CHAPTER – II SELECTION OF NOVEL HERBAL DRUG FOR THE...
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65 CHAPTER – II SELECTION OF NOVEL HERBAL DRUG FOR THE TREATMENT OF ALZHEIMER’S DISEASE AND COMPARISON OF THEIR EFFICACY WITH COMMERCIALLY AVAILABLE DRUGS
Transcript of CHAPTER – II SELECTION OF NOVEL HERBAL DRUG FOR THE...
65
CHAPTER – II
SELECTION OF NOVEL HERBAL DRUG FOR THE
TREATMENT OF ALZHEIMER’S DISEASE AND
COMPARISON OF THEIR EFFICACY WITH
COMMERCIALLY AVAILABLE DRUGS
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SELECTION OF NOVEL HERBAL DRUG FOR THE TREATMENT
OF ALZHEIMER’S DISEASE AND COMPARISON OF THEIR EFFICACY
WITH COMMERCIALLY AVAILABLE DRUGS
INTRODUCTION
Alzheimer’s disease (AD) is characterized as a progressive
neurodegenerative disorder associated with loss of neurons in distinct brain areas
and spinal cord. AD is considered as prominent cause of dementia in the aged
people. Memory is the ability of an individual to record the information and recall
it whenever needed. Dementia is a mental disorder characterized by loss of
intellectual ability judgment or abstract thinking, which invariably involves
impairment of memory. Stressful conditions are often associated with loss of
memory and cognitive functions which may lead to threats of AD.
AD is one of the internationally growing problem. Around 24 million
people have dementia. By 2040 the projection will be 81 million. About 50% have
the age related cognitive disorder Alzheimer’s disease (AD) (Ferri et al., 2005). In
Australia, AD affects about 1% (200,000) of the population.. This is expected to
increase to 2.8% by 2050 (Access 2005). The direct health costs spent for AD was
estimated at $ 5.6 billion (Access, 2003). These costs are projected to increase as
the population ages. The treatment of AD is a clinical challenge. Lot of drugs such
as cholinesterase inhibitors, N-methyl-d-aspartate antagonist (memantine), anti-
oxidant drugs, anti-inflammatory drugs and anti cholesterol drugs have been used
to control the symptoms of AD. But, these conventional western synthetic
medications offer symptomatic management in about one third of cases but do not
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prevent or halt the progression of AD. Consequently there is a need for new
therapies that can prevent or delay the onset of AD, slow the progression of AD or
manage the symptoms associated with AD.
Over the last century, natural products have been the principal source of
materials for new drug development. In the period 1989-1995, 61% of newly-
approved drugs (excluding biologics) were either directly derived from or
modelled from natural products (Cragg et al., 1997). However, less than 10% of
the World’s biodiversity has been tested for biological activity (Harvey, 2000).
So there remains considerable scope for natural products to produce new drugs in
the future. In China, traditional medicine employs over 12,000 individual species,
singly or in combined formulation for the management of a wide range of disorders
(Yan et al., 1999).
Longevity has been actively pursued in China for at least 2000 years via the
use of medicines and food stuffs (Needham and Lu, 2000). Although AD is a
modern disease entity, and has no direct analogue in the classical literature,
disorders of memory and cognitive deficit are referred throughout the classical and
modern herbal medicine literature. It is likely that some of these instances would
receive the modern diagnosis of AD. Modern clinical research suggests that some
traditional medicines may be of value in the management of dementia. The plant
Gingko biloba has received the most research. A number of studies suggest that
Gingko biloba can enhance memory (Diamond et al 2000; Kanowski and Hoerr
2003). Other traditional medicines and formulae have also received research
attention (Bent et al 2003; Jirong et al 2006).
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Traditionally herbal drugs have been used to enhance cognitive functions
and to alleviate other functions associated with AD. A number of medicinal plants
and medicines derived from these plants have shown memory enhancing properties
by virtue of their medicinal constituents. One of the mechanisms suggested to
dementia is decreased cholinergic activity in brain. Therefore cholinergic drugs (of
plant origin) like: muscarinic agonists (e.g. Arecoline, Pilocarpine etc.), nicotinic
agonists (e.g. Nicotine) and cholinesterase inhibitors (e.g. Buperzine) can be
employed for improving memory. Some other classes of drugs used in dementia
are: stimulants or nootropics (e.g. Piracetam, Amphetamine), putative cerebral
vasodilators (e.g. ergot alkaloids, Papavarine), calcium channel blocker (e.g.
Nimodipine). Since allopathic system of medicine provides radical cure, more
concentration is provided on natural products to cure dementia, and some excellent
results with certain plants, justified their use as memory enhancer (Jagdeep et al.,
2009; Jackson et al., 2009)
In this background, the present study was intended to screen novel anti-
Alzheimer’s, memory enhancing compounds form natural products. Apart from
this, intensive studies were targeted towards the efficacy determination of these
natural drugs and were compared with commercial frontline drugs. The natural
analogues such as Sida cordifolia, Thespecia populnea and Santalum album
extracts were tested with four frontline drugs, which are available in the market.
