Management of Cognitive Determinants in Senile Dementia of Alzheimer’s Type: Therapeutic Potential...
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Clinical Drug Investigation ISSN 1173-2563 Clin Drug InvestigDOI 10.1007/s40261-014-0235-9
Management of Cognitive Determinantsin Senile Dementia of Alzheimer’s Type:Therapeutic Potential of a Novel PolyherbalDrug Product
Ananya Sadhu, Prabhat Upadhyay,Aruna Agrawal, Kaliappan Ilango,Dipankar Karmakar, Gur Prit InderSingh & Govind Prasad Dubey
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ORIGINAL RESEARCH ARTICLE
Management of Cognitive Determinants in Senile Dementiaof Alzheimer’s Type: Therapeutic Potential of a Novel PolyherbalDrug Product
Ananya Sadhu • Prabhat Upadhyay • Aruna Agrawal •
Kaliappan Ilango • Dipankar Karmakar • Gur Prit Inder Singh •
Govind Prasad Dubey
� Springer International Publishing Switzerland 2014
Abstract
Background and Objective The enigmatic etiology of
neurodegenerative diseases poses a challenge for the
development of novel and efficient drugs. The objective of
the present study was to evaluate the efficacy of a polyherbal
(test) formulation on cognitive functions, inflammatory
markers and oxidative stress in healthy elderly as well as
senile dementia of Alzheimer’s type (SDAT) patients.
Method A randomized double-blind placebo- and active-
controlled clinical trial was performed in healthy elderly
subjects and SDAT patients with an age range of
60–75 years. The polyherbal test formulation along with a
placebo was given to healthy elderly subjects while the
SDAT patients received either the test formulation con-
taining extracts of Bacopa monnieri (whole plant), Hip-
pophae rhamnoides (leaves and fruits) and Dioscorea
bulbifera (bulbils) at a dose of 500 mg or donepezil drug
(Aricept) at a dose of 10 mg, twice daily, for a period of
12 months. After every three months, cognitive functions
were assessed by determining the mini mental state
examination (MMSE) score, digital symbol substitution
(DSS; subtest of the Wechsler Adult Intelligence Scale—
Revised), immediate and delayed word recall (digital
memory apparatus—Medicaid systems, Chandigarh,
India), attention span (Attention Span Apparatus—Medic-
aid systems, Chandigarh, India), functional activity ques-
tionnaire (FAQ) and depression (geriatric depression scale)
scores. Further inflammatory markers and level of oxida-
tive stress were analyzed using standard biochemical tests.
Results The trial was performed in 109 healthy subjects
and 123 SDAT patients of whom 97 healthy subjects and
104 SDAT patients completed the study. Administration of
the test formulation for a period of 12 months was effective
in improving cognitive functions in the SDAT patients,
when compared to the donepezil-treated group, as deter-
mined by the DSS (38.984 ± 3.016 vs 35.852 ± 4.906,
P = 0.0001), word recall immediate (3.594 ± 1.003 vs
2.794 ± 0.593, P \ 0.0001) and attention span (4.918 ±
1.239 vs 4.396 ± 0.913, P = 0.0208) scores. A significant
improvement in the FAQ (11.873 ± 2.751 vs
9.801 ± 1.458, P \ 0.0001) and depression (16.387 ±
2.116 vs 21.006 ± 2.778, P \ 0.0001) scores was also
observed, whereas no significant differences were observed
in the MMSE and word recall delayed scores. The level of
inflammation and oxidative stress was markedly reduced in
the SDAT patients treated with the test formulation when
compared to the donepezil-treated group indicating a likely
mechanism of action of the test formulation (homocysteine
30.22 ± 3.87 vs 44.73 ± 7.11 nmol/L, P \ 0.0001;
C-reactive protein [CRP] 4.751 ± 1.149 vs 5.887 ±
1.049 mg/L, P \ 0.0001; tumour necrosis factor alpha
[TNF–a] 1139.45 ± 198.87 vs 1598.77 ± 298.52 pg/ml,
P \ 0.0001; superoxide dismutase [SOD]
A. Sadhu (&) � P. Upadhyay � G. P. Dubey
Collabrative programme, Institute of Medical Science, Banaras
Hindu University, Varanasi, India
e-mail: [email protected]
A. Agrawal
Department of Kriya Sharir, Faculty of Ayurveda, IMS, BHU,
Varanasi, India
K. Ilango
Interdisciplinary School of Indian System of Medicine (ISISM),
SRM University, Kattankulathur 603203, Tamil Nadu, India
D. Karmakar
Arvind Remedies Ltd., 190, Poonamallee high road,
Aminjikarai, Chennai 600084, Tamil Nadu, India
G. P. I. Singh
Adesh University, Barnala Road, Bathinda, Punjab, India
Clin Drug Investig
DOI 10.1007/s40261-014-0235-9
Author's personal copy
1145.92 ± 228.75 vs 1296 ± 225.72 U/g Hb, P = 0.0013;
glutathione peroxidase [GPx] 20.78 ± 3.14 vs 25.99 ±
4.11 U/g Hb, P \ 0.0001; glutathione [GSH] 9.358 ±
2.139 vs 6.831 ± 1.139 U/g Hb, P \ 0.0001; thiobarbitu-
ric acid reactive substances [TBARS] 131.62 ± 29.68 vs
176.40 ± 68.11 nmol/g Hb, P \ 0.0001). Similarly, when
healthy elderly subjects treated with the test formulation
for 12 months were compared to the placebo group, a
significant (P \ 0.001) improvement in cognitive measures
(MMSE, DSS, word recall delayed but not immediate,
attention span, FAQ and depression scores) and a reduction
in inflammation (reduction in homocysteine, CRP, IL-6
and TNF-a levels) and oxidative stress levels (reduction in
SOD, GPx and TBARS and increase in GSH) was
observed. This indicated a protective effect of the test
formulation in managing cognitive decline associated with
the ageing process.
