It will be useful for doing projects in pharmacology

1. NEUROPROTECTIVE ACTIVITY OF CUCURBITA PEPO AGAINST
DIAZEPAM INDUCED AMNESIA AND ETHANOL INDUCED COGNITIVE
IMPAIRMENT IN RODENTS
B. VARA LAKSHMI (REG. NO. Y16BPH203006)
M. VENUSREE (REG. NO. Y16BPH203048)
P.H.S. VENKATESWAR (REG. NO. Y16BPH203053)
P. JAHNAVI (REG. NO. Y16BPH203056)
T. VIJAYA LAKSHMI (REG. NO Y16BPH203080)
UNDER THE GUIDANCE OF
MRS.KAKUNURI .LAKSHMI M. PHARM.,
ASSISTANT PROFESSOR.

2. ABSTRACT
OBJECTIVE: - The objective of this study was to identify the potential of Cucurbita pepo as protective and
therapeutic agent against alzheimer’s disease.
METHODS:-The learning and memory enhancing activity of Cucurbita pepo seed extract were investigated in rats
by using the ethanol induced cognitive impairment and diazepam induced amnesia and its effects on learning and
memory were examined by using Morris water maze (MWM) test and 8-radial arm maze (8-RAM) test.
RESULTS: - For Morris water maze test all groups showed significantly (P value is < 0.01 & < 0.05) decreases
transfer latency time at all periods as compared to ethanol and diazepam inducing group & for radial arm maze test all
groups showed significantly (P value is< 0.0001& < 0.001) decreased in the time taken to reach the paired arm and
number of entries in baited arm and non-baited arm as compared to ethanol inducing group. Therefore seed extracts of
Cucurbita pepo exhibited significantly learning and memory activity in Alzeimer’s disease.
CONCLUSION: - The present study suggests Cucurbita pepo that modifies the oxidative stress and is involved in
the protective effect against oxidative damage and neurodegenerative disease in rat.
KEY WORDS:
Cucurbita pepo, learning and memory activity, morris water maze, radial arm maze and alzeimer’s disease.

3. INTRODUCTION
NEURODEGENERATIVE DISEASES (NDs):
NDs are characterized by the progressive and irreversible loss of neurons in selective regions of the brain. In afflicted
individuals, symptoms typically manifest as memory loss, anxiety, and depression, which evolve to severe motor
dysfunction, profound cognitive deterioration, and loss of independent function. The risk of developing a ND rises sharply
with age; the number of people with a neurodegenerative disease is low at younger ages, but the prevalence of people
suffering from neurodegeneration doubles every 5 years after age 65.3 AD, the most common form of NDs, currently affects
about 10 percent of the population over age 65 and 47 percent of adults aged over 85 years. NDs typically involve a slow
decline in human function that results in an eventual need for constant care and assistance with the most basic activities of
daily life, consequently generating a substantial social and financial burden.(1)
EXAMPLES OF NEURODEGENERATIVE DISEASES (NDS):
Alzheimer’s disease (AD),
Parkinson’s disease (PD),
Amyotrophic lateral sclerosis,
Huntington’s disease, and
Friedreich’s ataxia,
They are devastating age-related conditions that have become one of the primary public health concerns of recent decades. (1)

4. PLANT PROFILE
TAXONOMICAL CLASSIFICATION:
Botanical name: Cucurbita Pepo
Kingdom: Plantae
Subkingdom: Tracheobionta
Class: Magnoliopsida
Subclass: Dilleniidae
Division: Magnoliophyta
Superdivision: Spermatophyta
Order: violales
Family: Cucurbitaceae
Genus: Cucurbita
Species: Pepo

5. PLAN OF WORK
COLLECTION OF CUCURBITA PEPO
POWDERED
SUBECTED TO EXTRACTION WITH POLAR AND NON POLAR SOLVENTS USING SOXHELT APPARATUS
DRYING
PRELIMINARY PHYTOCHEMICAL ANALYSIS
LEARNING AND MEMORY ACTIVTY IN RATS WITH ALZHEIMER’S DISEASE BY CUCURBITA PEPO L. SEED EXTRACT
RESULTS AND DISCUSSION