Different behavioral assay systems such as Hebbs William Maze, Elevated Plus
Maze and Two compartment passive avoidance test serve as an indicator to
determine the efficacy.
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MATERIALS AND METHODS
The commercially available frontline Alzheimer’s drugs such as Donepezil,
Celecoxib, Simvastatin and Vitamin E were selected for the memory enhancing
property studies. The drugs were obtained from local stockiest, Chennai, India. The
detailed description of the commercial drugs is given below.
DONEPEZIL
CHEMICAL NAME : (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-one hydrochloride
MOLECULAR WEIGHT : 415.96
PHYSICAL NATURE : Crystalline powder.
SOLUBILITY :
Freely soluble in chloroform, soluble in water and in glacial acetic acid, slightly soluble in ethanol and in acetonitrile and practically insoluble in ethyl acetate and in n-hexane.
CHEMICAL FORMULA : C24H29NO3HCl
SOURCE : Donepezil hydrochloride
CELECOXIB
CHEMICAL NAME : 4-[5-(4-methylphenyl)- 3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide
MOLECULAR WEIGHT : 381.38
PHYSICAL NATURE : Crystalline powder.
SOLUBILITY : Freely soluble in weak acids
CHEMICAL FORMULA : C17H14F3N3O2S
SOURCE : Diaryl-substituted pyrazole
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SIMVASTATIN
CHEMICAL NAME :
2,2-dimethyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]-1-naphthalenyl ester
MOLECULAR WEIGHT : 418.57
PHYSICAL NATURE : White to off-white, nonhygroscopic, crystalline powder, inactive lactone
SOLUBILITY : Practically insoluble in water, and freely soluble in chloroform, methanol and ethanol.
CHEMICAL FORMULA : C25H38O5
SOURCE : Derived synthetically from a fermentation product of Aspergillus terreus
VITAMIN E
CHEMICAL NAME : alpha-tocopherol
PHYSICAL NATURE : White amphorus
SOLUBILITY : fat-soluble antioxidant
NATURAL PRODUCTS
To evaluate the memory enhancing property in albino mice, the plant
materials such as Sida cordifolia, Thespesia populnea and Santalum album were
collected from Velimalai, Kanyakumari District, Tamil Nadu. The plants were
identified and authenticated by Prof. Dr. Jayaraman (Director, Plant Anatomy
Research Centre, National Institute of Herbal Science, Chennai, Ref. No.
PARC/2008/194), the name, group, location of collection and taxonomical status
are given below.
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TAXOMONY OF EXPERIMENTAL PLANTS
SIDA CORDIFOLIA
KINGDOM - Plantae
CLASS - Magnoliopsida
SUB CLASS - Rosidae
ORDER - Malvales
FAMILY - Malvaceae
GENUS - Sida
SPECIES - cordifolia
THESPESIA POPULNEA
KINGDOM - Plantae
CLASS - Magnoliopsida
SUB CLASS - Rosidae
ORDER - Malvales
FAMILY - Malvaceae
GENUS - Thespesia
SPECIES - populnea
SANTALUM ALBUM
KINGDOM - Plantae
DIVISION - Magnoliophyta
CLASS - Magnoliopsida
FAMILY - Sandalaceae
GENUS - Santalum
SPECIES - album
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COLLECTION AND IDENTIFICATION OF PLANT MATERIAL
The bulk collection of source material was carried out during July 2006.
Immediately after collection they were washed in distilled water to remove
epiphytes, sand and other exogenous matter. After draining of the water, the plants
were weighed and air dried under shade. After completing the drying process, they
were cut in to small pieces and dried in an incubator at 37°C. Completely dried
material was weighed and ground coarsely in a mechanical grinder.
EXTRACTION OF SECONDARY METABOLITES FROM PLANTS
PRELIMINARY ISOLATIONS
About 10 gm of coarsely powdered plant was packed in Whatman paper no.
1 filter paper and loaded in the Soxhlet apparatus. Methanol was taken as a solvent
to prepare crude extracts. The solvent was refluxed at approximately 65 °C and
extracted three times with distilled methanol. The other solvents such as acetone,
ether, toluene, dicholoro methane were also tried. Thus prepared extracts were
used for the primary screening. The highly active specimens were further scaled up
after the bulk extraction process.
BULK EXTRACTION PROCESS (BEP)
In this process about 500 gm of coarsely powdered plant materials were
refluxed 3 times in a 5 litre capacity round bottom flask in a water bath for about 6
hours using 3 liters of methanol. The extracts were filtered and concentrated to
recover the excess solvents in another distillation chamber. The concentrated
extract (about 100 ml) was again filtered though a watt man no.1 filter paper filled
with a Buchner funnel using suction procedure. Finally it was reduced to a thick
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oily crude extract in a rotary vacuum evaporator (JSGW-BUCHI Type) at 40°C,
collected in air tight plastic vials and stored in a refrigerator for further studies.
STANDARDISATION OF ROUTES OF ADMINISTERATION
In order to evaluate the suitable route of administration of the extract a pilot
study was carried out (Jackson et al., 2009). Based on that the oral administration
was chosen for further studies.
