals
Animals used in the study were laca mice of either sex (20-40g) bred in
Central Animal House facility of Panjab University, Chandigarh housed in cages with
food and water ad libitum and maintained on a natural light and dark cycle. The
experimental protocols were approved by the Panjab University Animal Ethical
committee.
3.2.3. Drugs
The drugs used in the present study were Pentylenetetrazole (PTZ) (Sigma,
USA), Diazepam (Sigma, USA). PTZ was dissolved in normal saline. Diazepam was
suspended in a drop of Tween 80 and volume was made up with sterile water. The test
drugs VVW02, VVDR03, V-PA and V-MA were also suspended in Tween-80.
Vehicle treated group received a drop of Tween-80 dissolved in sterile water and
acted as control group.
3.2.4. Experimental protocol and procedure
3.2.4.1. Anticonvulsant activity
Pentylenetetrazol (PTZ, Metrazol) is a central nervous system stimulant and
was synthesized in 1924. In 1926, Hildebrant showed its convulsant action. It
produces jerky type of clonic convulsions in mice. Pentylenetetrazole has been used
experimentally to study seizure phenomenon and to identify pharmaceuticals that may
control seizure susceptibility. The convulsive effect of this drug is considered to be
analogous to petit mal type of convulsions in man. It is considered as a GABA
antagonist but the mechanism of the epileptogenic action of PTZ at the cellular
neuronal level is still unclear.
For evaluating the effect of V. wallichii chemotypes on PTZ-induced
convulsions, animals were divided into following groups, each group consisting of six
animals.
Group 1: Vehicle treated
Group 2-4: VVDR03 (20, 40 and 80 mg/kg p.o.)
Group 5-7: VVW02 (20, 40 and 80 mg/kg p.o.)
Group 8-10: V-PA (20, 40 and 80 mg/kg p.o.)
Group 11-13: V-MA (20, 40 and 80 mg/kg p.o)
Group 14: Diazepam (1 mg/kg, i.p.)
Animals were injected pentylenetetrazol 80mg/kg intraperitoneally (i.p.) one
hour after the drug treatment. Onset of seizures, delay in death and the mortality were
noted after 1 hr in all the groups (Kulkarni, 1999).
3.2.4.2 . Measurement of anxiety levels
Elevated plus maze, is a novel test to study the anxiogenic and anxiolytic drug
effects in rodents (Kulkarni, 1999). Exposure of the animals to novel maze alley
evokes an approach-avoidance conflict which is stronger in open arm as compared to
enclosed arm. Rodents (rats and mice) have aversion for high and open arm, they
freeze, become immobile, defecate and show fear like movements. The plasma
cortisol level is also reported to be increased, as a true reflection of anxiety. Major
advantages of this test procedure are a) it is simple, fast and less time consuming b)
no prior training or noxious stimuli is required and c) it is predictable and reliable
procedure for studying anxiety response as well as antianxiety action of drugs.
The plus maze apparatus for mice consist of two open (16x5 cm) and two
closed arms (16x5x12 cm) and is placed at a height of 25 cm. Anxiety reduction in the
plus-maze is indicated by an increase in the proportion of time spent in the open arms
(time in open arms/total time in open or closed arms), and an increase in the
proportion of entries into the open arms (entries into open arms/total entries into open
or closed arms). Total number of arm entries and number of closed-arm entries are
usually employed as measures of general anxiety in rodents (Hogg, 1996).
Animals were divided into following thirteen groups, each group consisting of
six animals.
Group 1: Vehicle treated
Group 2-4: VVDR03 (10, 20 and 40 mg/kg p.o.)
Group 5-7: VVW02 (10, 20 and 40 mg/kg p.o.)
Group 8-10: V-PA (10, 20 and 40 mg/kg p.o.)
Group 11-13: V-MA (10, 20 and 40 mg/kg p.o)
The animals were then placed individually at the center of the maze with head
facing the open arm one hour after the treatment. During the 5 min test, the number of
entries in closed arms and the time spent in closed arm were recorded (Kulkarni,
1999). Similarly in a separate protocol the effect of above treatments were noted in
elevated plus maze after 14 days of daily dosing.
3.2.4.3. Rota rod test
The test was carried out using an apparatus consisting of a horizontal rod of 3
cm diameter which was made to rotate at a speed of 20 rpm. A pretest was carried out
and only those animals who demonstrated the ability to remain on the revolving rod
for at least 1 min were selected. Animals were divided into following groups, each
group consisting of six animals.
Group 1: Vehicle treated
Group 2-4: VVDR03 (10, 20 and 40 mg/kg p.o.)
Group 5-7: VVW02 (10, 20 and 40 mg/kg p.o.)
Group 8-10: V-PA (10, 20 and 40 mg/kg p.o.)
Group 11-13: V-MA (10, 20 and 40 mg/kg p.o.)
Group 14: Diazepam (1 m/kg, i.p.)
The drugs were given 1 h before the test and the time for which mice remained
on the revolving rod was noted and compared with the control group. Mouse unable
to remain on the rod at least for three min was considered as a positive test and the
time of its fall was recorded (Viswanatha et al., 2006).
3.2.4.4. Statistical analysis
Results were expressed as mean±SEM. The intergroup variation was measured
by one way analysis of variance (ANOVA) followed by Tukey’s test. Statistical
significance was considered at P<0.05. The statistical analysis was done using the
Jandel Sigma Statistical Software version 2.0.
3.3. RESULTS
3.3.1. Acute toxicity
The oral administration of V-PA, V-MA, VVDR03 and VVW02 at doses
ranging from 10-80mg/kg did not produce any lethal effect. No adverse effect or
mortality was detected in albino mice up to 2000mg/kg, p.o dose of VVDR03 and
VVW02 during 24 h observation period. Similarly no adverse effect or mortality was
detected in albino mice up to 1000mg/kg dose of V-PA and V-MA.3.3.2. Effect on
PTZ induced convulsions
In PTZ induced convulsions both the extracts and oils were found to have no
effect on onset of convulsions when PTZ was injected to the mice (data not shown).
VVW02 and VVDR03 were found to produce dose dependent effect in delaying onset
of death but the effect was significant at 80 mg/kg in comparison to control and other
groups (Table 3.1.). VVDR03 delayed the time by 40.8 % at 80 mg/kg while VVW02
increased the time for onset of death by 30.4 %.
In case of valerian oils a dose dependent response was obtained with V-PA
and an inverted U shaped curve was obtained with V-MA but only 40mg/kg dose
significantly delayed the onset of death with respect to control group. When V-PA
was evaluated for effect on PTZ-induced convulsions then only 80 mg/kg dose of V-
PA produced significant delay in onset of death (73.9 %) (Table 3.2.).
The delay in onset of death is similar for VVDR03 and VVW02 as there is no
statisitical significant difference between them. Similarly V-PA was found to be more
effective in delaying the onset of death than V-MA. And out of all four V-PA was
most effective in delaying onset of death after PTZ injection.
3.3.3. Effect on elevated plus maze
Oral administration of both the extracts and oils at doses 10, 20 & 40 mg/kg
were found to have no anxiolytic effect in plus maze after 1 hr of administration
(Table 3.3. & 3.4.). Similarly no anxiolytic effect was found when the above drugs
were administered daily for 14 days and then tested in elevated plus maze (Table 3.5.
& 3.6.).