6. LITERATURE REWEIW
Frederickb et.al, Chemical examination of pumpkin seed’ Chemical technology and analysis of oils, fats and waxes,
Vol11, 3rd edit., January 13, 1910.509.
R. pearlet.al, A study of the effects of Cucurbita pepo seeds on kidney excretion, Vol8, February4, 1922,192.
Carlskottsbergsgata et.al, Structure of Proto chlorophyll containing Plastids in the Inner Seed Coat of Pumpkin Seeds
{Cucurbita pepo)., 22, Goteborg,17 April, 1979.
D. C. Joshi et. Al, Physical properties of pumpkin seeds, C. Z. Nkosi et. al, Phytother, Res. 20, 25th April 2006,935-
940, J.agric,Engng Res. (1993) 54,19, November 1991,219-229.
C. Z. Nkosi et. al,Antioxidative Effects of Pumpkin Seed (Cucurbita pepo) Protein Isolate in CCl4- Induced Liver
Injury in Low-Protein Fed Rat, Phytother, Res. 20, 25th April 2006,935-940.
David G,et. al; Oil and Tocopherol Content and Composition of Pumpkin Seed Oil in 12 Cultivars,J. Agric, Food
Chem., Vol. 55,2007, No. 10.

7. AIM AND OBJECTIVES
AIM:
To evaluate the pharmacological screening of Cucurbita pepo seed extracts against neurodegenerative disorders in
rodents.
OBJECTIVES:
The objectives of present study are:
1. To carry out the polarity based Cucurbita pepo seed extraction using different solvents.
2. To carry out the preliminary phytochemical analysis of Cucurbita pepo seed extracts.
3. To screen the Cucurbita pepo seed extracts against ethanol- induced cognitive impairment and
diazepam induced amnesia (Morris water maze) and ethanol- induced cognitive impairment [8-
Arm radial maze (8-RAM)].

8. MATERIALS AND METHODS
DRUGS AND CHEMICALS:
For learning and memory activity in Alzheimer's disease:
Inducing agents -Ethanol - 60% (Ethanol was prepared as a 60% solution in distilled water and administered i.v at a
dose of 2.5 mg/ kg),
-Diazepam- was diluted in normal saline and administered i.v at a dose of (1 mg/kg).
-Ethanol- 20% (Ethanol was prepared as a 20% solution in sterile normal saline and administered subcutaneously at a
dose of 4.5 mg/ kg),
Standard drug- Donepezil hydrochloride (2.5 mg/kg, p.o)

9. FIGURE - 5: Selection of methods for learning and memory activity

10. MATERIALS AND METHODS
INVESTIGATION OF LEARNING AND MEMORY ACTIVITY IN ALZHEIMER’S MODEL BY USING
MORRIS WATER MAZE (MWM)
A. MORRIS WATER MAZE (MWM):
Evaluation parameters:
Transfer latency in sec

11. B. ETHANOL- INDUCED COGNITIVE IMPAIRMENT: (26)
Selection of dose and treatment period:
The learning and memory enhancing activity of the aqueous and ethanolic seed extracts of cucurbita pepo was
investigated using the ethanol- induced cognitive impairment [Ethanol (60%) is used to induce dementia like
condition in the dose 2.5 mg/kg administered i.p for 15 days). (25) The test animals were randomly chosen and divided
into four groups having five rats in each as follows:
Group I: Inducing Group- Ethanol (2.5 mg/kg was administered i.p for 15 days).
Group II: Standard Group -Donepezil hydrochloride(26) (2.5 mg/kg was administered orally for 15days) + Ethanol.
Group III: Test-I -Aqueous seed extract of cucurbita pepo [CPAE- 100mg/kg was administered orally for 15days) +
Ethanol.
Group IV: Test -II -Ethanolic seed extract of cucurbita pepo [CPEE- 100mg/kg was administered orally for 15 days)
+ Ethanol.