EXPERIMENTAL ANIMALS
Albino mice of either sex were procured from kings Institute, Guindy,
Chennai were used for the study. Mice weighing around (20±2) gm and aged 4-6
weeks were kept in polypropylene cages and maintained in fumigated animal
house at a room temperature of (27±2°C). All the animals have free access to food
and water and were housed in natural (12 hours light –dark cycle Food given to
mice consisted of wheat flour kneaded with water and mixed with a small amount
of refined vegetable oil. The animals were acclimatized for at least 5 days to the
laboratory conditions before behavioral experiments. Experiments were carried out
between 0900 hours and 1800 hours. The experimental protocol was approved by
the institutional animal ethics committee (IAEA) and the care of
laboratory animals was taken as per the guidelines of committee for the
purpose of control and supervision of experiments on animals (CPCSEA).
Ministry of Forest and Environment, Government of India. Registration No –
CPCSEA/ORG/CH/2008/Reg.No. 1143/365, IAEA No – 1143ac/07/CPCSEA
dated 28.02.2008.
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ACUTE TOXICITY STUDIES:
Acute toxicity studies were performed according to organization for
economic cooperation and development (OECD). Guidelines Ecobichon (1997).
Male albino mice selected by random sampling technique were employed in this
study. The animals were fasted for 4 hours with free access to water only. The
different doses of the extracts were administered orally and mortality if any was
observed for 3 days. If mortality was observed in two out of three animals, then the
dose administrated was considered as toxic dose. If the mortality was observed in
only one animal out of three animals then the same dose was repeated again to
confirm the toxic effect. If no mortality was observed then only higher doses of the
extracts were employed for further toxicity studies.
DRUG TREATMENT
The mice were divided in to different groups. Each group comprised of a
minimum of six animals. Extracts were administered orally for 15 successive days
to young mice. After 90 minutes of the administration of the last dose, mice were
exposed to the training session using two compartments passive avoidance
apparatus, Hebbs William maze apparatus and elevated plus maze apparatus.
Retention memory was recorded after 24 hours on the 16th day. Amnesia was
induced in separate groups of young mice using scopolamine 0.4 mg/kg after 90
minutes of the last dose of the drug administration. The animals were exposed to
the training session after 45 minutes of scopolamine injection. The memory
retention was measured after 24 hours. Donepezil was used as a standard drug and
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was injected for 7 days to positive control groups. Drug extracts were administered
orally for 15 days to separate groups for bio chemical studies.
VEHICLE
Extracts were suspended in 0.5 % w/v carboxymethylcellulose (CMC) and
given orally in a volume of 1 ml/100 gm of mouse. All control group animals
received vehicle 0.5% w/v CMC for 7 consecutive drugs.
ELEVATED PLUS MAZE
Elevated plus-maze served as the exteroceptive behavioral model to
evaluate memory in mice. The procedure, technique and end point for testing
memory was followed as per the parameters described by the investigators
working in the area of psychopharmacology (Parle et al., 2004; Parle and Dhingra
2003; Parle and Singh 2004; Parle et al., 2005). The elevated plus maze apparatus
for mice consisted of a central platform (10 cm × 10 cm) connected to two open
arms (50 cm × 10 cm) and two covered (enclosed) arms (50 cm × 40 cm × 10 cm)
and the maze was elevated to a height of 50 cm from the floor. On the first day (i.e.
15th day of drug treatment), each mouse was placed at the end of an open arm,
facing away from the central platform. Transfer latency (TL) was recorded on the
first day (training session) for each animal. TL was defined as the time (in seconds)
taken by the animal to move from the open arm into any one of the covered arms
with all its four legs. The mouse was allowed to explore the maze for another 2
min and then returned to its home cage. Retention of this learned-task (memory)
was examined 24h after the first day trial (i.e. 16th day, 24h after last dose).
Significant reduction in TL value of retention indicated improvement in memory.
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HEBB-WILLIAM MAZE
Hebb-Williams maze is an incentive based exteroceptive behavioural
model useful for measuring spatial working memory of Mice (Parle and Singh
2004). It consists of mainly three components. Animal chamber (or start box),
which is attached to the middle chamber (or exploratory area) and a reward
chamber at the other end of the maze in which the reward (food) is kept. All the
three components are provided with guillotine removable doors. On the first day
(i.e. 15th day of drug treatment), the mouse was placed in the animal chamber or
start box and the door was opened to facilitate the entry of the animal into the next
chamber. The door of start box was closed immediately after the animal moved
into the next chamber so as to prevent back-entry. Time taken by the animal to
reach the reward chamber (TRC) from the start box was recorded on first day
(training session) for each animal. Each animal was allowed to explore the maze
for 3 minutes with all the doors opened before returning to its home cage.
Retention of this learned task (memory) was examined 24 h after the first day trial
(i.e. 16th day, 24h after last dose) (Parle et al., 2005).
