Introduction:Hyperlipidemia is a metabolic disorder characterized by fluctuated blood lipid levels which can lead to several diseases.Learning is the ability to acquire new information and skills through experience whereas Memory is a process by which information acquired through learning is stored and retrieved. Objective:To evaluate the antihyperlipidemic activity and also nootropic activity of methanolic extract of Calycophyllum spruceanumbark Materialsand methods:Screening of methanolic extract of Calycophyllum spruceanum bark (MECSB)for antihyperlipidemic activity was done in high-fat diet Induced rats using atorvastatin as standard and extracts at a dose of 125, 250, and 500mg/kg. To screen nootropic activity rats were pretrained using Cook’s Pole Climbing Apparatus.scopolamine was used to induce amnesia in rats and piracetam as standard and extracts at a dose of 125, 250, and 500mg/kg. Results: Significantdecrease (p <0.5) in cholesterol, triglycerides, LDL, VLDL, and increase in HDL levels shown that the extract has Antihyperlipidemic activity. An increase in CAR (Conditioned Avoidance Response) and less time taken by the animal as jump response to avoid shock shows that the extract has Nootropic Activity. Key Words – Hyperlipidemia, Nootropic activity,Calycophyllum spruceanum, Cholesterol, pole climbing apparatus

1. EVALUATION OF ANTIHYPERLIPIDEMIC ACTIVITY OF CALYCOPHYLLUM
SPRUCEANUM
Dr. I. Veena Rani1
, G. Preeti Raj1
, K. Suneetha2
, Dr K Vanitha Prakash2
1.
Department of Pharmacology, SSJ College of Pharmacy, vattinagulpally, Gandipet,
Hyderabad- 500075
2.
Department of Pharmaceutical Analysis, SSJ College of Pharmacy, vattinagulpally,
Gandipet, Hyderabad- 500075
ABSTRACT
Introduction:Hyperlipidemia is a metabolic disorder characterized by fluctuated blood lipid
levels which can lead to several diseases.Learning is the ability to acquire new information and
skills through experience whereas Memory is a process by which information acquired through
learning is stored and retrieved.
Objective:To evaluate the antihyperlipidemic activity and also nootropic activity of methanolic
extract of Calycophyllum spruceanumbark
Materialsand methods:Screening of methanolic extract of Calycophyllum spruceanum bark
(MECSB)for antihyperlipidemic activity was done in high-fat diet Induced rats using atorvastatin
as standard and extracts at a dose of 125, 250, and 500mg/kg. To screen nootropic activity rats
were pretrained using Cook’s Pole Climbing Apparatus.scopolamine was used to induce amnesia
in rats and piracetam as standard and extracts at a dose of 125, 250, and 500mg/kg.
Results: Significantdecrease (p <0.5) in cholesterol, triglycerides, LDL, VLDL, and increase in
HDL levels shown that the extract has Antihyperlipidemic activity. An increase in CAR
(Conditioned Avoidance Response) and less time taken by the animal as jump response to avoid
shock shows that the extract has Nootropic Activity.
Key Words – Hyperlipidemia, Nootropic activity,Calycophyllum spruceanum, Cholesterol, pole
climbing apparatus.
INTRODUCTION
Hyperlipidemia is a metabolic disorder characterized by elevated levels of lipids like cholesterol,
triglycerides, lipoproteins1
like very-low-density lipoproteins, low-density lipoproteins with a
concomitant decrease of high-density lipoproteins2
. It can be classified into primary and
secondary, where primary hyperlipidemia is due to a genetic defect and secondary
hyperlipidemia is acquired3
. Hyperlipidemia also leads to many disorders like atherosclerosis4
,
myocardial infarction, coronary artery disease, and ischemic disease2
. The drugs which are used
to decrease hyperlipidemia are known as antihyperlipidemic drugs and they include statins, fibric
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2. acid derivatives, bile acid sequestrants and many more among which statins are used to the most
which are of course with some side effects5
. Different parameters like age, external factors like
alcohol and drugs leads to impairment of memory.Accumulation of LDL levels leads to
atherosclerosis, increased levels of triglycerides leads to coronary heart disease which are
regarded as major risk factors of hyperlipidemia6
.