12. C DIAZEPAM INDUCED AMNESIA:
SELECTION OF DOSE AND TREATMENT PERIOD:
The learning and memory enhancing activity of the aqueous and Ethanolic seed extracts of cucurbita pepo was
investigated using the [Diazepam is used to induce amnesia like condition in the dose 1 mg/kg administered i.p for 8
days).(27) The test animals were randomly chosen and divided into four groups having five rats in each as follows:
Group I: Inducing Group-Diazepam (1mg/kg was administered i.p for 8 days).
Group II: Standard Group-Donepezil hydrochloride (26) (2.5 mg/kg was administered orally for 8days).
Group III: Test-I- Aqueous seed extract of cucurbita pepo[CPAE- 100mg/kg was administered orally for 8days).
Group IV: Test –II- Ethanolic seed extract of cucurbita pepo [CPEE- 100mg/kg was administered orally for 8 days).

13. INVESTIGATION OF LEARNING AND MEMORY ACTIVITY IN ALZHEIMER’S MODEL BY USING 8-
ARM RADIAL MAZE (8-ARM/8-RAM)

14. B. RADIAL ARM MAZE:
Evaluation parameters:
No. of entries in baited arms and non-baited arms.
Time taken to reach the paired arm.

15. C. ETHANOL- INDUCED COGNITIVE IMPAIRMENT: (26)
Selection of dose and treatment period:
The learning and memory enhancing activity of the aqueous and ethanolic seed extracts of cucurbita pepo was
investigated using the ethanol- induced cognitive impairment [Ethanol (20%) is used to induce dementia like
condition in the dose 4.5 mg/kg administered s.c for 21 days) (26).The test animals were randomly chosen and divided
into four groups having five rats in each as follows:
Group I: Inducing Group-Ethanol (4.5 mg/kg was administered subcutaneously for 21 days).
Group II: Standard Group -Donepezil hydrochloride (26) (2.5 mg/kg was administered orally for 21 days) + Ethanol.
Group III: Test-I -Aqueous seed extract of cucurbita pepo[CPAE- 100mg/kg was administered orally for 21 days) +
Ethanol.
Group IV: Test -II -Ethanolic seed extract of cucurbita pepo [CPEE- 100mg/kg was administered orally for 21 days)
+ Ethanol.
All the treatment group animals received respective control, standard and test treatment 30 minutes prior to the
ethanol administration for 21 days of experimental period.

16. STATISTICAL ANALYSIS:
The values are expressed as mean± SEM. The statistical analysis was performed using one way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test. Comparisons were made between haloperidol group and
test/standard groups. P-values <0.05 was considered statistically significant. The statistical analysis was done by using
Graph pad prism version no: 6.0.

17. RESULTS AND DISCUSSION
Phytochemical
constituents
Aqueous seed extract of
Cucurbita pepo
Ethanolic seed extract of Cucurbita
pepo
Alkaloids - -
Carbohydrates + +
Flavonoids + +
Phenols + +
Saponins + +
Terpenoids - -
Sterols + +
Tannins + +
Proteins + +
Amino acids - -
Glycosides - -
Fixed oils and fatty
acids
+ +
Phytochemical screening of CPAE & CPEE

18. EFFECT OF SEED EXTRACTS OF Cucurbita pepo ON BEHAVIOURAL PARAMETERS i.e. MWM:
Animals treated with ethanol [2.5 mg/kg] alone for15 days showed a increase in transfer latency in seconds on 1st,7th& 15th days as well as
diazepam [1mg/kg] alone for 8 days showed a increase in transfer latency in seconds on 8th day & after 24 hrs i.e. 9th days.
TABLE NO.3: Effect of seed extracts of Cucurbita pepo on transfer latency (ethanol- induced cognitive impairment)
S.N
o.
Group Treatment
Transfer latency (In seconds)
1st
DAY 7th
DAY 15th
DAY
1. I Ethanol
24.2±2.3
5
27.6±3.89 27.8±4.04
2. II
Standard+eth
anol
5.6±1.46 4.6±1.20 2.6±0.67
3. III
CPAE+
ethanol
9.2±0.58 9.2±2.31 4.8±0.86
4. IV
CPEE+
ethanol
5.8±1.62 4.4±1.54 2.6±0.75

19. FIGURE-8: Effect of seed extracts of Cucurbita pepo on ethanol- induced cognitive impairment. Values are
expressed as Mean ± SEM, P < 0.01 vs. control (n = 5 animals).