TWO COMPARTMENT PASSIVE AVOIDANCE APPARATUS
The method of Buresova and Bure (1983) was used to assess the retention
of learned behavior. The apparatus consists of a square box with a floor grid of 50
x 50 cms and wooden walls of 35 cms height. This box was illuminated with 100
watts bulb. In the centre of one of the walls there is an opening of 6 x 6 cms which
can be opened or closed using a transparent plexy glass sliding door. This opening
leads to a small (15 x 15 cms) dark compartment provided with an electrified floor,
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which can be connected to a shock source, stimulator obtained from Hugo Sachs
Electronics, Germany having a maximum output of 100 mA. The animals were
placed in the illuminated chamber facing away from the entrance to the dark
compartment. The door was closed after the mouse entered the dark compartment
and 1 mA foot shock was delivered for a period of 2 sec. Then the animal was
returned to the home cage. 24 h later each animal was placed again in the
illuminated chamber as before for a maximum period of 180 sec. The latency taken
by the animal to enter the dark compartment was measured. Animals not entering
the dark compartment within this period received a latency time of 180 sec.
Absence of entry into the dark compartment indicated positive retention.
STATISTICAL ANALYSIS
To find out the variation in the experimental and the control group
ANOVA test was performed using COSTAT (release 2) software. The variation
results are given in the appropriate place.
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RESULTS
COLLECTION AND IDENTIFICATION OF ALZHEIMER’S DISEASE
PLANTS
The successful development of anti Alzheimer’s drugs from natural
products relies on continuous availability of the resource materials and collection
methods. In the present study, 3 different plants such as Sida cordifolia, Thespesia
populnea, and Santalum album were selected and collected from Velimalai, K.K
Dist, Tamil Nadu.. Velimalai was found to be an excellent area for the collection of
these medicinal plants. The status of these plants in this area is more or less same,
irrespective of the season. The name, group, local name, place of collection and net
weight of collection are presented in table 3.
Table 3. The name, place of collection and net weight of collection of the
medicinal plants
Species Local name Group Place of
collection
Net weight for
drug collection
(kg)
Sida cordifolia Aanan shrub Velimalai, K.K Dist,
Tamil Nadu. 1.000kg
Thespesia populnea Selanthi Tree -do- 1.250 kg
Santalum album Santhanam Tree -do- 1.500 kg
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PREPARATION OF CRUDE PLANT EXTRACT
PRIMARY ISOLATION
Three species of plants were used for primary isolation process. The
characteristic colours of the methonolic extract were presented in table 4. The
extracts of S. cordifolia was green in colour. The bark of T. populnea extract
produced yellow red colour. While the stem of Santalum album produced
characteristic yellow brown colour extract. Some coloration was noted in the bulk
extraction process. The quantity of yield of the extracts are presented in table 4.
Table 4. Characteristic features of the methanol extracts of medicinal plants
Species Dry colour Extract
colour
Quantity of extract
yield (g/kg)
Sida cordifolia Dry green Green 19
Thespesia populnea Reddish Brown Yellow red 28
Santalum album Yellowish brown Dark Brown 40
STANDARDIZATION OF ROUTE OF ADMINISTRATION
The results of route of administration specified that the parenteral routes as
Intra muscular and Intra venous (IM and IV) were not effective (Table 5). In the
case of intra muscular injection received groups, there was showed high
restlessness, whereas the orally administrated group gave the impression of relaxed
mood. So oral administration was preferred for further experiments.
Table. 5. The route of administration of plant extracts and its response in
experimental mouse
Route of Administration Response
Parenteral routes (IM and IV) Difficulty in administration Restlessness in the animal (due to pain)
Oral route relaxed mood
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ACUTE TOXICITY STUDIES
To determine the toxicity of the plant extracts in Albino mice, toxicity
studies were carried out and the results were shown in the table 6. Acute toxicity
bio assay were performed in albino mice against three medicinal plants. All the
doses (1mg, 2mg, 4mg, 8mg/kg body weight of animal) of Sida cordifolia,
Thespesia populnea, and Santalum album employed for oral toxicity studies were
found to be non toxic but mortality were noted in higher doses. Santalum album
produced 50% mortality in the doses of 120mg/kg body wt. Based on the
interpretation in the Fig. (12). The LD50 rate is approximately 40mg / kg body
weight. The sub lethal dose such as 1/10th, 1/20th and 1/40th of LD50 doses were
taken for further experiments.
