Int. Journal of Applied Sciences and Engineering Research, Vol. 5, Issue 4, 2016
© 2016 by the authors – Licensee IJASER- Under Creative Commons License 3.0
Research article
www.ijaser.com
[email protected]
ISSN 2277 – 9442
Evaluation of biochemical and phytochemical parameters in
germinating and nongerminating seeds of Cucurbita maxima
Kiran Kumar M, Mounika S.J, Pola Sudhakar, Sandeep B.V
Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India.
DOI: 10.6088/ijaser.05034
Abstract: Generally, plants and their seeds are used as nutritional and medicinal sources. Due to secondary
metabolites such as alkaloids, flavonoids, tannins and saponins, plants acquire medicinal properties. The
current study was aimed to evaluate the proximate composition and phytochemicals in germinating and
nongerminating seeds of Cucurbita maxima. Cucurbita maxima is a member of family Cucurbitaceae. Fruits
of Cucurbita consumed as vegetables. Specific extraction and estimation methods were used for
quantification and the results were statistically analysed with students’ F- test. Germination of Cucurbita
maxima seeds occurred within 5 days. Nongerminating seeds contain highest content of reducing sugar
(5.43±0.14 mg/gm), nonreducing sugars (360±20 mg/gm), starch (375±70 mg/gm), lipid (512±0.2 mg/gm)
and calorific value (642 K.Cal/100gm) than germinating (267.3 K.Cal/100gm) seeds. Whereas superior
amounts of proteins (150.3±0.7 mg/gm), DNA (0.663±0.054 mg/gm) and phytochemicals were found in
germinating seeds. Due to high content of phytochemicals highest antioxidant potential was found in
germinating seeds. From the results obtained in this study Cucurbita maxima (pumpkin) plants seeds play
very important role in the fields of medicine and pharmaceutical field.
Keywords: Antioxidants, phytochemicals, proximate composition, secondary metabolites.
1. Introduction
Humanity has been depending on plants not only for food but also as medicine since time immemorial due
to the in evident of scientific research of medicinal properties of plants are due to the presence of bioactive
secondary metabolites such as alkaloids, flavonoids, tannins and saponins. Sofowora (1982) Therefore
plants have major role in drug development. According to the previous findings phytochemicals have been
scientifically moved to have anticancer, antimicrobial properties. In developing countries people choose
the plants which are available at low cost for their nutritional and medicinal requirements Okigbo et al.
(2009).
In the present study the pumpkin seeds were taken to evaluate the phytochemicals and proximate
composition, due to their health promoting and nutritional properties. Cucurbita maximum is a
Cucurbitaceae family member, locally it is called as pumpkin. It is a perennial, climbing herb with tendrils
and it has flexible succulent stem with trifoliate leaves. Acquaah (2004) Size, shape, weight, colour of the
fruit may differ. It has hard rind, with a thick edible flesh and numerous seeds. Robinson and Walters (1997)
Achu et al. (2005) found that protein, fat, carbohydrate content of pumpkin varies in the range 20-40%,
44-53% and 7-10% correspondingly with different bioclimatic regions in Cameroon. Younis et al. (2000)
reported that Cucurbita pepo seeds are rich source of oil, carbohydrates and vitamin E, with linoleic, oleic
and palmitic and stearic acids are the major constituents of the oil. Seeds of pumpkin are also a rich source
of macro elements such as magnesium, phosphorus and potassium and have moderate amount of
—————————————
*Corresponding author (e-mail: [email protected])
Received on March, 2016; Published on August, 2016
341
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
microelements like calcium, manganese, copper and zinc. Lazos (1986). In ancient folk medicine pumpkin
seeds were used to eradicate intestinal parasites. Dutta et al. (2006) due to the presence of secondary
metabolite Cucurbitacin. Chung and ko (1976). Pumpkin seeds have significant amounts of L-Tryptophan,
so they were used as a remedy for depression. Eagles (1990). Consumption of pumpkin seeds can prevent
formation of stones in kidney by reducing the substance which enhance stone formation in urine and
increasing the levels of substances that inhibit stone formation. Suphakarn et al. (1987); Sophipat et al.
(1993). Cucurbita seed oil prevents benign prostatic hyperplasia due to the presence of Linoleic and oleic
acids. Carbin and Eliasson (1989), Carbin et al. (1990), Koch (1995) Schiebel and Friedrich (1998). In
recent days researchers have great attention towards underutilized crops like pumpkin to increase the
available resources, and to eradicate the deaths of malnutrition and in the formulation of value added
products. Due to these reasons pumpkin considered as “poor man’s” food. Chweya and Eyzaguirre (1999).