Conclusion The results of this study demonstrate the
therapeutic potential of this novel polyherbal formulation
for the management and treatment of SDAT.
Key Points
Administration of test formulation containing
extracts of Bacopa monnieri (whole plant),
Hippophae rhamnoides (leaves and fruits) and
Dioscorea bulbifera (bulbils) in both normal elderly
subjects and SDAT patients after 12 months of
treatment demonstrated significant improvements in
various cognitive and neuropsychiatric measures like
mini mental state examination (MMSE) score, digital
symbol substitution (DSS; subtest of the Wechsler
adult intelligence scale—revised), immediate and
delayed word recall, attention span, functional
activity questionnaire (FAQ) and depression
(geriatric depression scale) scores.
This was accompanied by a reduction in
inflammation and oxidative stress as determined by
the measurement of various markers such as SOD,
GPx, GSH, TBARS, IL-6, TNF-a, CRP and
homocysteine in the blood plasma.
These findings suggest that the test formulation is a
safe novel polyherbal drug product and has immense
therapeutic potential for the management and
treatment of neurodegenerative disorders. However,
in order to demonstrate the efficacy of the drug,
further studies are needed to analyze the levels of
CNS and brain biomarkers of neurodegeneration
along with Phase III clinical trials to explore and
assess the disease modifying potential of the drug.
1 Introduction
Neurodegeneration is defined by the progressive loss of
structure or function and death of neurons [1]. Neurode-
generative diseases are highly debilitating with a serious
detrimental impact on social, economic, and health con-
ditions, including family life. Such diseases often lead to
severe morbidity and even death. Neurodegeneration can
be found from the molecular to systemic levels of neuronal
circuitry. Defective energy metabolism, excitotoxicity and
oxidative damage have been implicated as crucial factors
for the manifestation of neurodegeneration [2, 3]. These
disorders can either be hereditary or sporadic leading to
progressive nervous system dysfunction. More than 600
disorders severely affect the nervous system, resulting in
the atrophy of central and peripheral structures of the
nervous system. Diseases such as Parkinson’s disease,
Huntington’s disease, Alzheimer’s disease (AD) and other
dementias, brain cancer, degenerative nerve diseases,
encephalitis, epilepsy, genetic brain disorders, head and
brain malformations, prion diseases, and others, occur as a
result of neurodegenerative processes [4].
The advent of modern healthcare facilities in the past
century has contributed towards a longer and healthier
lifespan of people. This has and will result in an increase in
the world’s aged population and consequently in an
increase in the number of people with non-communicable
diseases, including dementia. AD is the most common
form of dementia and has been estimated to contribute
towards 60–70 % of cases [5, 6]. In 2010, it was estimated
that the total number of people suffering from dementia
worldwide was 35.6 million and is projected to nearly
double every 20 years, to 65.7 million in 2030 and 115.4
million in 2050 [7]. The total number of new cases of
dementia each year worldwide is nearly 7.7 million,
implying one new case every four seconds. The greatest
risk for dementia is ageing [8]. Dementia in the population
older than 65 years of age is referred to as senile dementia.
In total, approximately 7 % of the population older than
65 years of age and 30 % of people older than 80 years are
thought to be affected by senile dementia of Alzheimer’s
type (SDAT) [9]. In India, since the population is under-
going a demographic transition, age-related diseases like
dementia are on the rise The Dementia India report in 2010
estimated that the number of people with dementia in India
is around 3.7 million and this number is set to double in the
next 20 years [10]. The disease manifests itself by dis-
turbing multiple higher cortical functions, including
memory, thinking, orientation, comprehension, calculation,
learning capacity, language and judgement [11–13].
The cause and progression of AD is not well understood.
Research identifying modifiable risk factors of dementia is
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in its infancy. Several studies show that the disease is
associated with plaques and tangles in the brain [14].
Current treatments only help with the symptoms of the
disease and there are no available treatments that can stop
or reverse its progression [15, 16]. As of 2014, more than
1,000 clinical trials have been, or are being, conducted to
test various compounds in AD [17]. In the meantime, pri-
mary prevention is mainly focused on mental stimulation,
exercise and a balanced diet as ways to delay cognitive
symptoms (though not brain pathology) in healthy older
individuals [18]. Since the aged population is increasing,
government, policy-makers and other stakeholders need to
address the impact of dementia as an increasing threat to
global health.
For thousands of years the traditional system of Indian
medicine has promoted the therapeutic potential of several
medicinal plants for the treatment and management of
neurodegenerative diseases such as AD, Parkinson’s,
dementias and other neuronal disorders. In Ayurveda, an
approach for the prevention and treatment of degenerative
diseases is known as Rasayana, and involves the use of
polyherbal preparations as rejuvenators or brain tonic [19].
Very few studies have been done to investigate the mode of
action of such formulations and to utilize ancient wisdom
for the development of novel therapeutics. In this study,
three plants namely, Bacopa monnieri, Hippophae
rhamnoides and Dioscorea bulbifera, with properties that
have a direct effect on learning and memory have been
selected to develop a novel polyherbal formulation for the
treatment and amelioration of neurodegenerative disorders
like SDAT.
B. monnieri Linn or Brahmi, is a plant in the family
Scrophulariaceae. It has been used in the Ayurvedic sys-
tem of medicine for centuries and is claimed to act as a
nerve tonic. Extracts of the plant have been extensively
used for the treatment of various neurological and neuro-
psychiatric diseases [20]. In the present formulation, an
extract of the arial part of the plant, especially stems and
leaves has been used. Bioactive molecules like bacoside
present in the extract act on different receptors responsible
for memory and cognition. Several studies have shown that
B. monnieri acts on the cholinergic system (prevents/slows
down the loss of acetylcholine), and regulates the glu-
tametergic and GABAergic receptors, thereby preventing
cognitive decline [21]. Positive effects of the plant towards
ameliorating neuroinflammation and promoting healthy
brain ageing have also been reported [22–24].