Table 6. Mortality rate of different plant extract administered mice
Extract Dose No. of Animal Dead % Mortality
Sida cordifolia
10 mg/Kg 1/8 12.5%
20 mg/Kg 2/8 25.0%
40 mg/Kg 4/8 50.0%
80 mg/Kg 6/8 75.0%
Thespesia populnea
10 mg/Kg 2/8 25.0%
20 mg/Kg 3/8 37.5%
40 mg/Kg 4/8 50.0%
80 mg/Kg 7/8 87.5%
Santalum album
10 mg/Kg 1/8 12.5%
20 mg/Kg 2/8 25.0%
40 mg/Kg 4/8 50.0%
80 mg/Kg 6/8 75.0%
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Fig. 12. Mortality percentage of mice adminstered with different
methanolic plant extracts
0
10
20
30
40
50
60
70
80
90
100
Dose (mg/kg body wt)
Mo
rtal
ity
%
S. codifolia T. populina S.album
TWO COMPARTMENT PASSIVE AVOIDANCE TEST
The results of two compartment passive avoidance test after the
administration of different drugs are shown in fig. 13 and table 7. It was noted that
the compounds positively influence the mice through increased step down latency
(SDL). The SDL was defined as the time (sec) taken by the mouse to step down
from two wooden platform of grid floor with all its paws on the grid floor. Based
on the results, all the drugs used in this study increased in SDL values in memory
over the control groups. SDL of Donepezil (4mg/Kg body wt.) administered
groups reflected long term memory of animals. The consistent memory increment
were noted in the group received S. album extract orally. The statistical analysis
made on the SDL level between the control and Donepezil and S. album
administrated group revealed that the variation was significant. The SDL values of
four commercial frontline drugs and three medicinal plants in mice are given in
table 7.
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Fig. 13. The SDL value of different doses of drug administered mice of Two
compartment passive avoidance test.
0
50
100
150
200
250
300
350
400S
tep
dow
n l
ate
ncy
1 2 4 8
Doses (mg/kg)
S. cordifolia T. populnea S. album Donepezil Celecoxib
Simvastatin Vitamin E
Table 7. The SDL value of different doses of drug administered mice of Two
compartment passive avoidance test
Drug Dose Activity (in sec)
Sida cordifolia
1mg/kg 119±5
2mg/kg 134±6
4mg/kg 149±7
8mg/kg 119±5
Thespesia populnea
1mg/kg 152±8
2mg/kg 168±7
4mg/kg 183±6
8mg/kg 153±7
Santalum album
1mg/kg 175±4
2mg/kg 285±5
4mg/kg 305±4
8mg/kg 175±5
Donepezil
1mg/kg 340±6
2mg/kg 359±5
4mg/kg 373±8
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8mg/kg 340±7
Celecoxib
1mg/kg 182±9
2mg/kg 197±7
4mg/kg 212±8
8mg/kg 182±8
Simvastin
1mg/kg 247±7
2mg/kg 259±8
4mg/kg 274±8
8mg/kg 247±5
Vitamin E
1mg/kg 211±7
2mg/kg 228±11
4mg/kg 242±9
8mg/kg 213±10
Vehicle Control 117±7
Two compartments Passive avoidance apparatus
Source of variation SS df Ms F
Main effect
Drug 751825 7 107403 1864***
Dose 10344 3 3448 60***
Interaction 1669 21 79 1.37NS
Error 3686 64 57
Total 767526 95
*** Highly significant, P<0.001, NS- Not Significant
Drug
4 3 6 7 5 2 1 8 LSD
65.1 92.4 127.3 165.5 193.1 224.1 253 357.3 6.18
Dose
4 3 2 1 LSD
175.2 175.5 ---------------------------------
188 200.4 4.37
Underlined means are not significant
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Based on the results Donepezil at a rate of 4mg/kg body wt. and S. album at
a rate of 4mg/kg body wt. enhanced memory functions than the other doses and
groups. Among these the donepezil (4mg/kg body wt.) administered groups
reversed the scopolamine induced memory loss within a short duration (305 sec).
The memory enhancement was three fold than the control. The S. album (4mg/kg
body wt) administered mice also showed good memory value followed by
Simvastatin 4 mg/kg (274 sec).
It was noted that the other medicinal plant extracts such as T. populnea and
S. cordifolia were showed good response than the control, but it was not
statistically significant. The lower doses of these extracts also showed more or less
same memory recovery with the control group. The other commercial frontline
drugs showed moderate memory recovery and are statistically significant.
HEBBS WILLIAM MAZE
Extraceptive behavioral changes of mice in terms of memory were studied
using Hebbs William maze. Time taken to reach reward chamber (TRC) by
dementia induced mice after the oral administration of different doses of four
frontline drugs (memory enhancing) and three medicinal plants are shown in
fig. 14 and table 8. Significant reduction in TRC value indicates improvement in
memory. The extracts of T. populnea and S. cordifolia did not show any significant
effect on TRC when compared with control groups. It clearly indicated that these
plants did not have much effect over induced dementia. Interestingly, the different
doses of S. album administered mice markedly reduced TRC when compared with
the control groups. It exhibited 161 sec to recover memory at 4mg/kg body weight,
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but the control group exhibited 411 sec. All the frontline drugs administered
groups reversed the amnesia induced by scopolamine even though the Donepezil
(D3) administered groups showed statistically significant TRC reduction than other
group. The variation of TRC value of this group (D3) was very high than the
control group. However the overall results show that the effective drug donepezil
(8mg/kg body wt) showed very least retention time within 119 sec. The results of
ANOVA displayed highly significant variation among the control and the effective
doses.
Table 8. The TRC value of different doses of drug administered mice Hebbs
William Maze test.