3.3.4. Effect on rota rod apparatus
All the doses (10, 20 and 40 mg/kg p.o) of VVDR03, VVW02, V-PA and V-
MA were found to have no effect on the time of fall in rota rod test suggesting that at
these doses valerian extracts and oils do not have muscle relaxant effect (Fig. 3.1.,
3.2., 3.3. & 3.4.). Diazepam the standard drug produced a significant decrease in time
of fall (49 sec) after intraperitoneal injection, depicting skeletal muscle relaxant
effect.
Table 3.1. Effect of different doses of VVDR03 and VW02 on convulsions induced by pentylenetetrazole. * denotes significance at p <0.05 in comparison to vehicle treated group
Treatment Dose (mg/kg) Onset of death (secs)
Vehicle 743 ±80
VVDR03 20 437±35
VVDR03 40 581 ±54
VVDR03 80 1257 ±140*
VVW02 20 566±50
VVW02 40 740.3±30
VVW02 80 1068±90*
Diazepam 1 No death
Table 3.2. Effect of different doses of V-PA and V-MA on convulsions induced by pentylenetetrazole. * denotes significance at p <0.05 in comparison to vehicle treated group
Treatment Dose (mg/kg) Onset of death (secs)
Vehicle 513±69
V-PA 20 323±67.9
V-PA 40 592.2±23
V-PA 80 1972±118*
V-MA 20 538±28
V-MA 40 735.7±39.6 *
V-MA 80 360±50
Diazepam 1 No death
Table 3.3. Effect of single administration different doses of VVDR03 and VVW02 on elevated plus maze
Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)
Vehicle 8±2 237.3±19.7
VVDR03 10 5±1.2 257±10.0
VVDR03 20 6±0.47 230±20.0
VVDR03 40 4±1 260±11.0
VVW02 10 3.8±1.39 265.2±14.7
VVW02 20 4±1.67 276±6.84
VVW02 40 4.4 ±1.83 248.4±42.4
Table 3.4. Effect of single administration of different doses of V-PA and V-MA on
elevated plus maze
Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)
Vehicle 5.3±0.3 211±42.8
V-PA 10 6.0±0.9 260±13
V-PA 20 5.3±1.7 262±23
V-PA 40 4.3± 1.3 251.3±31.4
V-MA 10 5.0±0.9 242±28
V-MA 20 4.6 ±1 237±48.5
V-MA 40 4± 1 271±3
Table 3.5. Effect of different doses of VVDR03 and VVW02 on elevated plus maze after 14 days of dosing.
Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)
Vehicle 8±2 237.3±19.7
VVDR03 10 4.2±1.7 222.6±13
VVDR03 20 6±2.1 211±9.8
VVDR03 40 9± 3.8 234±15.6
VVW02 10 5.2±0.47 209±14.3
VVW02 20 9.5±2.17 236±17.7
VVW02 40 6.6 ±1.8 242±16.1
Table 3.6. Effect of different doses of V-PA and V-MA in plus maze after 14 days of dosing Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)
Vehicle 3.6±1.2 230.7±19.7
V-PA 10 5±1.9 260±13.0
V-PA 20 3.4±0.7 278.2±9.8
V-PA 40 4.6±1.9 228.2±25.3
V-MA 10 6±1.2 222±28.0
V-MA 20 5±1.3 209.2±39.6
V-MA 40 7.2±2 257.8±10.6
0
50
100
150
200
250
300
350
Vehicle VVDR03(40)
VVDR03(20)
VVDR03(10)
Diazepam(1)
Tim
e of
fall
(sec
s)
0 min60 min
*
Fig. 3.1. Effect of single oral administration of VVDR03 (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading
0
50
100
150
200
250
300
350
Vehicle VVW02(40)
VVW02(20)
VVW02(10)
Diazepam(1)
Tim
e of
fall
(sec
s)
0 min60 min
*
Fig. 3.2. Effect of single oral administration of VVW02 (10, 20 & 40 mg/kg) on rota
rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading
0
50
100
150
200
250
300
350
Vehicle V-PA(40) V-PA(20) V-PA (10) Diazepam(1)
Tim
e of
fall
(sec
s)
0 min60 min
*
Fig. 3.3. Effect of single oral administration of V-PA (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading
0
50
100
150
200
250
300
350
Vehicle V-MA(40) V-MA(20) V-MA (10) Diazepam(1)
Tim
e of
fall
(sec
s)
0 min60 min
*
Fig. 3.4. Effect of single oral administration of V-MA (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading
3.4. DISCUSSION
The present study reports some neuropharmacological activities of valerian
extracts and oils in mice. Epilepsy is a common neurological disorder affecting about
0.5-1 % of the world’s population (Hackinski, 1998). The most popular and widely
used animal seizure models are the traditional Maximal Electroshock Seizures and
PTZ tests. Prevention of seizures induced by PTZ in laboratory animals is the most
commonly used preliminary screening test for characterizing potential anticonvulsant
drugs. Though the PTZ test predicts activity against absence seizures, the underlying
neuronal abnormality is poorly understand. Diminution of brain GABA level has been
reported after sub convulsive dose of PTZ (Ha et al., 2000). The MES test is
considered to be a predictor of likely therapeutic efficacy against generalized tonic-
clonic seizures. By contrast, the PTZ assay has been used preliminarily to evaluate
antiepileptic drugs. A large body of experimental evidences supports the involvement
of γ-Amino butyric acid (GABA) in seizures. GABA is the principal inhibitory
neurotransmitter in the cerebral cortex maintaining the inhibitory tone that
counterbalances neurons excitation. When this balance is disturbed, seizures ensue.
Reduction in GABA-mediated inhibitory activity of glutamate decarboxylase has
been reported in studies of human epileptic brain tissues (David, 2001). Glutamate
concentration increases before seizure onset and is found to be highest in the epileptic
hippocampus than non-epileptic hippocampus while GABA levels increased during
seizures was greater in non-epileptic hippocampus than in epileptic hippocampus
showing decreased levels of GABA in epilepsy (During and Spancer, 1993). The
GABAA receptor is responsible for most fast inhibitory neurotransmission in the
central nervous system. Consequently, this receptor has been targeted for the
pharmacological control of anxiety, sleep, and epilepsy. Numerous natural and
synthetic compounds interact with the GABAA receptor at distinct, yet incompletely
defined, sites. These compounds include barbiturates, benzodiazepines, neurosteroids
and picrotoxin (Sieghart, 1992; Smith and Olsen, 1995). GABAA receptor agonists as
well as drugs, which allosterically modulate the GABA receptor channel complex, are
therapeutically active against convulsive seizures (David, 2001).
In our study valerian extracts and oils were found to have delayed onset of
death in comparison to vehicle treated group while no effect was produced on onset of
convulsions and it failed to produce protection against mortality. There is an assertion
also that most drugs with anticonvulsant activity do not counteract pentylenetetrazole
seizures but only retard them (Loscher et al., 1991). We found that both VVDR03 and
VVW02 produced a dose dependent effect in delaying onset of death and the effect
being significant at 80 mg/kg dose in comparison to control group. But none of the
dose prolonged the time of onset of convulsions as compared to control group. The
delay in onset of death produced by valerian extracts may result from the effect of its
components on inhibitory neurotransmitter GABA (Menini et al., 1993; Santos et
al.,1994a, 1994 b). The effect produced by VVDR03 and VVW02 is comparable. It is
suggested that different constituents of valerian extract interact with the GABA
system in the brain like inhibition of GABA transaminase, interaction with
GABA/benzodiazepine receptors and interference in uptake and release of GABA in
synaptosomes (Morazzoni and Bombardell, 1995, Houghton, 1999). GABA is the
major inhibitory neurotransmitter in the brain and the inhibition of its
neurotransmission has thought to be an underlying factor in epilepsy (Olsen, 1981).