2. Materials and Methods
2.1 Chemicals
Chemicals and reagents (analytical grade) used for biochemical and phytochemical analysis were
purchased from sigma Aldrich. The experiments were performed at room temperature otherwise stated.
2.2 Sample collection
Fresh seeds of Cucurbita maxima were collected from the fields of Amalapuram, East Godavari dist,
Andhra Pradesh, India. Healthy seeds were cleaned by washing under tap water and used for the evaluation
of phytochemicals and biochemical analysis.
2.3 Seed germination
Washed healthy Cucurbita seeds and Petriplates were surface sterilized by using 30% sodium hypochloride
to prevent fungal contamination. Filter papers also sterilized in an autoclave. After surface sterilization,
seeds were placed on the whatman No.1 filter paper contained in Petriplates. Distilled water added to the
seed containing trays to wet the filter paper. Seeds were incubated at 280 C for 5 days. Growth of 2 cm
radical was considered as germination Mahmoud (2008).
3. Biochemical and Nutritional value quantification
3.1 Extraction and estimation of reducing sugars
Reducing sugars were estimated by the method of Miller (1972). 0.1 gm germinating and non germinating
seeds of Cucurbita maxima were homogenised separately in 80% hot ethanol and centrifuged at 5000g for
15 min at room temperature. The supernatant was evaporated by keeping in water bath at 800C and sugars
were dissolved by adding 10 ml distilled water. 1 ml of solution from both the samples were taken in
separate tubes and 3 ml of DNSA reagent was added, then boiled for 5 min in a water bath. After boiling 1
ml of Rochelle salt solution was added. The tubes were cooled to room temperature and measured the
intensity of dark red colour at 530 nm and calculated the concentration of reducing sugars from glucose
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
342
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
standard graph.
3.2 Extraction and estimation of Nonreducing sugars
Hedge and Hofreiter (1962) method was used to estimate the Nonreducing sugars. Germinating and
Nongerminating cucurbita seeds (0.25 gm) were hydrolysed separately by keeping in water bath for 3 hrs,
with 2.5 N Hcl (5 ml) and was neutralised with Na2CO3 after cooled it to room temperature. 0.1 ml extracts
were taken in separate test tubes and makeup to volume 1 ml with distilled water and 4 ml of anthrone was
added. The tubes were kept for boiling for 5 min. colour intensity was measured at 630 nm. Non reducing
sugars concentration was calculated from glucose standard graph.
3.3 Extraction and estimation of starch
Thayumanavan and Sadasivam (1984) method was used to estimate the starch by using anthrone method.
250 mg of fresh germinating and Nongerminating seeds were homogenised separately in 80% hot ethanol
to remove sugars. Residue was retained after centrifugation at 5000 X g for 15 min. The starch was
extracted by 52% perchloric acid at 0o C for 20 min. 0.1 ml of extract from germinating and
nongerminating seed extracts was taken in separate test tubes and the final volume was adjusted to 1 ml
with distilled water and 4 ml of anthrone was added. The tubes were kept for boiling for 5 min. The colour
intensity was measured at 630 nm. Starch concentration was calculated from the glucose standard graph.
3.4 Extraction and estimation of total protein
Ferreira et al (2002) method was used to extract the total protein by using poly vinyl pyrrolidone (PVP).
0.5 gm of germinating and nongerminating seeds were homogenised separately in 50 mM sodium
phosphate buffer containing 10% insoluble PVP and incubated at 400 C for overnight. Then the
homogenates were centrifuged at 14000 rpm for 20 min at 40 C. The supernatant was stored at -200C and
used for protein quantification. The total protein concentration was estimated by using Lowry et al (1951)
method.