Drug Dose Activity
Sida cordifolia
1mg/kg 353±8
2mg/kg 340±7
4mg/kg 324±7
8mg/kg 323±7
Thespesia populnea
1mg/kg 320±9
2mg/kg 304±9
4mg/kg 292S±9
8mg/kg 290±10
Santalum album
1mg/kg 190±12
2mg/kg 174±10
4mg/kg 161±9
8mg/kg 160±8
Donepezil
1mg/kg 150±10
2mg/kg 134±9
4mg/kg 120±8
8mg/kg 119±10
Celecoxib 1mg/kg 287±10
2mg/kg 271±8
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4mg/kg 253±11
8mg/kg 257±8
Simvastatin
1mg/kg 221±9
2mg/kg 205±10
4mg/kg 191±9
8mg/kg 190±9
Vitamin E
1mg/kg 254±8
2mg/kg 239±7
4mg/kg 223±9
8mg/kg 222±8
Vehicle 411±9
Hebbs William maze
Source of variation SS df Ms F
Main effect
Drug 694849.48 7 99264.21 950.05***
Dose 11853.03 3 3951.01 37.816***
Interaction 1234.71 21 58.79 0.56NS
Error 6686.66 64 104.47
Total 714623.9 95
*** Highly significant, P<0.001, NS- Not Significant
Drug
4 3 6 7 5 2 1 8 LSD
130.1 171.5 201.1 234.3 268.5 301.5 335 408.5 8.33
Dose
4 3 2 1 LSD
245.5 247.5 ---------------------------------
259.7 273.3 5.89
Underlined means are not significant
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Fig 14. The TRC value of different doses of drug administered mice Hebbs
William Maze test.
0
50
100
150
200
250
300
350
400T
ran
sfer
late
ncy
1 2 4 8
Doses (mg/kg)
S. cordifolia T. populnea S. album Donepezil Celecoxib
Simvastatin Vitamin E
ELEVATED PLUS MAZE
Transfer latency (TL) was defined as the time (sec) taken by the animal to
move from open arm in to one of the coveted arm with all its four legs. Significant
reductions in TL value of retention indicated improvement in memory. The
different doses of Donepezil (1, 2, 4, 8 mg/kg) showed dose dependent reduction
in TL in albino mice, when compared to the respective control group. The same
trend was noted in Santalum album administered groups indicating significant
improvement in memory. The dose 4 mg/kg Santalum album successfully
recovered memory deficits induced by scopolamine. The memory enhancement
noted in all doses of Vitamin E, Celecoxib and Simvastatin were not statistically
significant (Table 9 and Fig. 15).
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Table 9. The TL value of different doses of drug administered mice Elevated
Plus Maze test.
Drug Dose Activity
Sida cordifolia
1mg/kg 273±7
2mg/kg 256±5
4mg/kg 243±6
8mg/kg 242±5
Thespesia populnea
1mg/kg 242±7
2mg/kg 227±7
4mg/kg 214±6
8mg/kg 212±6
Santalum album
1mg/kg 112±8
2mg/kg 92±7
4mg/kg 82±7
8mg/kg 83±5
Donepezil
1mg/kg 82±9
2mg/kg 72±13
4mg/kg 53±7
8mg/kg 53±6
Celecoxib
1mg/kg 212±9
2mg/kg 197±9
4mg/kg 181±10
8mg/kg 181±9
Simvastatin
1mg/kg 145±6
2mg/kg 131±6
4mg/kg 116±4
8mg/kg 116±3
Vitamin E
1mg/kg 180±6
2mg/kg 170±7
4mg/kg 156±10
8mg/kg 155±9
Vehicle 357±6
89
Fig. 15. The TL value of different doses of drug in Elevated Plus Maze test.
0
50
100
150
200
250
300
Tran
sfer
la
ten
cy
1 2 4 8
Doses (mg/kg)
S. cordifolia T. populnea S. album Donepezil Celecoxib
Simvastatin Vitamin E
90
DISCUSSION
SELECTION AND COLLECTION OF PLANT MATERIAL
Over 30,000 plant species currently are used for medicinal purposes in the
world. (Manach et al 2005). These plant drugs provide numerous health benefits,
including antipsychotic, antifatigue, antidepressant, noxilytic, hypnotic, anti-
inflammatory, antioxidant, analgesic, anti neoplastic, anti arrhythmic, anti diabetic
and anti lipogenic effects. (Duncan et al., 2003; Nijveldt et al., 2001; Williams et
al., 2004; Williamson and Monach 2005).
The drug shows antidepressant, anti inflammatory, anti oxidant and anti
psychotic benefits may be particularly beneficial to Alzheimer’s disease patient in
the late stages of disease progression. Over the last 3 decades a diverse group of
natural products were chemically synthesized and tested for potent biological
activity. However, the number of compounds, which were taken, used for the
field/clinical trials is very less. In the light of earlier reports it could be described
that the failure of successful collection of concerned source materials in bulk was
the main reason.