Standard antiepileptic drugs, phenobarbitone and diazepam are thought to produce
their antiepileptic effects by enhancing GABA neurotransmission. This also may be
responsible for anticonvulsant and sedative action produced by V. wallichii.
Overproduction of NO has been linked to neurotoxicity during ischemia, some
forms of neurodegenerative brain diseases, and induction of seizures (Garthwaite,
1991). Nitric oxide (NO) has been linked to epileptic activity through the formation of
cyclic GMP (cGMP). Stimulation of the brain N-methyl-D-aspartate (NMDA)
receptors with glutamate or excitatory amino acids increases calcium influx, which
results in activation of cNOS and formation of NO. NO in turn activates guanylate
cyclase to synthesize cGMP, which is assumed to initiate seizures (Garthwaite, 1991).
NO modulates experimentally induced seizures in a complex manner. In addition to
stimulation of cGMP, NO has several other actions: it blocks NMDA receptors in a
negative feedback manner, thereby attenuating excitable activity (Manzoni et al.,
1992); promotes and suppresses glutamate release (Pelligrino et al., 1996) and reduces
the receptor activity of the inhibitory γ-aminobutyric acid (GABA) neurotransmitter
(Robello et al., 1996). PTZ induction of seizures may be related to the antagonistic
activity of the compound at the GABA-A receptor and to its activation of the NMDA
receptor (Kaputlu and Uzbay, 1997). Thus, the induced NO may augment the
capability of PTZ to induce seizures at both pathways. In case of valerian oils V-PA
produced a dose dependent effect and the results being significant at 80 mg/kg in
comparison to control group. V-MA produced an inverted U shaped curve in which
the effect produced at 40 mg/kg is significant in comparison to control group.
Valerian oil V-PA was found to be most effective in delaying onset of death in PTZ-
induced convulsions. Valerian oils may be effective because of terpenes present in
them as some researchers have reported anticonvulsant activity of monoterpenes
(Librowski et al., 2000). Therefore it seems that antiseizure profile of V. wallichii oils
may be related in part to terpenoids present in them like α-pinene, camphene and
terpineol (Arora and Arora, 1963) and the terpenes might be mediating anticonvulsant
effect by affecting nitric oxide pathway (Ahn et al., 2003). In another study done by
us we found that valerian oils produced an antidepressant effect mediated by NO
pathway and a similar inverted U-shaped activity curve or biphasic dose response
curve was observed with V-MA in producing antidepressant effect. Such biphasic
curve has been reported in other studies and it may be due to facilitatory and
inhibitory effect of NO on the NMDA receptor. In addition such mechanisms may
help explain why so many contradictory results have been found suggesting that NO
either facilitates or inhibits NMDA receptor mediated events such as seizures
(Rundfeldt et al., 1995), neurotoxicity (Weissman et al., 1992), anxiolysis and
nociception (Harkin et al., 1999).
The elevated plus maze (EPM) is a rodent model of anxiety that is used as a
screening test for putative anxiolytic compounds and as a general research tool in
neurobiological anxiety research. The test setting consists of a plus-shaped apparatus
with two open and two enclosed arms, each with an open roof, elevated 15 cm from
the floor. The model is based on rodents aversion for open spaces. This aversion leads
All the test drugs were administered orally. L-NAME was given half an hour
after administration of test drugs and then mice were allowed to swim in Porsolt
apparatus 30 minutes after intraperitoneal injection of L-NAME.
4.2.5. Statistical analysis
The data were expressed as mean± SEM of 6 animals. Results were analysed
statistically by One-way ANOVA followed by Tukey’s multiple comparison using
sigma stat software. The difference was considered statistically significant if p<0.05.
4.3. RESULTS
4.3.1. Acute toxicity
Same as in section 3.3.1. of chapter 3.
4.3.2. Effect on locomotor activity
None of the dose of VVDR03 significantly decreased the locomotor activity
while VVW02 at 40 mg/kg produced significant decrease (45.8%) in locomotor
activity (Table 4.1.). V-MA did not alter the spontaneous locomotor activity except at
dose 40 mg/kg which significantly produced 44.9 % decrease in this parameter (Table
4.2.). While 40 mg/kg dose of V-PA produced a decrease in locomotor activity but the
effect was not significant. So the results depict that none of the doses of VVDR03 and
V-PA altered locomotor activity in mice. Imipramine the standard drug was also
devoid of any effect on locomotor activity.
4.3.3. Effect of acute dosing of extracts in FST
When single oral administration of different doses of VVDR03 and VVW02
were studied for effect in FST, 40 mg/kg dose of both the extracts (Fig. 4.2. & Fig.
4.3.) significantly decreased immobility period as compared to vehicle treated group.
The effect was found to be more pronounced with VVW02 (57% decrease in
immobility) than VVDR03 (32.7 % decrease in immobility) (statistical significant)
and group treated with standard drug imipramine (36.2 % decrease).
4.3.4. Effect of acute dosing of oils in FST
Different doses of V-PA produced a dose response curve i.e on increasing the
dose response was increased (Fig. 4.4.). The effect being significant at 40 mg/kg
(57.6% decrease) and 20 mg/kg (46.9% decrease). Whereas different doses of V-MA
produced an inverted U shaped curve, the effect being significant at 40 mg/kg and 10
mg/kg dose (Fig. 4.5.). 10 mg/kg produced 39.7% decrease while 40 mg/kg dose
produced 58% decrease in immobility as compared to control group. When compared
statistically both the oils were found equally effective in producing antidepressant
effect.
4.3.5. Effect of NOS inhibitor L-NAME on the action of VVDR03 and VVW02
(10 mg/kg) in FST
L-NAME was combined with 10 mg/kg dose of VVW02 and VVDR03 and
the modulation by L-NAME was then studied after half and hour of L-NAME
injection. L-NAME per se did not have any effect on the immobility period in FST
neither it potentiated the effect of VVDR03 and VVW02 (Fig 4.6.). The combination
did not change the locomotor activity (results not shown).
4.3.6. Effect of NOS inhibitor L-NAME on the action of V-PA and V-MA (10
mg/kg) in FST
To assess the modulatory effect of NOS inhibitor (L-NAME) on FST, V-PA,
V-MA (10 mg/kg p.o) was combined with L-NAME (10 mg/kg i.p). L-NAME per se
did not have any effect on the immobility period in FST. However, when L-NAME
was given 30 min after V-PA and V-MA it significantly potentiated the effect of V-
PA and V-MA (Fig. 4.7.). The combination did not increase the locomotor activity
(results not shown).
Table 4.1. Effect of different doses of extracts (VVDR03 & VVW02) of Valeriana wallichii in actophotometer apparatus in mice. (*) show significance at P<0.05 vs. vehicle group. Value in parenthesis is % decrease in locomotor counts. Treatment Dose (mg/kg. p.o) Mean ambulatory scores
Vehicle 240 ±32.4
VVDR03 10 235±64
VVDR03 20 139.6±65.4
VVDR03 40 210 ±58.5
VVW02 10 188.75±10
VVW02 20 105.3±62.7
VVW02 40 139±21 (45.8) *
Imipramine 10 200 ±6
Table 4.2. Effect of different doses of oils (V-MA & V-PA) of Valeriana wallichii in actophotometer apparatus in mice. (*) show significance at P<0.05 vs. vehicle group. Value in parenthesis is % decrease in locomotor counts.