H. rhamnoides, belongs to the family Elaeagnaceae, and
is commonly known as seabuckthorn. An extract of the
leaves and fruit pulp has been used in this polyherbal
formulation. The fruit of H. rhamnoides is rich in different
flavonoids like vitamin P and quercetin, contains water-
and fat-soluble vitamins along with essential amino acids,
folic acid, various fatty acids, phytosterols, alpha tocoph-
erol and phenolic compounds. Studies have shown that the
fruit pulp extract has a potent anti-oxidant property [25],
which helps in protecting neurons from oxidative damage
that occurs for a variety of reasons. The high concentration
of folic acid in this plant can help in reducing elevated
levels of homocysteine, which is a marker for neurode-
generation [25, 26]. H. rhamnoides has also been shown to
target dopaminergic, serotonergic, and noradrenergic
receptors [27, 28].
D. bulbifera is a member of the Dioscoraceae family.
Tubers of Dioscorea have been used throughout the world
as a food and herbal medicine. The extract of the bulbil of
the plant was used in this formulation. D. bulbifera extracts
have shown hypolipidemic, anti-inflammatory, anti-hyper-
glycaemic and anti-obesity properties along with alpha-
glucosidase, amylase and lipase inhibitory effects, which
may help prevent neurodegeneration caused by hyper-
glycaemia, hyperlipoproteinaemia and obesity. The targets
of action are suggested to be pro-inflammatory cytokines-
CRP, TNF-a, IL-6, adipokines, adiponectin, leptin, resistin,
gherlin, and hyperinsulinaemia. Pharmacologically active
components of the Dioscorea species include diosgenin,
which is a steroidal saponin, and dioscin, a form of dios-
genin with sugars attached. Studies suggest that dietary
diosgenin may lower plasma cholesterol levels, reduce
blood sugar and decrease inflammation [29, 30].
The polyherbal formulation described here, an inter-
vention based on the traditional knowledge of Ayurvedic
system of medicine, is an attempt to provide a safe, effi-
cacious and cost-effective herbal drug to target multiple
aspects of neurodegeneration [31]. Although the precise
mechanisms of action of such herbal drugs have yet to be
determined, it is well known that this group of plants
possess strong antioxidant, anti-inflammatory and neuro-
protective properties (Table 1). In order to establish the
efficacy and potency of this novel formulation for the
prevention and treatment of SDAT, a double-blind pla-
cebo- and active-controlled randomized clinical trial was
carried out for a period of 12 months.
2 Subjects and Methods
2.1 Test Formulation Preparation
Film-coated tablets (500 mg) were prepared from an
extract containing a combination of three plants namely, B.
monnieri, H. rhamnoides and D. bulbifera combined with
excipients such as dibasic calcium phosphate and micro-
crystalline cellulose. The dried whole plant of B. monnieri,
dried fruit and leaves of H. rhamnoides and dried tuber of
D. bulbifera were utilized for extraction. The extraction
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was performed at a temperature of 70–80 �C. The pH of
the solution was maintained between 7–10. Each tablet
contained the following amount of extracts: 225 mg of B.
monnieri and 125 mg each of H. rhamnoides and D.
bulbifera. The placebo was prepared using dibasic calcium
phosphate and microcrystalline cellulose and replicated the
test drug in appearance, odour, and texture. Packaging and
randomization was performed, and then the trial was
conducted.
2.2 Participants
The study protocol was approved by the institution’s eth-
ical committee and all participants gave their written
informed consent. Each volunteer was requested to com-
plete a medical health questionnaire prior to participation.
All the subjects then underwent extensive medical evalu-
ation to ascertain subject suitability for entering the clinical
trial. The inclusion criteria for the SDAT subjects included
age[60 years, willingness to give informed consent and a
deterioration of memory along with at least three of the
following five complaints: poor orientation, poor judge-
ment and problem solving difficulties, trouble in the
functioning of community affairs, inability to function
independently in home and during hobbies and difficulties
in personal care. Patients were then assigned to the SDAT
group following clinical screening using the dementia rat-
ing scale-II [32]. Subjects suffering from any metabolic
disease, established neuropathy or tuberculosis were
excluded. SDAT patients receiving conventional treatment
were also excluded from the study.
The participants were divided into the following four
groups; Group A: healthy elderly subjects who were given
placebo, Group B: healthy elderly subjects who were given
the test formulation, Group C: SDAT patients who were
treated with a standard drug donepezil (Aricept 10 mg,
twice daily) and Group D: SDAT patients treated with the
test formulation.
2.3 Procedures and Treatments
This study was conducted for 12 months in a double-
blinded placebo- and active-controlled randomized man-
ner. The treatments or placebo were administered twice a
day, during the study period of 12 months. A random list of
numbers was determined by a computer-generated series
with the proper sequence applied to container labels and
supplied to participants in the order enrolled after being
randomly assigned to the various treatment groups. All
participants were assessed for baseline data of memory,
cognitive functions, and biochemical markers like homo-
cysteine, CRP (C-reactive protein), IL-6 (interleukin 6),
TNF-a (tumor necrosis factor alpha), SOD (superoxide
dismutase), GPx (glutathione peroxidase), GSH (glutathi-
one), and TBARS (thiobarbituric acid reactive substances).