20. TABLE NO.4: Effect of seed extracts of Cucurbita pepo on transfer latency (diazepam induced amnesia)
S.N
o.
Group Treatment
Transfer latency (In
seconds)
8TH
DAY
After 24 hours
(i.e.9TH
DAY)
1. I Diazepam 26.2±3.89 23±3.38
2. II
Standard+
diazepam
4.4±0.81 2.0±0.32
3. III
CPAE+
diazepam
9.6±2.40 6.6±1.12
4. IV
CPEE+
diazepam
5.6±0.40 2.2±0.80

21. FIGURE-9: Effect of seed extracts of Cucurbita pepo on diazepam induced amnesia. Values are expressed as Mean ±
SEM, P < 0.007 vs. control (n = 5 animals).

22. EFFECT OF SEED EXTRACTS OF Cucurbita pepo ON BEHAVIOURAL PARAMETERS i.e. 8-RAM:
Animals treated with ethanol [4.5 mg/kg] alone for 21 days showed an increase in time taken to reach paired arm & number
of entries in baited arms and non-baited arms in 1st,7th , 15th & 21 st days.
TABLE NO.5: Effect of seed extracts of Cucurbita pepo on time taken to reach paired arm (ethanol- induced cognitive
impairment)
S.N
o.
Gro
up
Treatment Time Taken to reach Paired arm (Sec)
1st
day 7th
day 14th
day 21st
day
1 I Ethanol
144.4±3.
66
134.2±3.
69
120.2±2
.65
103.2±2.98
2 II
Standard+eth
anol
76.2±0.9
6
66.6±1.9
8
57.8±0.
86
48.4±2.08
3 III
CPAE+
ethanol
121.2±5.
58
112.4±5.
97
98.2±2.
47
92.0±3.33
4 IV
CPEE+
ethanol
99.0±1.3
0
90.0±3.7
0
83.2±4.
54
69.2±2.95

23. FIGURE-10: Effect of seed extracts of Cucurbita pepo on Time taken to reach Paired arm. Values are expressed as
Mean ± SEM, P< 0.0001 vs. control (n = 5 animals).

24. TABLE NO.6: Effect of seed extracts of Cucurbita pepo on number of entries in baited arms and non-baited arms
(ethanol- induced cognitive impairment)
Gro
up
Treatment
Number of entries in baited arms and non-baited arms
B.A
N.B.
A
B.A
N.B.
A
B.A
N.B.
A
B.A N.B.A
I Ethanol
1.4±0.2
4
1.2±0.2
0
1.2±0.2
0
1.6±0.2
4
1.0±0.31 1.4±0.4
0.6±0.2
4
0.6±0.24
II
Standard+eth
anol
3.8±0.3
7
3.4±0.2
4
3.2±0.3
7
2.2±0.5
8
5.8±0.37
8.8±0.3
7
9.4±0.5
0
11.8±1.9
3
III
CPAE+
ethanol
2.2±0.3
7
2.4±0.4
0
2.6±0.2
4
4.4±0.5
0
5.4±0.50
6.8±0.8
0
5.8±1.0
2
6.2±0.96
IV
CPEE+
ethanol
6.4±0.5
0
7.0±1.3
0
7.0±1.3
0
11.0±0.
89
10.4±0.9
2
9.0±1.0
4
8.8±1.1
6 c

25. FIGURE-18: Effect of seed extracts of Cucurbita pepo on number of entries in baited arms and non-baited arms. Values are
expressed as Mean ± SEM, P < 0.0001 vs. control (n = 5 animals).