Treatment Dose (mg/kg. p.o) Mean ambulatory scores
Vehicle 414 ±13.6
V-PA 10 383 ±45.4
V-PA 20 415 ±62.9
V-PA 40 319 ±36
V-MA 10 391±40
V-MA 20 324± 20.3
V-MA 40 228 ±48.7*(44.9)
Imipramine 10 300 ±40
Fig. 4.2. Effect of valerian extract VVW02 on the immobility period induced by FST. VVW02 at different doses were administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.
Fig. 4.3. Effect of valerian extract VVDR03 on the immobility period induced by FST. VVDR03 at different doses were administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.
Fig. 4.4. Effect of V-PA on the immobility period induced by FST. V-PA at different doses was administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.
Fig. 4.5. Effect of V-MA on the immobility period induced by FST. V-MA at different doses was administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.
Fig. 4.6. Effect of 10 mg/kg dose of VVDR03 & VVW02 in combination with L-NAME (10mg/kg i.p.).
Fig. 4.7. Effect of 10 mg/kg dose of V-PA & V-MA in combination with L-NAME (10mg/kg i.p.). a denotes significance at P < 0.05 in comparison to L-NAME per se, b denotes significance at P < 0.05 in comparison to V-PA per se and c denotes significance at P < 0.05 in comparison to V-MA per se. # denotes significance at P < 0.05 in comparison to vehicle treated group.
4.4. DISCUSSION
One of the purposes of this study was to evaluate the antidepressant-like effect
of valeriana by an animal model for depression. We employed the forced swimming
test (FST) on mice. The immobility displayed by rodents when subjected to an
unavoidable stress such as forced swimming is thought to reflect state of despair or
lowered mood, which reflects depressive illness in humans. Additionally, immobility
time has been shown to be reduced by treatment with antidepressant drugs (Porsolt,
1981). The FST was designed to detect potential antidepressant compounds based on
the abilities of clinically effective antidepressants to reduce the immobility in FST
(Porsolt et al., 1977). Though the FST does not adequately reflect the symptomalogy
of human depression, it appears to have a higher predictive validity compared to other
animal models Additionally, it is sufficiently specific, since it discriminates
antidepressants from neuroleptics and anxiolytics (Borsini and Meli, 1988; Willner,
1984). The present study has shown that valerian oils and extracts posses
antidepressant effect in FST.
In our study DCM extracts of both the chemotypes (VVW02 and VVDR03) at
40mg/kg dose significantly reduced the immobility time in FST in acute study.
VVW02 was found to produce 57 % decrease in immobility at 40 mg/kg as compared
to vehicle treated control group (Fig. 4.2.) while 40 mg/kg dose of VVDR03 produced
32.7% decrease in immobility which is comparable to the effect produced by standard
drug imipramine (Fig. 4.3.). In case of valerian oils V-MA produced significant
decrease in immobility at dosage 10 and 40 mg/kg while 20 mg/kg did not affect the
immobility period (Fig.4.5.) and produced an inverted U shaped curve. V-PA in single
dose study produced a dose dependent effect i.e. 20 and 40 mg/kg dose of V-PA
produced a significant decrease in immobility as compared to group receiving 10
mg/kg and control group (Fig.4.4.). Psychostimulants, which exert an indiscriminate
motor stimulating activity, have previously been shown to have efficacy at decreasing
immobility in FST, but no antidepressant activity (Porsolt, 1981). So in order to
exclude a false positive result, we employed an additional test to check motor
stimulating activity of extracts and oils. In the present study VVW02 and V-MA
lowered the locomotor activity at 40 mg/kg dose only whereas no alteration was seen
with VVDR03 and V-PA. Therefore the reduction in immobility by valerian extract
time can be attributed to an inherent antidepressant like effect.
A study depicted antidepressant effect of kessyl glycol diacetate, a valepotriate
possibly due to blockade of monoamine uptake (Miller and Murray, 1998). Methanol
extracts of Japanese valerian (Valeriana fauriei) also exhibited strong antidepressant
activity in the forced swimming test in mice (Oshima and Matsuoka, 1995). The
active fraction was identified as a sesquiterpenoid called a kessyl alcohol and its
activity compared favorably to that of imipramine. These studies support earlier
findings of a Japanese valerian root extract which significantly inhibited immobility
induced by a forced swimming test in rats (Sakomoto et al., 1992). Another recent
study showed anti-anxiety and anti-depressant benefits of valerian but failed to show
sedative or muscle relaxant benefits (Hattesohl et al., 2008). A study done on
Japanese valerian confirms that valerian extract and imipramine (a tricyclic anti-
depressant) significantly inhibited immobility in FST and reversed reserpine-induced
in mice, a test which suggests antidepressant actions (Sakamoto et al., 1992).
During the past few years, nitric oxide (NO), an inorganic free radical has
emerged as an important signal and effector molecule in several biological processes
including vasodilatation, inflammation and neurotransmission (Garthwaite, 1991;
Lowenstein, 1994; Moncada, 1991). NO is produced in neurons from the L-arginine
by the calmodium-dependent enzyme. NO synthase and NO fulfill some of the most
important criteria for a neurotransmitter (Knowles, 1994; Dawson, 1994). An
important target of NO is soluble guanlyl cyclase which is activated by NO binding to
heme part of the enzyme, causing elevation in cGMP. It appears that in different
systems, NO may be able to modulate vesicular release of neurotransmitter in either
direction or not all depending on the coincident level of presynaptic activity and NO
concentration (Garthwaite, 1995). In order to explore the involvement of nitric oxide
(NO) signaling pathway in the antidepressant-like effect of valerian extracts and oils,
10 mg/kg dose of VVDR03, VVW02, V-PA and V-MA were combined with L-
NAME (10 mg/kg) (a selective nitric oxide synthase inhibitor) (Babbedge et al., 1993;
Re es et al., 1990) and then tested for alteration in immobility in FST. No potentiation
was found in case of valerian extracts. It was found that L-NAME per se did not alter
the immobility period compared to control whereas it potentiated the action of V-PA
and V-MA significantly. The potentiation was even significant to group receiving L-
NAME only. The L-NAME was devoid of locomotor stimulatory property at 10
mg/kg dose which is in accordance to earlier study (Harkin et al., 1999). Moreover, a
study depicts that stimulation of NO synthase not only antagonized the behavioral
effect of NO synthase inhibitors but also of the prototypic antidepressant, imipramine.
So it may be possible that one or more component of essential oil (sesquiterpenes) of
Valeriana might be acting as NOS inhibitor as various studies report inhibition of NO
synthesis in lipopolysac-charide (LPS)-activated RAW 264.7 cells by sesquiterpenes
(Ahn et al., 2003; Motai and Kitanaka, 2005). In other studies, also sesquiterpenoids
have been found to have suppressive effect on NOS and COX-2 activity (Lee et al.,
2002; Yoon et al., 2008).