In the present study, three medicinal plants were collected for the primary
bio-activity screening especially for anti Alzheimer’s disease properties, All these
plants were collected from velimalai, Kanyakumari Dist of Tamilnadu, India. In
order to ascertain the effective novel drug for the treatment of alzheimer’s disease,
different plant methanolic extract were used. In most of the experiment, Santalum
album produced significant results than the other plants such as Thespecia
populnea and Sida cordifolia.
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In this study the stem of S. album, bark of T. populnea and leaves of S.
cordifolia were used to screen the effective anti-alzheimer’s properties. The three
medicinal plants used in this study have been proven in Asian and modern herbal
pharmacoepoia for the treatment of various other diseases. Apart from this, these
plant extracts are used as herbal medicines by the local vaidyas to treat memory
loss. This information motivated this research to consider these plants for the
development of anti alzheimer’s drugs. In another study, the Ginkgo biloba leaves
have been used to treat memory loss. The leaves of Ginkgo biloba have been
recorded in Asian and modern herbal medicines as treatment for dysfunction of
heart and lungs (Defeudis and Drieu, 2000).
During the past decade, in vivo and in vitro experiments in mammalian
system and clinical studies in humans demonstrated that Ginko biloba exhibits a
range of biochemical and pharmacological effects. They include, free radical
scavenging activity (Lien et al., 1999), inhibitions of membrane lipid peroxides.
(Defeudis and Drieu 2000), Cognition enhancement particularly in rats and
alleviating symptoms in the experimental animals (Blavet, 1992; Winter 1991,
1998), anti- platelet activating factor activity contributing to improvements in
cerebral insufficiency (Smith et al., 1996), enhancing neuronal spasticity (Gobil
and Packe,r 2002) anti inflammatory effects (Oberpichler et al., 1990) and anti
apoptotic activities in neuronal cells (Blastianetto et al., 2000, Luo et al., 2002,
Smith et al., 2002). Ginko biloba extract is considered as medicine of the future
among traditional medicines (Berline, 2002). These plants seem to act as medicine
92
at all levels of life from molecules, cells tissues, to the entire organisms (Christen
and Maixent 2002).
Nevertheless, in other studies T. populnea was used for anti inflammatory
(Benhaim et al., 1994), anti fertility effects in rat (Ghosh and Bhattacharya) and
memory enhancement activity (Vasudevan and Parle, 2007a). Parle and Vasudevan
(2007 B) also investigated the effect of albana, an Ayurvedic herbal mineral
preparation for memory and brain choline esterase activity. In general the herbal
drugs used to treat Alzeheimer’s disease, were not proven statistically. The
scientific results related to anti Alzheimer’s drugs of natural origin and other
formulations were scanty. In the present study new efforts are being taken to
explore the scientific properties behind these three drugs. Apart from this, the
efficacy of these plants were compared with the commercial frontline drugs like
Donepezil, Vitamin E, Celecoxib and Simvastatin.
EXTRACTION
In the primary screening, the methonolic solvent system was used to extract
the active principle from the medicinal plants. However, methanol is used for the
extraction of polar active principles. According to earlier reports the methanol
extracts of these plants showed effective response than the other solvents (Jackson
et al., 2009). The other reports also indicated that the methonolic extracts showed
better efficacy than the other (Ratnasooriya et al., 1990 , Pettit et al., 1990,
Ramesh et al., 1999). This development of appropriate solvent systems could be
recorded as important step for drug development. Jackson et al. (2009) reported
that among the other extracts of S. album, the methanolic extract showed
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maximum memory enhancement activity. So, in the present study methanol was
preferred as a solvent for further bulk extractive process. Nag and De (2008) also
used different parts of Shankhpushpi methanolic extract to check the memory
function in albino mice. Although, (Taranalli and Cheeramkuzhi, 2000) found that
the ethanolic extracts of root parts of shankhpushpi significantly produced memory
retention.
The methanolic extracts of S. cordifolia, T. Populena and S. album efficacy
were checked with the commercial frontline anti Alzheimer’s drugs such as
Donepezil, Celecoxib, Vitamin E and Simvastatin. Donepezil, a cholinesterase
inhibitor (AchEIs) and mainstay for treating Alzhemeir’s disease (Cummings,
2004) Donepezil is one among the four approved drugs of United States Food &
Drug Administrations (USFDA) for the treatment of Alzheimer’s diseases. Despite
the therapeutical rationale for neuro protective mechanisms, the use of anti
inflammatory drugs showed better function and cognitive status over Alzheimer’s
disease patients. The drug celecoxib was well tolerated and the patients did not
report more adverse effects in this study. The growing evidence for the existence
of oxidative stress and the accumulation of free radical in the brain of
Alzheiemer’s patients (Grundman 2000, Christen 2000) has let to the notion of anti
oxidant as a potential treatment. The main antioxidant strategy has been the
treatment with α tocopherol (Vitamin E).
ACUTE TOXIC BIO ASSAY
The toxic potentials and lethal dose of medicinal plant extracts were
evaluated in albino mice. In accordance with the present study, crude extracts of T.