3.5 Extraction and estimation of total lipid
Bligh and Dyer (1959) method was used for the extraction of total lipids from the germinating and
nongerminating seeds of cucurbita. 1 gm seeds were homogenised separately with 3.75 ml methanol:
chloroform (2:1V/V) and 1 ml of 1 mM EDTA in 0.15 M acetic acid was added. Homogenate was
transferred to new glass tube and the homogenizer rinsed with 1.25 ml of chloroform and transferred to the
tube, finally 1.25 ml of 0.88% Kcl was added and centrifuged at 3000 rpm for 2 min. The lower phase was
transferred to new tube. The total lipid concentration was estimated by the method of Kinght et al. (1972)
by using phosphovanillin. Three test tubes were labelled as blank, standard and test. 0.1 ml extract was
taken respectively in 3 test tubes. 2 ml of concentrated H2SO4 was added to all the test tubes and boiled for
10 min. after cooled to room temperature. 10 ml phosphovanillin was added and incubated at 370C for 15
min. The optical density was measured at 540 nm. Concentration of lipid was calculated by the formula
given below
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
343
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
X 500
3.6 Extraction and Estimation of Total DNA
The germinating and non germinating seed extracts were prepared by 0.5gms of tissue placed in a mortar
and homogenized with 2ml of extraction buffer. The homogenate transferred to a 2ml eppendorf tube. An
equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to the tubes and mixed well by
gently shaking the tubes. The tubes centrifuged at room temperature for 15mins at 15000rpm. The upper
aqueous phase was collected in a new tube and adds equal volume of chloroform: isoamyl alcohol (24:1).
The upper aqueous phase obtained after centrifugation at room temperature for 10min at 15000rpm was
transferred to a new tube. DNA was precipitated from the solution by adding 0.1ml of 3M sodium acetate
(pH-7.0) and 0.7ml of iso propanol and allowed to incubate 15mins at room temperature. The tubes were
centrifuged at 40C for 15mins at 15000rpm. The DNA pellet was washed twice with 70% ethanol and then
very briefly with 100% ethanol and air dried. DNA was dissolved in TE buffer. To remove RNA 5µl of
DNase free RNase added to the DNA. Romana et al. (2014). DNA was quantified by diphenylamine test
Gendimenico (1988). 3ml of sample was taken in a test tube and 4ml of DPA was added and allowed to
incubate for20 min at 360C. Colour intensity was measured at 595 nm wavelength in spectrophotometer
and calculated the concentration of DNA from graph plotted using purified calf thymus DNA as a standard.
3.7 Estimation of nutritive value
After estimation of protein, fat and carbohydrate, the nutritive value was calculated by the following
formula. Nutritive value (Kcal per 100 g) = 4 (Protein %) + 9 (Fat %) + 4 (Carbohydrate %).
3.8 Phytochemical and antioxidant Activity Analysis
3.8.1 Methanolic extract preparation
Methanol was used as the solvent for the preparation of seed extracts Farrukh et al. (2006). 10 gm
germinating and nongerminating seeds were separately soaked in methanol for 10 days at room
temperature under dark conditions with stirring at regular intervals. The final extracts were filtered using
whatman No.1 filter paper. The filterate was concentrated under reduced pressure at 600C using rotary
evaporator and stored at 40C. Stock solution was prepared by the dissolving extracts in methanol. Stock
solution was used to estimate the phytochemicals.
3.8.2 Estimation of Alkaloids
Sreevidya and Mehrotra (2003) methodology was used to estimate the total alkaloid content. Standard
solution was prepared by dissolving 5mg of boldine and leaf extract separately in 5ml of warm distilled
water and pH was adjusted to 2-2.5 with 0.01 M Hcl and 2ml of DR (Drangendorff’s Reagent) was added,
formed orange precipitate was centrifuged at 5000 rpm for 15 min. Again DR was added to check complete
precipitation. 2 ml of sodium sulphide was added to the residue to form a brownish black precipitate,
centrifuged at 5000 rpm for 15 min. complete precipitation was checked by adding 1% sodium sulphide
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
344
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
again. The remaining residue was dissolved in 2 ml of HNO3 with warming and sonication and the final
volume makeup to 10 ml with distilled water. Finally 5 ml of thiourea was added to the above solution to
form a yellow bismuth complex. The absorbance was measured at 435 nm. The amount of bismuth in the
boldine solution or extract was measured from the calibration curve of bismuth nitrate. The results were
expressed as mg of boldine equivalents per gm of extract.
3.8.3 Estimation of Flavonoids
Total flavonoids content was estimated by the method of Bao et al. (2005). 1 ml distilled water and 75 µl of
5% Na2NO3 was added to the 1 ml of seed extract. 75 µl of 10% AlCl3.H2O and 0.5 ml of 1 M NaOH were
added with 5 min interval. The solution was thoroughly mixed and incubated for 15 min. The absorbance
was measured at 510nm. The total flavonoids content was calculated using standard quercetin calibration
curve. The results were expressed as mg of quercetin equivalents (QE) per gm of extract
3.8.4 Estimation of Phenolics
The amounts of total phenolics were determined according to the method of Javanmardi et al. (2003). For
200 µl of test sample, 1 ml of Folin ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) were added.