An inverted U-shaped activity curve or biphasic dose response curve observed
with V-MA has been reported in previous studies and it may be due to facilitatory and
inhibitory effect of NO on the NMDA receptor. NO regulates NMDA receptor
activity in a biphasic manner playing both a positive (via activation of guanylate
cyclase) and negative (via feedback effects on the NMDA receptor resulting in
decreased NMDA receptor and NO synthase activity) modulatory role in NMDA
receptor mediated events (Lei et al., 1992; Manzoni and Bockaert., 1993). In addition
such mechanisms may help explain why so many contradictory results have been
found suggesting that NO either facilitates or inhibits NMDA receptor mediated
events such as seizures (Rundfeldt et al., 1995), nociception, neurotoxicity (Weissman
et al., 1992) and anxiolysis (Harkin et al., 1999). Moreover, a study depicts that NO
has a dual role in the modulation of depression (Da Silva et al., 2000).
conditions for the experiment were +0.800 V, sensitivity ranges from 1 to 100 nA.
Separation was carried out at a flow rate of 1 ml/min. Samples (20µl) were injected
manually. Brain samples were homogenized in homogenizing solution containing
0.1M perchloric acid. After that samples were centrifuged at 24,000×g for 15 min.
The supernatant was further filtered through 0.25µm nylon filters before injecting in
the HPLC injection pump. Data were recorded and analyzed with the help of
Empower® software provided by Waters® (Beyer et al., 2002).
5.2.5. Statistical analysis
The data were expressed as mean± SEM of 6 animals. Results were analysed
statistically by One-way ANOVA followed by Tukey’s multiple comparison using
Jandel Sigma Stat Software. The difference was considered statistically significant at
p<0.05.
5.3. RESULTS
5.3.1. Acute toxicity
Same as in section 3.3.1 of chapter 3.
5.3.2. Effect of extracts in FST after 14 days of dosing
VVDR03 and VVW02 were found to produce a dose dependent response
curve in chronic study. 20 and 40 mg/kg dose of both the extracts decreased
immobility period significantly as compared to control group. The decrease being
30.8% and 55.9 % with VVW02 (Fig. 5.1) while VVDR03 (Fig. 5.2) produced 17.2
% and 45 % decrease in immobility in FST.
5.3.3. Effect of oils in FST after 14 days of dosing
Only 20 mg/kg dose of V-PA produced a significant decrease in immobility in
FST (Fig. 5.3.) as compared to vehicle treated group. V-PA (20 mg/kg) produced 69.6
% decrease which is much more marked than the effect produced by 40 mg/kg dose of
V-PA. 40 mg/kg dose of V-MA produced a significant decrease (70.9%) in
immobility while 20 mg/kg V-MA has no significant effect in FST (Fig. 5.4.).
5.3.4. Effect of extracts and oils on the neurotransmitter levels in mice brain
When checked for the alterations in the neurotransmitter levels after 14 days
of dosing 20 and 40 mg/kg dose of VVW02 increased norepinephrine levels dose
dependently and significantly. No alteration in dopamine levels is seen with any dose
of VVW02 (Fig. 5.5.). In case of VVDR03 both 20 and 40 mg/kg increased the
norepinephrine levels in whole brain significantly. The alterations being dose
dependent. None of the dose altered dopamine levels as compared to control except
20 mg/kg (Fig. 5.6.). 40 mg/kg and 20 mg/kg of V-PA increased norepinehrine levels
by 48.5 % and 84.96% respectively but the effect is significant at 20mg/kg dose (Fig.
5.7.). Similarly V-MA at 40 mg/kg produced a marked increase in norepinehrine
levels (Fig. 5.8.) while 20 mg/kg did not affect norepinehrine levels. None of the dose
of V-PA and V-MA altered dopamine levels in brain.
0
50
10 0
150
2 0 0
2 50
3 0 0
V ehicle V V W 0 2( 10 )
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
Mea
n im
mob
ility
per
iod
(sec
)
*
*
Fig. 5.1. Effect of VVW02 on the immobility period induced by FST. VVW02 at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.
0
50
10 0
150
2 0 0
2 50
3 0 0
V ehicle V V D R 0 3( 10 )
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
Mea
n im
mob
ility
per
iod
(sec
)
*
*
Fig. 5.2. Effect of VVDR03 on the immobility period induced by FST. VVDR03 at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.
0
50
10 0
150
2 0 0
2 50
V ehicle V - PA ( 10 ) V - PA ( 2 0 ) V - PA ( 4 0 )
Mea
n im
mob
ility
per
iod
(sec
)
* *
Fig. 5.3. Effect of V-PA on the immobility period induced by FST. V-PA at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.
0
50
10 0
150
2 0 0
2 50
V ehicle V - M A ( 10 ) V - M A ( 2 0 ) V - M A ( 4 0 )Mea
n im
mob
ility
per
iod
(sec
)
*
Fig.5.4. Effect of V-MA on the immobility period induced by FST. V-MA at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.
Fig. 5.5. Effect of different doses of VVW02 on the alteration in neurotransmitter levels in the mouse whole brain. VVW02 was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.
Fig. 5.6. Effect of different doses of VVDR03 on the alteration in neurotransmitter levels in the mouse whole brain. VVDR03 was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.
Fig. 5.7. Effect of different doses of V-PA on the alteration in neurotransmitter levels in the mouse whole brain. V-PA was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.
Fig. 5.8. Effect of different doses of V-MA on the alteration in neurotransmitter levels in the mouse whole brain. V-MA was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group
5.4. DISCUSSION
In this chronic study repeated administration of VVW02 and VVDR03 for 14
days produced a dose dependent response curve (Fig. 5.1 & 5.2). The effect being
significant at 20 and 40 mg/kg for both the extracts and out of them VVW02 (Maaliol
chemotype) was found to more effective than VVDR03 (Patchouli alcohol
chemotype).
In case of oils, V-PA produced a U shaped curve and the effect was found to
be significant at 20mg/kg in comparison to control (Fig. 5.3). Such U-shaped activity
curve may be due to multiple receptor action and has also been reported for some
herbal medicines (Butterweck et al., 2000, Butterweck et al., 2001 & Sakakibara et
al., 2006). V-MA produced a dose dependent response curve and the effect being
significant at 40 mg/kg (Fig. 5.4).
When checked for the alterations in the neurotransmitter levels after 14 days
of dosing, 20 and 40 mg/kg dose of VVW02 increased norepinephrine levels dose
dependently and significantly. No alteration in dopamine levels was seen with any
dose of VVW02 (Fig. 5.5). Similarly 20 and 40 mg/kg dose of VVDR03 increased the
norepinephrine levels in whole brain significantly and dose dependently (Fig. 5.6).
None of the dose altered dopamine levels as compared to control except 20 mg/kg of
VVDR03. 20 mg/kg of V-PA increased norepinehrine levels significantly and V-MA
at 40 mg/kg produced a marked increase in norepinehrine levels (Fig. 5.7 & 5.8).
From the previous study done by us we found that a NOS inhibitor (L-NAME)
potentiated the antidepressant effect of valerian oils in FST depicting the alteration of
NO level in mice brain by valerian oils leading to antidepressant effect. So it appears
that in different systems, NO may be able to modulate vesicular release of
neurotransmitter in either direction or not all depending on the coincident level of
presynaptic activity and NO concentration (Garthwaite, 1995). There are other studies
which also show that blockade of nitric oxide synthesis elevates plasma levels of
catecholamines and their metabolites at rest and during stress in rats (Richard et al,
1997). So from our results it can be speculated that the level of nitric oxide synthase
(NOS) inhibition elicited by valerian oil leads to a critical NO concentration which in
turn, alters the vesicular release of neurotransmitter like norepinephrine involved in
depression and we can say that the nitric oxide signaling pathway is involved in the
antidepressant-like effect of V. wallichii oils. While in case of valerian extracts no
potentiation was seen with L-NAME which suggests that some other pathway is
involved in antidepressant effect of extracts (VVDR03 and VVW02).