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Populnea showed significant feeding deterrent. The feeding activity was reported
in clathria species. (Wright et al 1999, Kanaaya 1990, Selvin 2002) the feeding
deterrent activity was also determined by dendrilla (Baker et al., 1995) but the
other two extracts not showed any feeding deterrent activity. In testing these
extracts did not produce any mortality even in higher dose during experimental
period. It clearly indicated that these three plants are not toxic to the mice. Based
on the earlier reports the stress free administration was recommended in the animal
experiment. The oral route of administration was preferred in this study to avoid
anonpuious stress in the cultured mice.
DOSE OF ADMINSTERATION
The effective, eco friendly, cheap and easily available drugs were selected
and screened and were given more priority for the development of modern anti-
Alzheimer’s drug. For the effective treatment of diseases, the dose of
administration and route of administration is very important. The challenge is very
high in the administration of natural medicine. There are differences in the
property of herbal drugs including significant variation across batches of the drugs
since the bioactivity varies considerably due to their differences in growth
environment. Although herbal drugs promise significant health benefits, they have
been found to be effective when administered to treat complex diseases like
Alzheimer’s diseases. In the present study 28 different doses were screened to
determine their efficacy. The measuring indexes such as two compartment Passive
avoidance apparatus, Hebbs William Maze and Elevated plus maze were used.
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Behavioral symptoms are very common in the course of Alzheimer’s
diseases. These behavioral changes include verbal and physical aggression,
agitation, hallucinations, delusions, sleep disturbance, depression, distractibility,
apathy, aberrant motor behavior and wandering. These are similar behavioral
symptoms which decrease the quality of life for patients and care givers and
increase the likelihood of institutionalization. When possible, the first step in
treatment includes evaluation and treatment of factors (such as pain or fever) that
may have triggered or contributed to the symptoms. Non pharmacological
interventions such as music, light, exercise or relaxation should also be considered.
However in the present study, the herbal drugs were compared for their efficacy
with other frontline drugs in different doses. The memory regain property after the
induction of dementia by scopolamine was studied to choose the better memory
enhancing drugs. The memory evaluation assay systems like Two Compartment
passive avoidance appratus, Hebbs William Maze Apparatus Elevated Plus Maze
apparatus were performed after the administration of different dose of drugs. The
same kind of experiments were carried out by (Vasudevan and Parle 2007) in T.
populnea. He compared the young and aged male wister rats treated orally with
200 and 400 mg/kg and found remarkable activity on 8th day (elevated plus maze)
the same trends were also noted in Hebbs William maze experiments. It was
clearly indicating that T. populnea showed significant improvement in memory.
But in the present study, Donepezil shows better memory enhancement than the
others followed by S. album in very low doses (4 mg/kg body weight).
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In the present experiment no behavioral and physiological changes
occurred except T. populnea. For this preliminary screening short term experiment
were carried out. Depressive symptoms and major depressive episodes commonly
occur during the course of dementia and can affect up to 50 % of patients with
Alzheimer’s disease. Apathy is even more frequent and it is often a diagnostic
challenge to distinguish Apathy from true depression. Many studies have tested the
efficacy of anti depressants in treating the depression associated with Alzheimer’s
disease. Accessing these studies in the aggregate yields conflicting results. Some
trials have shown same benefit with sertraline, Paroxetine, Fluoxetine, citalopram,
Imipramine, amytriptyline, clomypramine and mocdobemide in Alzheimer’s
disease patients. with depression. Others have found no difference between active
drug and placebo. Possible explanation for the diversions regarding treatment
benefits includes small sample size in some clinical trial, limited duration of
treatment different criteria and methods for measuring efficacy etc.
In practice depressive symptoms in Alzheimer’s diseases are usually treated
with selective serotonin reuptake inhibitors (SSRI). It is necessary to begin with
low doses and increasing the dose, based on clinical response and side effect.
Tricyclie antidepressants have similar efficacy but are used less often because they
have prominent anti cholinergic side effects, which have the potential to worsen
memory and counteract the AchEI given as a treatment. Nortryptiline has the
mildest anti cholinergic effects and was the rational choice before the development
of the SSRIS.
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Anti psychotic drugs are widely used to treat neuropsychiatric behavioral
symptoms in Alzheimer’s disease including delusions and hallucinations when
these symptoms become disturbing to the patient or disruptive to the environment.
Few randomized trials have evaluated their efficacy.
Risperidone, Haloperidol and Olazepine have been showed to be effective
when compared to placebo in Alzheimer’s diseases. Small open label non
randomized trials have shown that Quetiapine may provide some beneficial effect
in Alzheimer’s disease patients. There are no published data on other newer drugs
such as Sertindole, Aripiprazole or Ziprasidone. Two trials directly compared
rispenridone versus haloperidol. Although there were no differences in global
neuropsychiatric scores, rispenridone was significantly better in suppressing
aggressiveness. The groups administered with Donepezil 4 mg/kg body weight
showed very quick memory regain than the other group. It might be because of the
dose specific memory enhancement nature. This research is followed by S. album 4
mg/kg body weight which produced moderate activity next to control. The same
trend was noted in all different experiments groups of Two Compartment Passive
avoidance apparatus, Hebbs Williams Maze and Elevated plus maze apparatus.