The healthy subjects and patients were given the drugs, to
be taken independently at home. After every 3 months they
were assessed according to the above-mentioned parame-
ters, throughout the 12 months of treatment. The code for
study allocation was only broken when the last participant
completed the entire follow-up. The staff involved in the
collection of the study’s endpoints were instructed to fol-
low a rigorous protocol and not to discuss any issues
related to the use of medication. Reviews for compliance
with medication and side effects were performed inde-
pendently by the investigators, who were also blinded to
group allocation. Compliance was determined by the col-
lection of unused tablets at each follow-up. Subjects were
asked to call the study centre if they experienced any
medical problems during the study period. At the end of the
study, they were also asked about adverse events. The
following tests and measurements were drawn at baseline
Table 1 A summary of the medicinal properties of plants used in the test formulation
Plant name Part of the plant used Major bioactive
component
Therapeutic properties/targets References
Bacopa monnieri
Family:
Scrophulariaceae
Whole plant
Dose: 450 mg/day
Bacoside Regulates cholinergic system, antioxidant
and cytoprotective effect, acts on
oxidative stress markers, memory
enhancer and nerve tonic
[20, 22, 23, 50–52]
Hippophae rhamnoides
Family: Elaeagnaceae
Leaves and fruits
Dose: 250 mg/day
Quercetin Anti-inflammatory, anti-oxidant,
immunomodulatory properties,
treatment of hypertension and
hyperlipidemia, anti-radiation,
cardioprotective and anti-atherogenic
property
[25–28, 53–58]
Dioscorea bulbifera
Family: Dioscoraceae
Bulbils
Dose: 250 mg/day
Diosgenin Anti-inflammatory, analgesic,
antihyperlipidemic, antihyperglycemic,
antioxidant and anthelmintic activity
[29, 30, 59–62]
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and at follow-up visits and compared to see whether any
changes suggested adverse events: physical examination
(height, weight, body mass, ponderal, obesity and waist to
hip ratio index, blood pressure, pulse, fundoscopy), blood
examination (haemoglobin, white blood cell count, differ-
ential count of neutrophils, lymphocytes, eosinophils,
monocytes and basophils, red blood cell count, erythrocyte
sedimentation rate, platelet count, fasting and post-prandial
blood sugar, HbA1c, IL-6, TNF-a, high-sensitivity CRP,
adiponectin, homocysteine, serum alkaline phosphatase,
total protein and serum albumin), urine test (urinary van-
illomandelic acid, 17 ketosteroides and glucocorticoid),
cardiology test (electrocardiography and chest X-ray) and
liver function test (serum glutamic-oxaloacetic transami-
nase, glutamic-pyruvic transaminase, bilirubin and alkaline
phosphatase).
2.4 Analysis of Cognitive Functions
Cognitive function was assessed following structured per-
formance tests, which included mental status, verbal
memory, complex psychomotor skills, and attention/exec-
utive functions. Mini Mental State Examination (MMSE)
was used to assess mental status [33]. Memory scores were
tested using the digital memory apparatus (Medicaid sys-
tems, Chandigarh, India) device for both immediate and
delayed memory performance. Complex psychomotor skill
was examined using the digital symbol substitution (DSS)
test, which is a sub-test of the Wechsler Adult Intelligence
Scale—Revised [34], and has a score range of 0–93.
Attention span scores were obtained using the electronic
device—Attention Span Apparatus (Medicaid systems,
Chandigarh, India). A well-trained psychologist/technical
person administered all four tests in the same order to all
the study patients. Depression was assessed by the Geri-
atric Depression Scale-15 (GDS-15) [35], which is a global
test for depression with scores ranging from 0 to 15.
Functional Activity Questionnaire (FAQ) scores were also
obtained to test cognitive function of the participants [36,
37].
2.5 Analysis of Biochemical Markers
Venous blood was collected in vacutainer tubes and sub-
jected to centrifugation at 1500 g for 15 min; the plasma
from the samples were then collected and stored at -80 �C
until they were assayed. The blood plasma level of
homocysteine was determined by a high-performance
liquid chromatography (HPLC) method. CRP, IL-6 and
TNF-a levels were detected in the blood plasma as markers
for inflammation by the ELISA method using test kits.
SOD and GPx activity, and GSH and TBARS levels were
measured as markers for oxidative stress. SOD activity was
estimated by the method of Misra and Fridovich (1971),
GPx activity was estimated by the method of Rotruck et al.
(1973). GSH levels as well as TBARS levels were detected
in the blood by colorimetric assays [38–43].
2.6 Statistical Analysis
All data are expressed as mean ± SD. The unpaired stu-
dent t test was performed to compare the results obtained
from the different groups. All statistical analysis was done
using the Graph pad prism ver. 2.0. Per-protocol analysis
was performed and statistical significance was regarded at
P \ 0.05. All data available from the follow-up studies
were included in the analysis, irrespective of whether the
participant attended all the follow-up visits.
3 Results
3.1 Demographic Data of Subjects for Clinical
Complaints
A total of 109 healthy elderly volunteers and 123 SDAT
patients with an age range of 60–75 years were recruited,
out of whom 97 healthy subjects and 104 SDAT patients
completed the study. The baseline demographic data of the
subjects for clinical complaints are presented in Table 2.
When percentages of participants with complaints or the
mean score in each of these parameters were compared
among the healthy elderly group and within the SDAT
group, no significant differences were observed. However,
the SDAT subjects showed significant differences in these
parameters compared to the healthy elderly subjects. These
included elevated levels of homocysteine, CRP, IL-6, TNF-
a and higher percentage of aggression, sleep disturbance,
communicate difficulty, depression and psychotic features.
The drop-out rates were lower in the test formulation
groups when compared to the corresponding placebo or
donepezil group, All of the drop-outs were lost to follow-
up.
3.2 Effect of the Test Formulation
on Neuropsychological Parameters
The effects of the test formulation on several neuropsycho-
logical parameters like memory, mental status, complex
psychomotor skills, and attention/executive functions are
shown in Table 3. After 12 months of treatment, results from
the healthy elderly group who received the test formulation
were compared to the group which only received the placebo.
Statistical analysis revealed that the test formulation-treated
group showed significant improvements over the placebo-
treated group in their MMSE score (P = 0.0003), DSS score
Management of Cognitive Determinants in Senile Dementia
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(P = 0.0008), delayed word recall score (P \ 0.0001),
attention span (P \ 0.0001), FAQ score (P = 0.001) and
depression score (P \ 0.0001). However, word recall
immediate score showed no significant changes (P\0.1500)
(Table 3). These results demonstrate the potential of the test
formulation to augment the cognitive deficits due to aging.