Biochemical theories of mood disorders have focused on the biogenic amines
but the importance of gamma butyric acid (GABA) in the neurochemical
pathophysiology of depression has also been demonstrated (Petty, 1995). Clinical data
also indicate that a decreased GABA function accompanies depression and there are
accumulating evidences which implicates a GABAergic dysfunction in depression
(Kubacka et al., 2006). There are various studies showing that the constituents present
in valeriana enhance GABA levels (Morazzoni and Bombardell, 1995, Houghton,
1999). So we can hypothesize that valerian extracts might be altering norpeinephrine
levels in brain by enhancing GABA neurotransmission.
CHAPTER-6
Studies on Analgesic Activity of Valeriana wallichii chemotypes
6.1. INTRODUCTION
Pain is a sensorial modality which is universally understood as a signal of
disease and it is the most common symptom requiring treatment with analgesic
agents. So pain often has a protective function. Throughout history, man has used
many different forms of therapy for the relief of pain, among them; medicinal herbs
are highlighted due to their wide popular use. In the relief of pain, opiates are
generally considered to act on the central nervous system exercising their effects
through three opioid receptors (µ, κ and δ) and such drugs are especially important for
the treatment of chronic pain. Although morphine has reigned for centuries as the king
of painkillers, its rule hasn’t been totally benign. There are concerns about its
addictive properties and side effects, which include respiratory depression,
drowsiness, decreased gastrointestinal motility, nausea and several alterations of the
endocrine and autonomic nervous system. Natural products in general and medicinal
plants in particular, are believed to be an important source of new chemical
substances with potential therapeutic efficacy. Taking into account the most important
analgesic, prototypes (e.g. salicylic acid and morphine) were originally derived from
the plant sources. The study of plant species traditionally used as pain killers should
still be seen as a fruitful research strategy in the search of new analgesic and anti-
inflammatory drugs.
Valeriana wallichii or Indian Valerian is a plant that steeped in history. Its
original use in herbal medicine today is as sedative and calming agent. Valepotriates
are responsible for the sedative action of the plant. It is used as a carminative and
antispasmodic in hysteria and similar nervous manifestations. Spasmolytic effect of
the V. wallichii has been used in different gastrointestinal disorders such as diarrhea
and abdominal spasm (Holmes, 1989). The studies depict that mild myorelaxant
action of Valeriana is attributed to the valepotriates component of the herb (Dunaev et
al., 1987). The valepotriates isovaltrate and valtrate, and the essential oil compound
valeranone were observed to suppress the rhythmic contractions in a closed part of the
guinea-pig ileum in vivo (Hazelhoff et al., 1982). The same compounds and
didrovaltrate relaxed potassium stimulated contractures and inhibited BaCl2
contractions in guinea-pig ileum preparations in vitro. The inhibition of muscle
contractions by the valium chemicals valeranone and didrovaltrate were as potent as
papaverine. There is a report showing analgesic effect of dried leaves of V. jatamansi
at a dose of 2 mg (Shrivastava and Sisodia, 1970). It has potential ethnomedicinal
uses like V. officinalis but till date there is no evidence-based clinical report. In this
context, we aimed to investigate the medicinal plant, V. wallichii for its analgesic
effect.
6.2. MATERIAL AND METHODS
6.2.1. Plant material and its extraction
Same as in section 3.2.1. of chapter 3.
6.2.2. Animals
Same as in section 3.2.2. of chapter 3.
6.2.3. Drugs
Aspirin was procured from Panacea Biotech, Lalru, India. To the test drugs
(VVW02, VVDR03, V-PA and V-MA) one drop of Tween-80 was added and then
volume was made up with distilled water. Solution of aspirin was also prepared using
Tween-80.
6.2.4. Procedure
Effect of valerian extracts and oils in Acetic acid-induced writhing
Acetic acid (1%) was injected i.p. in mice and the writhing response
characterized by abdominal constriction and hind limb stretching was counted for 10
min (Kulkarni, 1999). V-PA, V-MA, VVDR03 and VVW02 at doses of 20, 40 and 80
mg/kg were administered orally to mice 1 hr before the test. Aspirin 100mg/kg, p.o.
was used as a standard for comparison while vehicle treated group was kept as
control. The number of writhings in these groups were recorded and compared with
control and standard groups.
Effect of valerian extracts and oils on tail-flick latency
Analgesia was assessed with analgesiometer (Techno Electronics, India).
Basal reaction time of animals to radiant heat was recorded by placing the tip (last 1-2
cm) of the tail on the radiant heat source. The tail withdrawal from the heat (flicking
response) was taken as the end point. The animals, which showed flicking response
within 3-5 secs, were selected for the study. A cut off period of 12 secs was observed
to avoid damage to the tail (Kulkarni, 1999). The measurement of tail flick latency
was recorded at 15, 30, 60, 120 and 180 min after administration of V-PA, V-MA,
VVDR03 and VVW02 (20, 40 and 80 mg/kg).
Effect of valerian extracts and oils in combination with aspirin in acetic acid
writhing
For mechanistic study sub-effective dose i.e 20 mg/kg dose of VVDR03,
VVW02, V-PA and V-MA was combined with 5 mg/kg dose of aspirin and was then
studied in acetic acid induced writhing. Aspirin was given intraperitoneally 30
minutes after the administration of test drugs and acetic acid was injected 30 minutes
after aspirin. Per se effect of 5 mg/kg dose of aspirin was also studied in acetic acid
writhing.
6.2.5. Statistical analysis
The data were expressed as mean± SEM of 6 animals. Results were analysed
statistically by One-way ANOVA followed by Tukey’s multiple comparison using
sigma stat software. The difference was considered significant at p<0.05.
6.3. RESULTS
Acute toxicity
Same as in section 3.3.1 of chapter 3.
Effect of valerian extracts and oils in Acetic acid-induced writhing
Different doses of VVDR03 and VVW02 dose dependently inhibited the
number of writhings. VVDR03 produced significant inhibition of writhing at 80mg/kg
(54.3%) and 40 mg/kg (29.8%) (Fig. 6.1.). While VVW02 produced significant effect
only at 80 mg/kg and the percentage inhibition with it was found to be 29.8% (Fig
6.2.). The standard drug aspirin at 100mg/kg dose significantly inhibited the writhing
movements (64.9%) and the results were found to be highly significant (p<0.05) in
comparison to control group.
Similarly V-MA and V-PA at doses 20, 40 and 80 mg/kg, p.o. produced dose
dependent inhibition. V-PA at doses 80 and 40 mg/kg produced significant inhibition
of writhings i.e. 50% and 31.1% respectively (Fig. 6.3.). V-MA produced significant
inhibition in number of writhings at 80 mg/kg (63.6 %) and 40 mg/kg (27.2 %) as
compared to control group (Fig. 6.4.).