Along with this dyslipidemia leads to many neurological disorders like Alzheimer’s disease
where there are plaque formations in the brain7
.
Hence nootropic activity is also screened for the Calycophyllum spruceanum
Cognitive Disorder is a disruption or impairment at higher level functioning if brain. This
disorder is due to Delirium, Dementia or Amnesia.There are many types of chemicals that act as
neurotransmitters in human body. Acetyl choline is the main transmitter responsible for learning
and memory. Reduction in acetyl choline causes loss of cognitive function which is seen in
Alzemers. Nicotine, a tertiary amine and nicotinic agents improve working memory function.
The hippocampus and the amygdale are found to be responsible for memory. Apart from this
decrease in histamine, decrease in vasopressin also affects memory4
.Also Glutamate5
, Serotonin,
Dopamine8
are associated. Therefore drugs which increase these chemicals are given to the
patients to increase cognitive function. On the other hand memory can be increased or
maintained providing with drugs. Drugs which are used to increase the cognitive function of the
brain are recognized as nootropics which are also known as smart drugs.
Piracetam was the first Nootropic drug discovered. Other Nootropics include Aniracetam,
Nefiracetam, Pramiracetam, Fosracetam, Nebracetam and Oxiracetam. Investigations are lasting
on in order to find out more significant and potent drug.
Calycophyllum spruceanum is an Amazonian tree which has a specific ability to shed its bark
and regenerate every year for which the tree is known as ‘Tree of Youth”8. So far, only one
study has addressed the chemical composition of C. spruceanum bark. Zuleta focused on the
seco-iridoid fraction of an ethanol extract of dried stem barks9
. Methanolic extract of
Calycophyllum spruceanum bark (MECSB) was found to contain different chemical constituents
like phenolic compounds, tannins, phytosterols, alcohols, fixed oils, glycosides, flavonoids,
saponins and coumarins which are confirmed by phytochemical screening10
and was proved to
have antioxidant and anti-peroxidases potential11
, as anti inflammatory12
,
MATERIALS AND METHODS
Drugs and Chemicals – Atorvastatin, Methanol, diethyl ether, sodium cholate, phenol
cholesterol, cholesterol oxidase, peroxidase, 4-aminoantipyrine, cholesterol standard, triglyceride
standard, sodium phosphate buffer, hydrogen peroxide, triglyceride enzyme mixture, HDL
cholesterol mixture, accelerator, ascorbate oxidase, PEG 6000, stabilizer.
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3. Plant Collection, Authentification and Extraction - Bark of Calycophyllum spruceanum, used
for the present study is collected from local area identified and authenticated by Madhavshetty,
Assistant professor, Department of Botany in Sri Venkateshwara University at Tirupathi.
Specimen sample of the plant was preserved in the herbarium section of Tirupathi bearing a
voucher No. 2101 belonging to the family Rubiaceae.
Extraction Process- The desired method of extraction process is the soxhlet extraction method.
Bark of Calycophyllum spruceanum is collected. It is cleaned and washed using distilled water
and is dried under shade. The bark is then ground into fine powder and is sieved. Around
100gms of the powdered bark is kept for soxhlation using 1000ml of methanol as solvent. The
extraction process is continued until the solvent in the soxhlet turns pale or colorless. The filtrate
is concentrated and the extract is used. For further use it is stored in air tight container at 4°c in
refrigerator10
.
Animals –Male Albino rats weighing from 300gms –to 360gms were used. The study was
approved by Institutional Animal Ethics Committee with proposal no.
1488/PO/Re/S/11/CPCSEA/06/2018. Animals were acclimatized to the laboratory conditions
prior to the experiment,given with normal laboratory diet and water ad libitum.
Experimental Procedure for Antihyperlipidemic activity
Induction of hyperlipidemia – Hyperlipidemia is induced using High Fat Diet.