Similarly, results from the SDAT group treated with the test
formulation for 12 months were compared to the SDAT
group treated with donepezil. Analysis showed that the test
formulation treatment was either similar or significantly
better in the improvement of neuropsychological parameters
such as their MMSE (P = 0.9375), DSS (P = 0.0001), word
recall immediate (P \ 0.0001) word recall delayed
(P = 0.052), attention span (P = 0.0208), FAQ (P \0.0001) and depression (P\0.0001) scores (Table 3). These
results indicate a beneficial role of the novel herbal test
formulation for the treatment of SDAT.
3.3 Effect of the Test Formulation on Biochemical
and Inflammatory Markers
In this study the levels of inflammatory cytokines, TNF-aand IL-6, were measured in healthy elderly subjects and
SDAT patients before and after 12 months of treatment
(Table 4). Statistical analysis revealed a significant reduc-
tion in the levels of both TNF-a (P \ 0.0001) and IL-6
(P \ 0.0001) in the healthy elderly subjects following
treatment with the test formulation for 12 months when
compared to the placebo group. Similar analysis done on
SDAT patients treated either with donepezil or the test
formulation showed a significant reduction in the levels of
TNF-a (P \ 0.0001) in the test formulation group. The
levels of IL-6 was lowered in both groups following
treatment and although lower levels were observed in the
test formulation group when compared to the donepezil
group, the result was not found to be significant
(P = 0.2052). A significant decrease in the levels of
another inflammatory marker, CRP (P \ 0.0001), was
observed in the healthy elderly subjects after 12 months of
treatment with the test formulation, when compared to
placebo treated group (Table 4). A similar decrease in the
CRP (P \ 0.0001) levels was observed in SDAT patients
treated with the test formulation when compared to do-
nepezil. Moreover, it is well documented that the elevated
levels of homocysteine are associated with neurodegener-
ative disorders [43]. In the current study, treatment with the
test formulation resulted in a significant decrease in the
levels of homocysteine (P \ 0.0001) in both the healthy
elderly and SDAT participants after 12 months of treat-
ment with the test formulation (Table 4) when compared to
the corresponding control groups (placebo/donepezil).
3.4 Effect of the Test Formulation on Oxidative Stress
In the present study the effects of the test formulation on
antioxidants like SOD, GPx, GSH and TBARS were also
studied (Table 5). A comparison between the plasma
antioxidant activity of the test formulation and placebo-
treated healthy elderly groups showed a significant reduc-
tion in the SOD (P \ 0.0001) and GPx activity (P \0.0001) in the test formulation group. In addition to this,
lower TBARS (P\0.0001) and higher GSH (P\0.0001)
levels were also observed in the test formulation group
indicating a reduction in reactive oxygen species.
Table 2 Demographic data of study subjects
Characteristics Group A ? B (N = 109) Group C ? D (N = 123)
Number of subject recruited 109 123
Number of subjects completed trial 97 104
Aggression (%) 24 62
Sleep disturbance (%) 20 71
Communication difficulty (%) None reported 79
Depression (%) 25 68
Psychotic features (%) 9 67
Homocysteine (nmol/L) 21.99 ± 2.85 38.42 ± 4.16
CRP (mg/L) 2.38 ± 0.76 6.23 ± 1.09
IL-6 (pg/ml) 2.23 ± 0.41 4.12 ± 1.06
TNF-a (pg/ml) 483.80 ± 85.64 1124.06 ± 105.87
Values are expressed as mean ± SD unless specified otherwise
The number of subjects: 109 healthy subjects and 123 patients were recruited, of whom 97 healthy subjects and 104 patients completed the study.
Group A ? B (N = 109) were healthy elderly subjects and were treated with either placebo or test formulation, while group C ? D (N = 123)
were SDAT patients treated with donepezil or test formulation. Data for aggression, sleep disturbance, communication difficulties, depression
scores and psychotic features are presented as the percentage of subjects with these clinical complaints
CRP C-reactive protein, IL-6 interleukin 6, TNF-a tumor necrosis factor alpha
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s
Init
ial
Aft
er3
mo
nth
sA
fter
6m
on
ths
Aft
er
12
mo
nth
s
MM
SE
17
.48
±3
.72
16
.93
±3
.75
16
.45
±5
.11
14
.90
±4
.32
16
.93
±3
.71
17
.45
±4
.36
17
.87
±3
.91
18
.14
±4
.02
0.0
00
3
DS
S4
6.3
4±