The test tubes were allowed to stand for 30 min. Absorbance measured at 765 nm. The total phenolics
content was expressed as mg of gallic acid equivalents per gm of extract.
3.8.5 Estimation of Tannins
The total tannins were estimated by the method of Folin ciocalteu (1927). 6.25 ml of water, 0.5ml of Folin
ciocalteu reagent and 1.5 ml of 20% sodium carbonate were added to the 1 ml of seed extract and the
solution was incubated for 1 hour. The absorbance of sample was measured at 725 nm. The results were
expressed as mg of tannic acid equivalents per gm of extract.
3.8.6 Estimation of total antioxidant activity
The total antioxidant potential assay was measured by the method of Prieto et al. (1999). Reagent solution
containing 0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate was added to the
methanolic extracts of germinating and nongerminating seeds and the solution was incubated at 950C for
90 min. The mixture was cooled to room temperature. The colour intensity of the solution was measured at
695 nm. The total antioxidant activity was expressed as ascorbic acid equivalents (AAE).
3.8.7 Statistical analysis
The results of in vitro studies were given as Mean ± Standard Deviation (SD) obtained from three
independent experiments, and analyzed with Student’s f-test for paired data and a ‘p’ value less than 0.05
was considered as significant difference in the analysis.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
345
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
4. Results and discussion
4.1 Seed germination
Germination of Cucurbita maxima seeds occurred within 5 days. The radical was 5-6 cm long and the
plumule 11-12 cm long, at this stage seeds were collected for biomolecular and phytochemical analysis.
Cucurbita maxima seeds easily grow under normal conditions than other seeds. Our findings agree with the
earlier reports. Subin and steffy (2013) reported that the roots and shoots of cucurbita maxima seeds grew
upto 12.08 ± 2.08 cm and 15.67±1.62 cm. Findings of Fazlali (2013) revealed that the naked seeds of
pumpkin show 66.22 mm root growth under normal conditions. Imbibition of water and utilization of
reserve food materials are the important physiological and biochemical changes during the seed
germination. Enzymes, hormones and ions have a key role in seed germination for catabolic and anabolic
process.
Figure 1: Germination of Cucurbita maxima seeds
4.2 Biomolecular analysis and nutritional value
Germinating and nongerminating seeds of Cucurbita maxima showed a large variation among the
concentrations of biomolecules. Nongerminating seeds show highest content of reducing sugars than
germinating seeds. Nongerminating seeds of C.maxima have 5.43±0.14 mg/gm of reducing sugars where
as germinating seeds have 4.97±0.18 mg/gm. Highest content of nonreducing sugars were also found in
nongerminated seeds (380±20 mg/gm) Where as germinated seeds shows only 230±40 mg/gm. Greater
amounts of starch was found in nongerminating seeds that is 375±20 mg/gm, germinated seeds have
225±20 mg/gm. protein content was rich in germinating seeds of C.maxima. They have 150.3±0.7 mg/gm
protein, nongerminated seeds have only 70±0.63 mg/gm. Germinated seeds of cucurbita maxima have
128.2±0.1 mg/gm of lipid, where as nongerminated seeds have 512.0.2mg/gm of lipid. Nongerminated
seeds have four fold rich lipid concentrations than germinating seeds. High concentrations of DNA were
found in germinating seeds. Germinated seeds have 0.663±0.054 mg/gm of DNA where as
Nongerminating seeds have high 0.495±0.006mg/gm of DNA. In biomolecular analysis, nongermiating
seeds of Cucurbita maxima have more amounts of carbohydrates (38.5 %), lipids (51.2 %) and less
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
346
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
amounts of proteins (7 %), DNA (0.05%) whereas germinated seeds have low content of carbohydrate
(23.5%), lipids (12.8%) and high content of protein (15%), DNA (0.063%). Our results show similarity
with the findings of earlier reports. Kim (2012) reported that Cucurbita maxima seed have 129.08±8.25
mg/gm carbohydrate, 274.85±10.04 mg/gm proteins and 524.34±1.32 mg/gm lipids. In these results lipid
content is almost similar with our findings but protein content was low and carbohydrates increases
slightly in our results. Amoo (2004) reported that the crude protein, carbohydrate and fat content of
Cucurbita maxima are 14.31±0.01%, 24.0.12% and 52.13±0.13% respectively. These values are nearly
equal to our findings in nongerminating seeds. Our findings also show similarity with the reports of Amoo
and Moza (1999) in Bauchinia rasemosa seeds.