Effect of valerian extracts and oils on tail flick latency
In tail flick model different doses of VVDR03 (Table 6.1.) and V-PA (Table
6.3.) showed no significant increase in tail flick latency at different time interval when
compared to the predrug reaction time. But both VVW02 (Table 6.2.) and V-MA
(Table 6.4.) at 80 mg/kg dose produced significant increase in tail flick latency after
two hours of drug administration in comparison to predrug reaction. Similarly the
standard drug aspirin produced significant increase in tail flick latency after one and
two hours of drug administration.
Effect of valerian extracts in combination with aspirin in acetic acid writhing
Aspirin (5 mg/kg i.p) produced significant inhibition of writhings as compared
to vehicle treated group. But when 20 mg/kg dose of VVDR03 and VVW02 was
given in combination with aspirin (5 mg/kg) no potentiation was seen.
Effect of valerian oils in combination with aspirin in acetic acid writhing
V-PA (20 mg/kg) per se was found to have no effect on acetic acid writhing.
Aspirin per se produced 35% inhibition of writhing as compared to vehicle treated
group while when aspirin was given in combination with V-PA (20 mg/kg)
percentage inhibition was found to be 61.7% (Fig. 6.7.). Similarly V-MA in
combination with aspirin produced 52.4 % decrease in number of writhings (Fig.
6.8.). Thus the results show that aspirin potentiated the action of V-MA and V-PA in
acetic acid induced writhing.
0
5
10
15
2 0
2 5
3 0
3 5
4 0
4 5
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
V V D R 0 3( 8 0 )
V ehicle A sp ir in
Num
ber o
f writ
hing
s
*
*
*
Fig. 6.1. Effect of different doses of VVDR03 in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group
0
5
1015
2 0
2 5
3 0
3 54 0
4 5
50
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
V V W 0 2( 8 0 )
V ehicle A sp ir in
Num
ber o
f writ
hing
s
*
*
Fig. 6.2. Effect of different doses of VVW02 in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group
0
5
10
15
2 0
2 5
3 0
3 5
4 0
4 5
V - PA( 2 0 )
V - PA( 4 0 )
V - PA( 8 0 )
V ehicle A sp ir in
Num
ber o
f writ
hing
s
* *
*
Fig. 6.3. Effect of different doses of V-PA in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group.
0
5
10
15
2 0
2 5
3 0
3 5
4 0
4 5
V - M A( 2 0 )
V - M A( 4 0 )
V - M A( 8 0 )
V ehicle A sp ir in
Num
ber o
f writ
hing
s
*
*
*
Fig. 6.4. Effect of different doses of V-MA in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group. Table 6.1. Effect of different doses of VVDR03 in tail flick latency in mice. (*) denotes significance at P<0.05 versus predrug reaction Tail flick latency in seconds
Fig. 6.5. Effect of aspirin (5 mg/kg, i.p) in combination with VVDR03 (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to VVDR03 per se.
05
1015
2 02 53 03 54 04 550
V V W 0 2( 2 0 )
V V W 0 2( 2 0 ) +A sp
( 5)
A sp ( 5) V ehicle
Num
ber o
f writ
hing
s
*, a *
Fig. 6.6. Effect of aspirin (5 mg/kg, i.p) in combination with VVW02 (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to VVW02 per se.
0
5
10
15
2 0
2 5
3 0
3 5
4 0
4 5
V - PA ( 2 0 ) V - PA( 2 0 ) +A sp
( 5)
A sp ( 5) V ehicle
Num
ber o
f writ
hing
s
*, a, b
*, a
Fig. 6.7. Effect of aspirin (5 mg/kg, i.p) in combination with V-PA (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to V-PA per se and (b) denotes significance at P<0.05 as compared to Aspirin per se.
0
5
10
15
2 0
2 5
3 0
3 5
4 0
4 5
V - M A( 2 0 ) )
V - M A( 2 0 ) +A sp
( 5)
A sp ( 5) V ehicle
Num
ber o
f writ
hing
s
*, a, b *, a
.Fig. 6.8. Effect of aspirin (5 mg/kg, i.p) in combination with V-MA (20 mg/kg, p.o) on acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to V-MA per se and (b) denotes significance at P<0.05 as compared to Aspirin per se.
6.4. DISCUSSION
In this study, analgesic activity of valerian extract and oils were investigated
in both acetic acid-induced writhing and tail flick models in mice. The acetic acid-
induced writhing reaction in mice, described as a typical model for inflammatory pain,
has long been used as a screening tool for the assessment of analgesic or anti-
inflammatory properties of new agents (Collier et al., 1968; Khandelwal, 2007). This
method presents a good sensitivity; however, it shows poor specificity, leaving scope
for the misinterpretation of results. This can be avoided by complementing the test
with other models of nociception and by a performance motor test. Acetic acid causes
inflammatory pain by inducing capillary permeability (Amico-Roxas et al, 1984) and
liberating endogenous substances that excite pain nerve endings (Raj, 1996). The
abdominal constriction is related to sensitization of nociceptive receptors to
prostaglandins, bradykinin and substance-P (Huo et al, 2007, Singh and Majumdar,
1995). The nociceptive response caused by acetic acid is also dependent on the release
of TNF-α, interleukin-8 via modulation of macrophages and mast cells localized in
the peritoneal cavity (Ikeda et al., 2001). Because the writhing test usually lasts for
less than an hour, it is too short to cover central prostaglandin-dependent sensitization,
which requires spinal COX-2 induction (Hanns, 2007) however it primarily involves
COX-1 derived prostanoids and thus non-selective COX inhibitor offers a better
antinociceptive effect as compared to selective COX-2 inhibitors (Kulkarni and Jain,
2005).
The results suggest that in acetic acid writhing model VVDR03 produced
significant effect at 40 and 80 mg/kg while VVW02 was found to produce significant
effect at only 80 mg/kg. Both V-MA and V-PA produced significant effect in acetic
acid induced writhing at 40 and 80 mg/kg dose. The highest dose of V-MA tested (80
mg/kg) produced 63.6 % inhibition whereas that of V-PA produced 50 % inhibition.
Statistical comparison shows that both the oils are equally effective in producing
antinociceptive in acetic acid writhing. Aspirin also inhibited the number of writhings
significantly and the effect produced by 80 mg/kg V-MA is comparable to that of 100
mg/kg aspirin (64.9%).
Tail flick is an acute spinally mediated reflex to noxious thermal stimuli. The
fast rising pain in the tail flick gives rise to rapid tail withdrawal at the lowest possible
threshold for pain before it reaches to higher level. In the tail flick model, none of the
doses of V-PA and VVDR03 were found to have effect on tail flick latency when
compared to the predrug reaction time while 80 mg/kg dose of V-MA and VVW02
produced significant increase in tail flick latency after two hours. This indicates that
higher center is also involved in antinociceptive effect of the maaliol chemotype but
at higher doses.
The present study demonstrated that both valerian extracts and oils were found
to be more effective as peripheral analgesics. When studied for mechanism of action
in acetic acid writhing model aspirin potentiated the action of valerian oils while no
potentiation was seen with extracts. NSAIDs can inhibit COX in peripheral tissues
and, therefore, interfere with the mechanism of transduction of primary afferent
nociceptors (Fields, 1987). So the mechanism of analgesic effect of valerian oil could
probably be due to blockade of the effect or the release of endogenous substances that
excite pain nerve endings similar to that of indomethacin and other NSAIDs.