9.4
94
7.0
2±
8.9
34
5.9
4±
7.4
84
1.3
2±
8.6
34
3.8
7±
11
.06
46
.91
±1
0.7
74
7.8
2±
10
.96
49
.01
±1
2.1
30
.00
08
Wo
rdre
call
imm
edia
te
4.8
20
±1
.25
34
.36
2±
1.0
19
3.9
93
±0
.82
3.7
18
±0
.96
4.9
32
±1
.36
44
.55
6±
1.4
52
3.9
94
±0
.97
34
.01
2±
1.0
04
0.1
50
0
Wo
rdre
call
del
ayed
3.5
96
±0
.85
3.1
95
±0
.82
63
.01
7±
0.9
37
3.1
28
±0
.75
93
.75
1±
1.0
03
3.5
54
±0
.88
93
.87
8±
0.4
48
4.0
02
±0
.75
4\
0.0
00
1
Att
enti
on
span
7.3
51
±2
.07
17
.11
2±
2.2
91
7.3
12
±1
.79
16
.87
4±
1.1
19
8.7
34
±2
.11
48
.85
2±
2.7
72
9.2
43
±2
.73
49
.38
2±
1.9
42
\0
.00
01
FA
Q1
6.0
97
±2
.88
51
6.1
06
±3
.21
71
5.9
13
±2
.55
71
5.2
13
±2
.99
31
5.8
51
±3
.17
21
6.1
04
±2
.90
11
7.8
04
±2
.85
61
7.3
16
±3
.01
40
.00
10
Dep
ress
ion
12
.01
3±
2.7
14
12
.68
3±
2.6
55
13
.18
4±
3.0
03
13
.08
7±
2.8
56
11
.69
4±
2.4
52
9.8
83
±2
.90
11
0.1
45
±3
.00
49
.18
4±
3.1
14
\0
.00
01
Par
amet
erG
rou
pC
(N=
43
)G
rou
pD
(N=
61
)P
val
ue
(co
mp
aris
on
bet
wee
nG
rou
p
Cv
s.D
afte
r
12
mo
nth
s
Init
ial
Aft
er3
mo
nth
sA
fter
6m
on
ths
Aft
er1
2m
on
ths
Init
ial
Aft
er3
mo
nth
sA
fter
6m
on
ths
Aft
er1
2m
on
ths
MM
SE
7.0
19
±1
.31
66
.99
4±
2.1
28
7.9
04
±2
.71
87
.88
2±
1.9
56
6.0
14
±1
.21
27
.22
4±
2.1
04
7.8
02
±2
.11
47
.91
4±
2.1
06
0.9
37
5
DS
S3
4.8
59
±5
.38
43
5.7
33
±6
.11
73
5.9
86
±5
.75
23
5.8
52
±4
.90
63
6.4
95
±4
.31
03
8.1
12
±3
.72
13
7.9
36
±2
.89
43
8.9
84
±3
.01
60
.00
01
Wo
rdre
call
imm
edia
te2
.77
4±
0.6
21
2.5
37
±0
.39
82
.82
8±
0.7
84
2.7
94
±0
.59
32
.98
4±
0.7
12
3.1
04
±0
.80
23
.22
8±
1.0
42
3.5
94
±1
.00
3\
0.0
00
1
Wo
rdre
call
del
ayed
1.8
56
±0
.22
91
.90
6±
0.4
13
2.0
11
±0
.39
71
.88
4±
0.5
97
1.7
59
±0
.45
21
.91
4±
0.2
34
2.1
12
±0
.30
12
.04
9±
0.2
29
0.0
52
0
Att
enti
on
span
3.9
04
±0
.83
63
.88
7±
0.9
14
4.1
12
±0
.78
54
.39
6±
0.9
13
4.0
14
±1
.01
03
.93
6±
1.1
14
4.3
94
±1
.22
14
.91
8±
1.2
39
0.0
20
8
FA
Q8
.93
4±
2.1
12
9.2
92
±1
.75
89
.49
0±
1.3
94
9.8
01
±1
.45
88
.11
2±
1.4
42
9.4
56
±1
.59
31
1.6
22
±2
.11
21
1.8
73
±2
.75
1\
0.0
00
1
Dep
ress
ion
20
.82
1±
3.7
56
18
.77
5±
2.4
83
19
.25
8±
3.1
46
21
.00
6±
2.7
78
19
.42
6±
2.1
10
17
.85
6±
1.7
58
16
.91
4±
2.0
12
16
.38
7±
2.1
16
\0
.00
01
Val
ues
are
exp
ress
edas
mea
n±
SD
Gro
up
A(N
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1)
no
rmal
aged
trea
ted
wit
hp
lace
bo
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rou
pB
(N=
56
)n
orm
alag
edtr
eate
dw
ith
test
form
ula
tio
n,g
rou
pC
(N=
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)S
DA
Tp
atie
nts
trea
ted
wit
hd
on
epez
ilan
dg
rou
pD
(N=
61
)
SD
AT
pat
ien
tstr
eate
dw
ith
test
form
ula
tio
n.
Sta
tist
ical
anal
ysi
sh
ave
bee
nd
on
eto
com
par
esc
ore
saf
ter
12
mo
nth
str
eatm
ent
bet
wee
ng
rou
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vs.
Ban
dg
rou
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vs
D
MM
SE
Min
im
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lst
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dig
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sym
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un
ctio
nal
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vit
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nn
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Management of Cognitive Determinants in Senile Dementia
Author's personal copy
Ta
ble
4E
ffec
to
fte
stfo
rmu
lati
on
on
lev
els
of
infl
amm
ato
rym
ark
ers
Par
amet
erG
roup
A(N
=41)
Gro
up
B(N
=56)
Pval
ue
(com
par
ison
bet
wee
nG
roup
Avs.
Baf
ter
12
month
sIn
itia
lA
fter
3m
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sA
fter
6m
onth
sA
fter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
s
Hom
ocy
stei
ne
(nm
ol/
L)
21.0
13
±4.1
09
20.9
93
±3.7
82
23.1
04
±2.9
94
24.8
13
±3.1
06
19.3
20
±3.1
04
16.3
59
±2.9
11
15.4
84
±2.2
16
12.8
23
±2.1
04
\0.0
001
CR
P(m
g/L
)3.8
57
±0.7
52
4.1
16
±0.8
94
4.7
01
±1.0
22
4.6
12
±1.1
06
4.9
32
±1.0
12
3.7
51
±1.1
42
3.1
24
±1.2
24
3.0
93
±1.0
04
\0.0
001
IL-6
(pg/m
l)2.8
41
±0.3
16
2.6
63
±0.4
45
3.0
04
±0.6
91
2.9
41
±0.5
83
2.1
03
±0.7
51
2.0
82
±0.6
83
1.7
58
±0.7
14
1.3
32
±0.8
01
\0.0
001
TN
F-a
(pg/m
l)693.8
84
±84.0
42
806.7
52
±95.1
13
775.9
42
±110.8
15
811.6
93
±131.6
85
791.4
20
±102.4
52
682.8
24
±98.7
73
642.1
01
±121.7
52
584.7
30
±114.9
02
\0.0
001
Par
amet
erG
roup
C(N
=43)
Gro
up
D(N
=61)
Pval
ue
(com
par
ison
bet
wee
nG
roup
Cvs.