Table 1: Composition of High Fat Diet
S.no. Ingredients Amount (gm/kg)
1 Milk powder 100
2 Refined flour 610
3 Sugar 50
4 Butter 16
5 Salts 14
6 Vitamins 20
7 Fibres 10
8 Cholesterol 10
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4. Grouping of animals
The animals are divided into six groups under which each group contains five animals. They are
divided in such a way that Group1 are given with normal saline(control), Group 2(negative
control) are left untreated, Group 3 are treated with the (Standard) Atorvastatin10mg/kg and
Group 4,5 and 6 (T1,T2,T3) are treated with 125, 250 and 500mg/kg doses of methanolic extract
of calycophyllum spruceanum respectively.All the animals are fed with the high fat diet for 28
days.From 14th
day onwards group1 animals are given with normal vehicle, Doses are given
based on the body weight of the rat through oral route, using oral gavage.On the 28th
day blood
samples are collected through retro orbital sinus puncture and they are examined for serum lipid
levels13
.
Parameters estimated are total cholesterol, triglycerides (GPO-PAP method)14
, HDL (High
density lipoproteins)1
, LDL, VLDL.
Experimental procedure for Nootropic activity
Acclimatization to Cook’s Pole Climbing Apparatus14
Cook’s pole climbing apparatus is one of the majorly used instruments involved in evaluating
neurological activity of the animal. In order to find out the cognitive functioning, the animal is
subjected to conditioned avoidance response. It is a learning concept where the animal is trained
to climb up the pole in order to escape from the shock. For that each rat is allowed to get
acclimatized for 2min to the cook’s pole climbing apparatus.Then the animals are exposed to a
buzzer noise.After 5sec of buzzer the animals are subjected to mild shock.The animals now try to
escape from shock by climbing the pole.As soon as rat climbs the pole, buzzer and shock were
switched off.At least 10 trials at 1 min gap for 10 days are performed.The rats avoiding the shock
in all 10 trials are considered to develop CAR and they are used for further process.
Grouping of animals
The animals which developed CAR are divided into six groups under which each group contains
five animals. Animals are then induced with scopolamine (antimuscarinic agent which inhibits
the cholinergic transmission) 0.5mg/kg, through intraperitoneal route to produce amnesia, where
the memory is partially lost. Group1 are given normal saline, Group 2 are left untreated, Group 3
are treated with the standard piracetam and Group 4,5 and 6 are treated with 125, 250 and
500mg/kg of methanolic extract of calycophyllum spruceanum respectively.
Now the animals are again placed in the apparatus, buzzer is given with shock 10 sec later. The
animals which have high CAR jumped onto the pole immediately after the buzzer before shock.
In addition to this the other two parameters unconditional response and no response are
calculated. Unconditional response is the action of the animal to climb up the pole after giving
the shock. Animals which show no action even after giving shock are considered to show no
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5. response. Trials are performed one hour later and at 24 hours after inducing scopolamine.
Sessions are continued for 8 days and on 9th
day the mean values of the CAR are calculated15
.
STATISTICAL ANALYSIS
Values are expressed as Mean ± SD and results are analyzed by one way ANOVA followed by
Dunnet’s multiple comparison tests to find out the significance. ***p < 0.001, **p< 0.01 and *p
<0.5.