The amounts of fats and carbohydrate content of Cucurbita maxima obtained in this work are close
agreement with the report (38.92% fat and 24.30% carbohydrates) of Ojiako and Lgwe (2007). Karaye
(2013) reported that Cucurbita maxima have 28.68% which is nearer to our findings. Silou et al. (1999)
was observed the protein content varies from 13% (C.lanatus) to 34% (C.pepo) in the seeds of Cucurbita
from and 16.9% of protein found in the seeds of L.siceraria from niger. Mercy (2005) reported that the
protein and lipid content of Cucurbita maxima was 32.98 and 51.65 g/100gm dry weight. The above
findings show similarity with our observations. Our protein results also show similarity with the findings
of Kershaw and Hackett (1987) for oil seeds such as cotton (6.46%), peanuts (4.58%), palm kernel (5.31%),
sesame (4.60%), soya bean (11.07%) and sun flower (6.58%). Fokou et al. (2004) studies revealed that the
seeds are good source of proteins (C.mannii, 41.6%) and lipids (C.sativus, 57.34%). Kinkela and Bezard
(1993) stated that the lipid content of C.pepo and L.siceraria are 50.81 and 50.08% respectively, which are
very near to our findings in nongerminated seeds of Cucurbita maxima.
Our results were also similar to Murkovic et al. (1996) observed C.pepo seeds have 41.8 to 54.9% oil
content. Idouraine et al. (1996) also showed that the oil content of C.pepo is 34.5 to 43.6%. kinkela (1990)
stated that due to greater levels of oil content, Cucurbita seeds were considered as good sources of
vegetable oils. Significant variation was found in calorific value between germinating and Nongerminating
seeds of Cucurbita maxima. Nongerminated seeds have high calorific value (642 K.Cal/100 gm) than
nongerminating seeds (267.3 K.Cal/ 100 gm).Carbohydrates and lipids are the high energy yielding
compounds. Due to high content of carbohydrates and lipids nongerminating seeds of Cucurbita maxima
have high calorific value. Germinating seeds are rich in proteins and deficient with carbohydrates and lipid
hence they have low calorific value than germinating seeds.
Figure 2: Proximate composition of germinating and nongerminating seeds of Cucurbita maxima
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
347
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
Figure 3: Nutritive value of germinating and nongerminating seeds of Cucurbita maxima
4.3 Phytochemical and total antioxidant potential assay
Germinating seeds of Cucurbita maxima shows highest concentrations of alkaloids (180±0.05mg/gm) than
nongerminating seeds (60±0.08 mg/gm). Greater concentrations of flavonoids (0.412±0.00 mg/gm) were
found in germinating seeds. Nongermiating seeds have 0.34±0.03 mg/gm of flavonoids. More amounts of
phenolics were observed in germinating seeds (0.3±0.02 mg/gm) where as nongerminating seeds have
0.183±0.02 mg/gm. Tannins were observed as 1.95±0.14 mg/gm in germinating seeds and nongerminating
seeds have 0.34±0.03 mg/gm of tannins. Highest Antioxidant potential (0.53±0.09 mg/gm) was found in
germinating seeds of Cucurbita maxima. Nongerminating seeds have 0.43±0.08 mg/gm antioxidant
potential.
The results of phytochemical analysis of Cucurbita maxima seeds showed predictable presence of
alkaloids, flavonoids, phenols and tannins which may be responsible for many pharmacological actions.