Daf
ter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
s
Hom
ocy
stei
ne
(nm
ol/
L)
41.9
8±
6.9
040.9
0±
5.8
847.0
1±
6.1
344.7
3±
7.1
139.7
5±
6.3
933.4
5±
5.1
131.8
2±
5.9
030.2
2±
3.8
7\
0.0
001
CR
P(m
g/L
)6.2
25
±1.3
08
6.1
03
±1.4
16
5.9
85
±0.8
85
5.8
87
±1.0
49
6.1
02
±1.2
21
5.8
11
±1.7
23
5.0
49
±1.3
01
4.7
51
±1.1
49
\0.0
001
IL-6
(pg/m
l)4.9
85
±1.0
24
4.5
93
±1.2
21
4.1
16
±1.3
16
3.8
54
±0.8
59
5.3
94
±1.3
10
4.8
52
±1.2
26
3.8
85
±1.1
39
3.6
12
±1.0
14
0.2
052
TN
F-a
(pg/m
l)1618.7
7±
316.8
11498.9
0±
243.9
41483.8
5±
346.1
81598.7
7±
298.5
21402.7
5±
288.4
21291.8
2±
204.9
71235.7
5±
301.2
21139.4
5±
198.8
7\
0.0
001
Val
ues
are
expre
ssed
asm
ean
±S
D
Gro
up
A(N
=41)
norm
alag
edtr
eate
dw
ith
pla
cebo,
gro
up
B(N
=56)
norm
alag
edtr
eate
dw
ith
test
form
ula
tion,
gro
up
C(N
=43)
of
SD
AT
pat
ients
trea
ted
wit
hdonep
ezil
and
gro
up
D(N
=61)
SD
AT
pat
ients
trea
ted
wit
hte
stfo
rmula
tion.
Sta
tist
ical
anal
ysi
shav
ebee
ndone
toco
mpar
esc
ore
saf
ter
12
month
str
eatm
ent
of
gro
up
Avs.
Ban
dgro
up
Cvs
D
CR
PC
-rea
ctiv
epro
tein
,IL
-6In
terl
eukin
6,
TN
F-a
Tum
or
nec
rosi
sfa
ctor
alpha
A. Sadhu et al.
Author's personal copy
Ta
ble
5E
ffec
to
fte
stfo
rmu
lati
on
on
ox
idat
ive
stre
ssm
ark
ers
Par
amet
erG
roup
A(N
=41)
Gro
up
B(N
=56)
Pval
ue
(com
par
ison
bet
wee
nG
roup
Avs.
Baf
ter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
s
SO
D(U
/gH
b)
981.7
9±
201.8
41084.9
0±
193.7
31091.4
3±
216.7
81189.3
6±
189.5
7938.7
5±
09.2
2851.6
8±
214.7
5832.4
5±
282.9
0738.7
3±
125.0
1\
0.0
001
GP
x(U
/gH
b)
17.9
46
±3.1
04
18.7
35
±4.0
06
18.9
38
±3.9
13
19.7
75
±3.9
14
18.9
13
±2.1
02
17.3
32
±2.7
14
17.1
04
±2.1
72
16.1
42
±3.0
14
\0.0
001
GS
H(U
/gH
b)
8.2
14
±1.2
11
8.1
16
±1.3
16
7.3
54
±1.7
18
7.1
39
±1.9
08
8.9
42
±2.1
10
9.0
62
±1.7
58
9.8
58
±1.3
42
9.8
68
±1.4
51
\0.0
001
TB
AR
S(n
mol/
gH
b)
129.7
5±
31.8
1138.1
4±
35.4
9141.0
1±
26.7
5139.9
4±
34.8
2134.7
2±
29.5
5122.8
5±
31.7
8108.0
2±
41.6
2106.6
4±
34.9
0\
0.0
001
Par
amet
erG
roup
C(N
=43)
Gro
up
D(N
=61)
Pval
ue
(com
par
ison
bet
wee
nG
roup
Cvs.
Daf
ter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
sIn
itia
lA
fter
3m
onth
sA
fter
6m
onth
sA
fter
12
month
s
SO
D(U
/gH
b)
1416.9
8±
312.8
81386.3
9±
295.8
71345.8
7±
188.9
91296
±225.7
21312.7
5±
214.8
21268.9
1±
301.6
21185.3
3±
198.4
51145.9
2±
228.7
50.0
013
GP
x(U
/gH
b)
27.1
1±
3.3
525.9
3±
4.0
624.8
7±
3.7
825.9
9±
4.1
126.1
1±
5.0
225.6
3±
3.9
121.9
5±
2.8
520.7
8±
3.1
4\
0.0
001
GS
H(U
/gH
b)
7.4
83
±2.1
13
7.8
85
±1.4
58
7.1
43
±2.1
39
6.8
31
±1.1
39
7.0
12
±1.7
14
9.1
02
±1.7
72
9.8
21
±2.0
14
9.3
58
±2.1
39
\0.0
001
TB
AR
S(n
mol/
gH
b)
148.3
2±
59.3
4183.1
4±
62.7
7188.0
1±
59.4
9176.4
0±
68.1
1178.4
5±
65.3
5152.7
5±
59.6
2129.5
8±
38.4
5131.6
2±
29.6
8\
0.0
001
aV
alues
are
expre
ssed
asm
ean
±S
D
Gro
up
A(N
=41)
norm
alag
edtr
eate
dw
ith
pla
cebo,
gro
up
B(N
=56)
norm
alag
edtr
eate
dw
ith
test
form
ula
tion,
gro
up
C(N
=43)
SD
AT
pat
ients
trea
ted
wit
hdonep
ezil
and
gro
up
D(N
=61)
SD
AT
pat
ients
trea
ted
wit
hte
stfo
rmula
tion.
Dat
ais
repre
sente
das
mea
n±
SD
and
stat
isti
cal
anal
ysi
shas
bee
ndone
toco
mpar
esc
ore
saf
ter
12
month
str
eatm
ent
of
gro
up
Avs.