There are few reports on analgesic activity of Valeriana wallichii (Cao and
Hong, 1994; Schultz and Eckstein, 1962; Shrivastava and Sisodia, 1970). There is
another report which demonstrates analgesic activity of the volatile oil of Valeriana
amurensis in acetic acid writhing (Wu et al., 2007). There are many studies reporting
analgesic activity of essential oils and sesquiterpenes (Golshani et al, 2004; Santos et
al, 1997; Sayyah et al, 2002; Sayyah et al, 2003; Koudou et al, 2005; Ahmed et al,
1997). A recent report presents natural terpene 1,8-cineole for its analgesic effect
(Santos and Rao, 2000). In a study β-caryophyllene inhibited in vitro formation of
PGE1 (Burstein et al, 1975) and sesquiterpenoids have been found to have suppressive
effect on iNOS and COX-2 activity (Lee et al., 2002; Yoon et al., 2008). Anti-
inflammatory effect has been reported for limonene and terpineol (Duke and
Beckstrom, 1996). The limonene has been tested in the lipopolysaccharide (LPS)-
induced pleurisy model and when administered orally was able to inhibit the LPS-
induced inflammation including cell migration and inhibiting the production of nitric
oxide, gamma-interferon and IL-4 (Souza, 2003). The inhibitory effect of terpineol, a
volatile terpenoid alcohol on the compound action potential of rat sciatic nerve has
been reported (Moreira, 2001).
Few studies on Indian valerian have demonstrated that it contain flavonoid
compounds (Marder et al., 2003; Tang and Liu, 2003). Flavonoids are known to target
prostaglandins which are involved in the late phase of acute inflammation and pain
perception (Rajnarayana et al., 2001). Iridoids found in many medicinal plants exhibit
a wide range of bioactivities including cardiovascular, antiphepatotoxic, chlorectic,
et al., 1991). Superoxide anions undergo dismutation which causes the production of
hydrogen peroxide (Hochstein and Atallah, 1988; Riley and Behrman, 1991).
Superoxide anion is normally involved in inflammatory conditions. Formation of
superoxide anion radical leads to a cascade of other ROS (Grisham, 1992).
Superoxide dismutates to hydrogen peroxide (H2O2) and oxygen. It is less reactive
when compared to the other free radicals. But it leads to the production of hydroxyl
and peroxynitrite radicals by combination with hydrogen peroxide and nitric oxide
radical respectively. The pathological effects of superoxide anion are indirect in the
sense that it is the subsequently formed hydroxyl and peroxynitrite radicals which are
involved in different pathological conditions like cancer, cardiac, renal ischemia,
atherosclerosis, diabetes etc. Thus if, superoxide anion production is controlled or
scavenged, the formation of hydroxyl and peroxynitrite radicals can be limited, such
that, the diseases caused by them can sufficiently be reduced.
In our study none of the dose of VVW02 (Fig. 8.2.) altered brain SOD levels.
While VVDR03 at 40 mg/kg produced significant increase in SOD levels as
compared to control group (Fig. 8.4.). Similarly when valerian oils V-PA and V-MA
were studied for modulation of SOD levels in brain no effect was found when
compared with control group (Fig. 8.6. & 8.8.).
00 .1
0 .20 .3
0 .40 .50 .6
0 .70 .8
0 .91
V V W 0 2( 10 )
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
V ehicle
nmol
es/m
g pr
0
0 .0 0 2
0 .0 0 4
0 .0 0 6
0 .0 0 8
0 .0 1
0 .0 12
0 .0 14
0 .0 16
0 .0 18
V V W 0 2( 10 )
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
V ehicle
mic
rom
oles
/mg
pr
(a) (b)
Fig 8.1. Effect of different doses of VVW02 on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain
0
2
4
6
8
10
12
14
16
18
V V W 0 2( 10 )
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
V ehicle
mic
rom
oles
of
H2O
2 de
com
pose
d/m
in/m
g p
r
0
0 .5
1
1.5
2
2 .5
3
V V W 0 2( 10 )
V V W 0 2( 2 0 )
V V W 0 2( 4 0 )
V ehicle
un
its/
mg
pr
(a) (b)
Fig. 8.2. Effect of different doses of VVW02 on a) Catalase level in mice brain b) Superoxide dismutase level in mice brain
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
V V D R 0 3( 10 )
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
V ehicle
nmol
es/m
g pr * a
0
0 .0 0 5
0 .0 1
0 .0 15
0 .0 2
0 .0 2 5
0 .0 3
V V D R 0 3( 10 )
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
V ehicle
mic
rom
oles
/mg
pr * a b
(a) (b)
Fig. 8.3. Effect of different doses of VVDR03 on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain. Results are significant at P<0.05. (*) versus vehicle group, a versus VVDR03 (20 mg/kg), b versus VVDR03 (10 mg/kg)
02
46
810
1214
1618
2 0
V V D R 0 3( 10 )
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
V ehicle
mic
rom
oles
of H
2O2
deco
mpo
sed/
mim
/mg
pr
**
*
00 .5
11.5
22 .5
3
3 .54
4 .55
V V D R 0 3( 10 )
V V D R 0 3( 2 0 )
V V D R 0 3( 4 0 )
V ehicle
units
/mg
pr
* a b
(a) (b)
Fig. 8.4. Effect of different doses of VVDR03 on a) Catalase level in mice brain b) Superoxide dismutase level in mice brain. Results are significant at P<0.05. * versus vehicle group, a versus VVDR03 (20 mg/kg) b versus VVDR03 (10 mg/kg)
2 . 8
2 . 9
3
3 . 1
3 . 2
3 . 3
3 . 4
3 . 5
3 . 6
V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e
nmol
es/m
g pr
0 .0 12
0 .0 12 5
0 .0 13
0 .0 13 5
0 .0 14
0 .0 14 5
0 .0 15
V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e
mic
rom
oles
/mg
pr
(a) (b)
Fig. 8.5. Effect of different doses of V-PA on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain
2 .6
2 .7
2 .8
2 .9
3
3 .1
3 .2
3 .3
3 .4
3 .5
V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e
mic
rom
oles
of H
2O2
deco
mpo
sed/
min
/mg
pr
0
0 .5
1
1.5
2
2 .5
3
3 .5
4
4 .5
5
V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e
units
/mg
pr
(a) (b)
Fig. 8.6. Effect of different doses of V-PA on a) Catalase level in mice brain b) Superoxide dismutase level in mice
0
0 .5
1
1.5
2
2 .5
V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e
nm
ole
s/m
g p
r
0
0 .0 0 1
0 .0 0 2
0 .0 0 3
0 .0 0 4
0 .0 0 5
0 .0 0 6
0 .0 0 7
0 .0 0 8
V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e
mic
rom
oles
/mg
pr
(a) (b)
Fig. 8.7. Effect of different doses of V-MA on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain
0
0 . 5
1
1. 5
2
2 . 5
V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e
mic
rom
oles
of H
2O2
deco
mpo
sed/
min
/mg
pr
0
0 .5
1
1.5
2
2 .5
3
3 .5
4
4 .5
V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e
units
/mg
pr
(a) (b)
Fig. 8.8. Effect of different doses of V-MA on a) Catalase level in mice brain b) Superoxide dismutase level in mice
8.4. DISCUSSION
Antioxidants are radical scavengers which protect the human body against free
radicals that may cause pathological conditions such as ischemia, anaemia, asthma,