26. SUMMARY AND CONCLUSION
In the present investigation, Cucurbita pepo posses the presence of carbohydrates, tannins, flavonoids, phenols, sterols and
proteins
Cucurbita pepo showed cholinesterase inhibitor mechanism at an effective dose of 100 mg/kg against ethanol- induced
cognitive impairment & diazepam induced amnesia in rats.
Cucurbita pepo ethanolic extract showed comparatively significant effect exerted to standard drug donepezil hydrochloride
in the finding of transfer latency in sec (i.e. learning and memory activity). Transfer latency was recorded after administration
of ethanol & diazepam at different days and graphs were ploted according to the results obtained. This effect is attributed to
its ability to improve the levels of the acetylcholine that are decreased in the Alzheimer’s disease.
Cucurbita pepo showed cholinesterase inhibitor mechanism at an effective dose of 100 mg/kg against ethanol- induced
cognitive impairment.
Cucurbita pepo ethanolic extract showed comparatively significant effect exerted to standard drug donepezil hydrochloride
in the finding of time taken to reach paired arm (sec) & number of entries in baited arms and non-baited arms (i.e. learning
and memory activity). Time taken to reach paired arm (sec) & number of entries in baited arms and non-baited arms was
recorded after administration of ethanol at different days and graphs were ploted according to the results obtained.
Therefore finally suggests ethanolic extract of Cucurbita pepo show same effects exerted to the standard drugs.

27. REFERENCE
 Mukesh yadav,et. al; Medicinal and biological potential of pumpkin: An updated review; Nutrition Research Reviews (2010), 23, 184–190.
 Mohamed Makni, et. al; Anti diabetic Effect of Flax and Pumpkin Seed Mixture Powder: Effect on Hyperlipidemia and Antioxidant Status In
Alloxan Diabetic Rats. Journal of Diabetes and Its Complications 25 (2011) 339–345.
 Sali Aliu*, et. al; Nutritive and Mineral Composition in a Collection of Cucurbita Pepo l Grown in Kosovo. Food and Nutrition Sciences, March
22nd, 2012, 2012,3, 634-638.
 Abuelgassim O, et. al; The Effect of Pumpkin (Cucurbita Pepo l) Seeds And l-Arginine Supplementation on Serum Lipid Concentrations in
Atherogenic Rats; 1 Afr J. Tradit Complement Altern Med. (2012) 131 9(1): 131-13.
 Elinge C. M.1*, et. al; Proximate, Mineral and Anti-nutrient Composition of Pumpkin (Cucurbita pepoL) Seeds Extract; International Journal of
Plant Research 2012, 2(5): 146-150.
 Agnieszka Nawirska-Olszań ska a et. al; Characteristics of antioxidant activity and composition ofpumpkin seed oils in 12 Cultivars; Food
Chemistry ,5 February 139 (2013) 155–161.
 Torobiova O. A.a, et. al; Analysis of the components of pumpkin seed oil in suppositories and the possibility of its use in pharmaceuticals. Journal
of chemical and pharmaceutical Research, 2014,6(5): 1106 -1116.
 Shahangir biswas, et. al; Cytotoxicity and Phytochemical Studies of Pumpkin Seed (Cucurbita Maxima Linn.) Extract. Journal of Biomedical and
Pharmaceutical Research 3 (2) 2014, 29-36.
 Young Hye Cho, et. al; Effect of Pumpkin Seed Oil on Hair Growth in Men with Androgenetic Alopecia: A Randomized, Double-Blind, Placebo-
Controlled Trial. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine, Volume 2014, 7pages.
 Raphael Kwiri, et. al; International Journal of Nutrition and Food Sciences 2014; 3(4): 279-283.
 Raphael Kwiri,et. al;Proximate composition of pumpkin gourd (Cucurbita pepo) seeds from Zimbabwe; International Journal of Nutrition and
Food Sciences 2014; 3(4): 279-283.

28. FOR WATCHING OUR PRESENTATION…..WE HOPE YOU ENJOYED IT.