RESULTS
PHYTOCHEMICAL CONSTITUENTS
Table No.2
PHYTO CHEMICAL CONSTITUENTS METHANOLIC EXTRACT of CP
Carbohydrates -ve
starch -ve
Proteins and amino acids -ve
Phenolic compounds and tannins +ve
Phytosterols +ve
Alkaloids +ve
Fixed oils +ve
Glycosides +ve
Flavonoids +ve
Saponins +ve
Coumarins +ve
(-) indicates absence (+) indicates presence
Table No.3 – Body weights of the rats before dosing and after dosing
GROUP WEIGHT GAIN
DAY 0 DAY 14 DAY 28
Control 340 ± 3.10 350 ± 3.36 350 ± 3.12
Negative control 340 ± 3.32 350 ± 3.35 360 ± 5.22
Standard 360 ± 8.00 370 ± 3.33 320 ± 5.00
T1 300 ± 7.00 320 ± 3.00 290 ± 1.23
T2 340 ± 3.00 360 ± 5.26 340 ± 2.56
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6. T3 360 ± 7.15 390 ± 6.23 375 ± 2.33
Table No. 4 Estimation of Lipid profile
TREATMENT
GROUPS
Cholesterol TG HDL LDL VLDL
Control
67.4± 9.94 66.7 ± 6.27 39.8 ± 7.16 19.2 ± 4.32 13.4 ± 2.41
Negative control
114 ± 14.5 269 ± 25.5 19 ± 2.92 26.2 ± 5.76 31.6 ± 6.29
Standard
82.8 ± 4.32***
119 ±
9.55***
28.2 ± 3.11**
17.2 ± 1.64*
21.0 ±
2.35***
T1
102 ± 7.54 243 ± 12.5*
20.06 ±
3.65*
21.5 ± 6.74* 25.8 ±
2.59*
T2
94.8 ± 10.6*
188 ±
5.50**
27.2 ± 3.03*
17.8 ± 2.28*
23.9 ±
1.88**
T3
81.8 ± 4.32***
115 ±
9.68***
29 ± 2.24**
14.6 ± 4.62**
19.6 ±
1.14***
Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by
Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, total cholesterol
level is compared with negative control
Table No. 5 - CAR responses after 4hrs of drugs inducing
TREATMENT
MEAN ± SD
CAR UR NR
Control 26.70 ± 0.60 0.17 ± 0.33 0.00 ± 0.00
Negative control 4.0 ± 2.01 10.20 ± 2.00 18.60 ± 4.82
Standard 16.0 ± 3.2** 9.8 ± 4.0* 3.9 ± 0.2*
T 1 23.00 ± 2.00* 4.03 ± 1.20 0.50 ± 0.20
T 2 25.00 ± 1.26** 4.68 ± 2.00* 1.56 ± 0.1*
T 3 26 ± 5*** 5 ± 2* 2 ± 1*
Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by
Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, total cholesterol
level is compared with negative control
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7. Table No. 6 - CAR responses after 24hrs of drugs inducing
TREATMENT
MEAN ± SD
CAR UR NR
Control 27.80 ± 0.26 0.19 ± 3.56 0.00 ± 0.00
Negative control 1.7 ± 3.2 7.6 ± 1.8 15.0 ± 3.3
Standard 23.5 ± 1.6 4.8 ± 2.0 1.8 ± 1.0
T 1 24.60 ± 2.10 1.89 ± 0.20 0.10 ± 0.45
T 2 26.0 ± 2.7 2.1 ± 1.3 0.2 ± 0.9
T 3 28.6 ± 2.6 3.2 ± 1.3 0.4 ± 0.7
Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by
Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, total cholesterol
level is compared with negative control
Discussion
Hyperlipidemia is a metabolic disorder characterized by fluctuated blood lipid levels mostly
elevated levels of lipids like cholesterol, triglycerides or elevated levels of lipoproteins like LDL,
VLDL with concomitant decrease in HDL levels.High Density Lipoproteins are considered as
good lipoproteins where they carry the lipids back to liver which get excreted in bile, leading to
no accumulation of lipids and fats. VLDL carries triglycerides which are used by body cells in
the form of energy. Remaining VLDL is converted into LDL. LDL carries cholesterol all over
the body used for synthesizing many substances. High amounts of cholesterol leads to plaque
formation. Increased levels of lipids lead to many disorders which is life threatening and so
hyperlipidemia is considered as a major risk factor for several diseases.