Among the phytochemicals, alkaloids were found in large concentrations. Alkaloids and their derivatives
are used as therapeutic agents. Oseni and Okoye (2013) reported that the Citrullus lanatus seed has
33.7±0.4% alkaloid content and 0.16 mg/ml of phenol content. Our results show lower concentrations of
phytochemicals than Citullus lanatus. Marles and Farnsworth (1995) reported that the phytochemical and
pharcological studies with different solvent extracts of Cucurbita maxima contain significant amounts of
carbohydrates, flavonoids, tannins, phenolics and Saponins. Okaka et al. (1992) stated that alkaloids affect
glucagons and thyroid stimulating hormones. The quantitative estimation of flavonoids revealed that the
concentrations of flavonoids are slightly increases in germinating seeds of Cucurbita maxima. Hour (1980)
stated that flavonoids are involved in protection against allergies, inflammation, free radicals, microbes,
viruses and tumours. Flavonoids are water soluble antioxidant and free radical scavengers which prevent
oxidative cell damage and have anti cancer activity Sodipo (2000). Phenolics have reactive oxygen
scavenging ability due to their electron donating capability. Phenolic compounds and their antioxidant
ability play a vital role in disease prevention in plants and animals such as Alzheimer, Parkinson and cancer.
From the preliminary phytochemical studies of cucurbita maxima seeds extracts the total phenolic content
shows large variation between germinating and Nongerminating seeds. Germinating seeds have superior
amounts of phenolic compounds. Tannins have antiviral Lu, Liu et al. (2004), antibacterial Akiyama et al.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
348
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
(2001) and antiparasitic effects Kolodziej et al. (2005). Souza et al. (2007) discovered that the tannins
isolated from the bark have anti inflammatory and anti ulcer activity in rodents and strong antioxidant
activity. All the above results were in accordance with the previous reports of Geetha and Geetha (2014)
Due to the large quantity of phytochemicals in germinating seeds, they show greater antioxidant potentials
than nongerminating seeds. At the time of germination stored food materials catabolised and produce
secondary metabolites hence phytochemicals are rich in germinating seeds. Habibur (2013) studies
revealed that methanolic extracts of Citrullus lanatus seeds showed maximum antioxidant potential.
Table 1: Phytochemical concentration in germinating and nongerminating seeds of Cucurbita
maxima
S.No
Parameter
Germinating seeds
Nongerminating seeds
1.
Alkaloids
180±0.05
60±0.08
2.
Flavonoids
0.412±0.00
0.34±0.03
3.
Phenolics
0.3±0.02
0.183±0.02
4.
Tannins
1.95±0.14
0.34±0.03
All Values represents the mean ± SD of triplicates
P<0.05 was considered as significant difference
Figure 4: Total antioxidant potential in germinating and nongerminating seeds of Cucurbita maxima
5. Conclusion
From the results of this study Cucurbita maxima seeds were considered as rich sources of fats,
carbohydrates and proteins. They could decrease the problem of malnutrition and phytochemical study
revealed that pumpkin seeds were rich in alkaloids, which play an important role in medicine to cure
diseases. Due to potentials of these species, their cultivation should be encouraged.
Acknowledgments
The authors acknowledge the facilities made available by the Department of Biotechnology, Andhra
University, Visakhapatnam and University Grants Commission, Govt. of India for The financial support.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
349
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
6. References
1. Acquaah, G. 2014. Horticulture, principles and practices, 3rd edition. Prentice hall, Upper saddle
River, NJ.
2. Akiyama, H., Kazuyasu, F., Yamasaki, O., Oono, T., Iwatsuki, K. 2001. Antibacterial action of
several tannins against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy, 48(48),
487-491.
3. Amoo, I.A., Eleyinmi, A.F., Ilelaboye, N.O.A., Akoja, S.S. 2004. Characterisation of oil extracted
from gourd (Cucurbita maxima) seed. Journal of Food, Agriculture and Environment, 2(2), 38- 39.
4. Amoo, I.A. and Moza, L. 1999. Extraction and Physicochemical properties of oil from Bauhinia
racemoza seeds. Riv. Ital.Sostanze Grasse, 76, 399-400.
5. Bao,J., Cay, Y., Sun, M., Warg, G. and Corke, H. 2005. Anthocyanins, flavonol and free radical
scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and
stability. Journal of Agricultural and Food Chemistry, 53(6), 2327-2332.
6. Bligh,E.G., Dyer, W.J. 1959. A Rapid method of total lipid extraction and purification. Canadian
Journal of Biochemistry and Physiology, 37(8),911-917.
7. Carbin, B.E., Eliasson, R. 1989.Treatment by Cucurbicin in benign prostatic hyperplasia. Swed J
Biol Med, 2, 7–9.
8. Carbin, B.E., Larsson, B., Lindahl, O. 1990. Treatment of benign prostatic hyperplasia with
phytosterols. Br J Urol, 66, 639–641.