Ban
dgro
up
Cvs
D
SO
Dsu
per
oxid
edis
muta
se,
GP
xglu
tath
ione
per
oxid
ase,
GSH
glu
tath
ione,
TB
AR
Sth
iobar
bit
uri
cac
idre
acti
ve
subst
ance
s
Management of Cognitive Determinants in Senile Dementia
Author's personal copy
Similarly, when the SDAT group treated with donepezil
was compared to the test formulation treated group, a
marked reduction in SOD activity (P \ 0.0013), GPx
activity (P \ 0.0001) and TBARS levels (P \ 0.0001),
along with an increase in GSH levels (P \ 0.0001) was
observed in the latter (Table 5). These results demonstrate
the ability of the test formulation to reduce oxidative stress
in both healthy elderly subjects as well as SDAT patients.
3.5 Safety and Adverse Events
The percentage of trial subjects completing the study
were placebo, 80.3 %, test formulation, 96.5 % for
healthy subjects, donepezil (10 mg twice daily), 74.1 %,
and test formulation, 93.8 % for SDAT patients. The
percentage of patients who discontinued the study were
placebo, 19.6 %, test formulation, 3.4 % for healthy
subjects, donepezil (10 mg twice daily), 24.8 %, and test
formulation, 6.15 % for SDAT patients (Fig. 1). All drop-
outs were lost to follow-up. The drop-out rates for the test
formulation-treated groups were lower than the corre-
sponding placebo or donepezil groups indicating that the
test formulation is safe and well tolerated among trial
subjects. Adverse events reported in the trial included
nausea, constipation and/or drowsiness were transient and
mild in severity.
Fig. 1 Flow chart of the
subjects who participated in the
clinical trial
A. Sadhu et al.
Author's personal copy
4 Discussion
The pharmaceutical industry is facing a serious challenge
in the drug discovery and development process for neuro-
degenerative diseases [44]. Drugs are becoming extremely
expensive, riskier and inefficient; a shift from the pre-
dominant single-target approach towards a multi-target one
could help address this. In recent years the Ayurvedic
system of medicine in India has gathered increasing rec-
ognition as a potential mode of treatment for diseases. This
system of medicine is based on reverse pharmacology and
thus provides an efficient platform for drug development
[45, 46].
Several medicinal plants have been extensively used in
Ayurvedic medicines as rejuvenators (which prevent dis-
eases), that have been shown to slow the process of
ageing and related disorders. For centuries, Ayurvedic
therapeutics have been prescribed for neurodegenerative
diseases, including dementias, and there are extensive
data available on the history of human use from both a
safety and efficacy perspective [47, 48]. Studies on
mechanisms of action of such therapeutics have demon-
strated their ability to increase levels of nerve growth
factors, neurotrophic factors and to reduce inflammation
and oxidative damage [31].
The present study demonstrates the potential of a
polyherbal formulation for the treatment and prevention of
neurodegenerative disorders with special reference to
SDAT. This test formulation is a combination of three
plants, namely B. monnieri, H. rhamnoides and D. bulbif-
era. These plants were selected based on their potential to
target multiple factors associated with the onset and pro-
gression of neurodegeneration, including oxidative stress,
inflammation and hyperhomocysteinaemia [21, 24, 30].
These plants contain several bioactive molecules such as
bacoside, quercetin, diosgenin and folic acid which provide
neuroprotective effects by acting on multiple targets like
glutamatergic, GABAergic, dopaminergic, noradrenergic
and serotonergic receptors along with oxidative stress
markers, pro-inflammatory cytokines: TNF-a, IL-6, CRP
and homocysteine [22, 26, 27, 29]. In this exploratory
double–blind placebo- and active-controlled study,
administration of the test drug in healthy elderly subjects
led to significant improvements in cognitive functions and
a reduction in markers for inflammation and oxidative
stress. Consequently, SDAT patients treated with the test
formulation also showed a significant improvement and
amelioration of their symptoms. In order to further
understand the effect of this drug, in depth molecular and
pharmacological studies in animals and humans are nee-
ded, to corroborate the mechanism of action that could
extend beyond its anti-inflammatory and anti-oxidant
effects. The effect of this formulation on the levels of
b-amyloid and tau (including phospho tau) in the brain or
CSF as well as on the accumulation of plaques and tangles
in the brain that are commonly associated with Alzheimer’s
disease warrants investigation in order to assess the dis-
ease-modifying potential of this drug [49]. In this direction,
multicentric (Phase III) clinical trials need to be performed
for a longer period of time to substantiate the therapeutic
potential of the test formulation.
5 Conclusion
Administration of a polyherbal formulation containing
extracts of B. monnieri (whole plant), H. rhamnoides
(leaves and fruits) and D. bulbifera (bulbils) for a period of
12 months resulted in significant improvement of cognitive
functions as well as reduction in inflammation and oxida-
tive stress related to neurodegeneration in healthy elderly
subjects in comparison to placebo and in SDAT patients in
comparison to donepezil.
Acknowledgments This study was supported in part by UGC Grant
No. Dev./D/CPBM/2015, AYUSH and DST India. Additional support
was provided by SRM University, Chennai and Adesh University
Punjab. The tablets were manufactured by Varanasi Bioresearch at no
cost. The clinical study was performed at Banaras Hindu University,
Varanasi, SRM University, Chennai and Adesh University, Punjab,
India. Thanks to the clinical investigators Prof. V N Mishra and Prof.
Malvin George for their help and support in conducting the clinical
trial. No competing financial interests exist for any authors. All
authors declare no conflicts of interest. The clinical trial titled ‘‘Pre-
vention and management of age related neurodegenerative disorders-
an Ayurvedic intervention’’, was registered (No. K.11022/10/2009-
DCC) with Dept. of AYUSH (Ministry of health and family welfare,
Govt. of India).
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