Learning and memory are considered as psychological process, where learning is
ability to acquire new information and memory is a process by which the learnt information is
stored and Cognition means processing of information. For information to be passed, stored and
memorized several neurotransmitters are involved like Glutamate, Acetyl choline, serotonin,
Dopamine. Depletion in these neurochemicals, excessive stimulation of these chemicals or
damage to the nerve cells leads to CNS disorders. Cognitive impairment occurs through many
disorders like delirium, dementia or amnesia or less oxygen supply to brain. Alteration in
neurochemicals or increase in oxygen supply to brain leads to cognitive enhancement.
Flavonoids, alkaloids, triterpinoids, saponins, phenols, monoterpene, xanthenes and
isothiocyanate are the chemical constituents responsible for this activity [70]
.
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8. In order to find out the medicine with less adverse effects following experiment is
done to find out the antihyperlipidemic activity and nootropic activity. Calycophyllum
spruceanum is an Amazonian tree where Phytochemical screening was done and the results are
there in Table no 2. Hyperlipidemia was induced using high fat dietfor 28 days, where dosing
starts from day 14 to day 28. Methanolic extract of Calycophyllum spruceanum bark (MECSB)
is dissolved in distilled water and is given to the animals for 14 days through oral route. The
amount of the extract given is based on the body weight of the animal. On 28th
day blood
samples are collected from retro orbital plexus by anesthetizing the animal using di ethyl ether to
estimate blood lipid levels and the results are compared with the standard statin atorvastatin.High
fat diet increased the lipid levels in the animals. The elevated blood lipid levels are estimated by
calculating the total cholesterol, triglycerides, HDL, LDL and VLDL.On administration of the
extract there is a significant decrease in the lipid levels.From table 3 it can be explained that
atorvastatin (as known) have significant effect on decreasing the cholesterol, Triglycerides, LDL
and VLDL an da significant increase in HDL. High dose of MECSB is more significant in
decreasing LDL levels.
To find out the nootropic activity animals werepretrained with Cook’s pole
climbing apparatus. Scopolamine was used as an inducing agent which blocks the action of
Acetylcholine, thereby decreasing cognitive function. The standard Nootropic drug piracetam is
used to compare the significance.At the same time MECSB when given to the animals induced
with scopolamine there is a significant increase in conditioned avoidance response (CAR). In
table 3 there is a significant increase in CAR at 4hrs, more than the standard. Effect of the extract
is more at high dose, also at 24hrs. Plants have flavonoids and these are the main class of
secondary metabolite. Flavanones,flavanols and anthocyanins are known to show significant
potential on cognitive abilities19-22
.
CONCLUSON
In the present investigation the Antihyperlipidemic and Nootropic activity of Calycophyllum
spruceanum were screened. High fat diet is induced to male albino rats were divided into groups
to perform Antihyperlipidemic activity. High fat diet is given for 28 days all through the
investigation. Dosing starts from day 14 and is continued up to 28 days. Parameters like
cholesterol, triglycerides, HDL, LDL and VLDL are estimated and mean values are calculated
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9. where MECSB had shown significant decrease in cholesterol, triglycerides, LDL, VLDL with an
increase in HDL.
In order to find out the Nootropic activity, the animals are first trained on the Cook’s pole
climbing apparatus with 2min acclimatization, buzzer followed by shock not more than 10 sec.
Animals jump onto the pole immediately after the shock to escape from shock. 10 trials are made
on each animal with 1 min time interval. Animals which escape the shock in all 10 trials are
known to develop CAR. After scopolamine induction (0.5mg/kg) doses are given and trials are
made immediately after 4hrs and 24 hrs consecutively for 8 days and mean values are calculated
on 9th
day. MECSB has shown significant increase in CAR more than standard without any side
effects.
The present findings show presence of flavonoids and saponins has effect in lowering blood lipid
levels and presence of saponins has an effect in increasing learning and memory as reported.
Based on the statistical analysis it can be concluded that Calycophyllum spruceanum bark has
Antihyperlipidemic, Nootropic activity which can be used in treatment as it is safe and potent.
However further investigation has to be made in order to find out the exact chemical constituent
responsible for the effect and mechanism.
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