9. Chung, W.C., Ko, B.C. 1976. Treatment of Taenia saginata infection with mixture of areca nuts
and pumpkin seeds. Chung Hua Min Kuo Wei Sheng Wu Hsueh Tsa Chih, 9, 31–35.
10. Chweya, J.A. and Eyzaguirre eds, P.B. 1999. The Biodiversity of Traditional Leafy Vegetables.
International Plant Genetic Resources Institute (IPGRI), Rome.
11. Dutta, K., Schuur, E.A.G., Neff, J.C. and Zimov, S.A. 2006. Potential carbon release from
permafrost soils of Northeastern Siberia, Glob. Change Biol, 12,(12), 2336–2351.
12. Eagles, J.M., Gibson, I., Bremner, M.H., Clunie, F., Ebmeier, K.P. and Smith, N.C. 1990. Obstetric
complications in DSM- III schizophrenics and their sublings. Lancet 335, 1139-1141.
13. Farrukh, A., Ahmad, I., Mehmood, Z. 2006. Antioxidant and Free Radical Scavenging Properties
of Twelve Traditionally Used Indian Medicinal Plants”. Turk J Biol, 30, 177-183.
14. Fazlali, R., Asli, D.E. and Moradi, P. 2013. The effect of seed priming by ascorbic acid on
bioactive compounds of naked Seed pumpkin (Cucurbita pepovar. styriaca) under salinity stress.
Intl J Farm & Alli Sci, 2(17), 587-590.
15. Ferreira, R.R., Fornazier, R.F., Vitoria, A.P., Lea, P.J and Azevedo1, R.A. 2002. Changes in
antioxidant enzyme activities in soybean under cadmium stress. J. Plant Nutr, 25(2), 327-342.
16. Fokou, E., Achu, M.B., Tchouanguep, M.F. 2004. Preliminary Nutritional Evaluation of Five
Species of Egusi Seeds in Cameroon. Afr. J. Food Agric. Nutr. Develop. (AJFAND), 4(1), 1-11.
17. Folin, C. and Ciocalteu, V. 1927. Tyrosine and tryptophan determination in protein. J. Biol.Chem,
73, 627- 650.
18. Geetha, T.S. and Geetha, N. 2014. Phytochemical Screening, Quantitative Analysis of Primary and
Secondary Metabolites of Cymbopogan citratus (DC) stapf. leaves from Kodaikanal hills,
Tamilnadu. Int.J.PharmTech Res, 6(2), 521-529.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
350
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
19. Gendimenico, G.J., Bouquin, P.L., Tramposch, K.M. 1988. Diphenylamine-colorimetric method
for DNA assay, A shortened procedure by incubating samples at 50°C. Analytical Biochemistry,
173, 45-4.
20. Habibur, R., Manjula, K., Anoosha, T., Nagaveni, K., Chinna, E. M., Dipankar, B. 2013. In-Vitro
Anti-Oxidant Activity of Citrullus lanatus Seed Extracts. Asian J Pharm Clin Res, 6(3), 152-157.
21. Hedge, J.E. and Hofreiter, B.T. 1962. In, Methods in Carbohydrate Chemistry. 17, (Eds.,) Whistler
RL and BeMiller JN. Academic Press, New York, p. 420.
22. Hour, S.S., Ahmed, E.M. and Carter, R.D. 1980. Concentration of watermelon juice. J. Food Sci,
45, 718-719.
23. Javanmardi, J., Stushnoff, C., locke, E. and Vivanco, J.M. 2003. Antioxidant activity and total
phenolic content of Iranian Ocimum accessions. Food Chem, 83, 547-550.
24. Karaye, I.U., Aliero, A.A., Muhamma, S. and Bilbis, S. 2012. Comparative evaluation of amino
acid composition and volatile organic compounds of selected Nigerian cucurbit seeds. Pakistan
Journal of Nutrition, 11(12), 1161–1165.
25. Kershaw, S.J., Hackett, F. 1987. Comparison of Three Standard Solvent Extraction Procedures for
the Determination of Oil Content in Commercial Oilseed Samples. J. Sci. Food Agric, 40(3),
233-244.
26. Kim, M.Y., Kim, E.J., Kim, Y.N., Choi, C., Le, B.H. 2012. Comparison of the chemical
compositions and nutritive values of various pumpkin (Cucurbitaceae) species and parts, Nutrition
Research and Practice, 6(1), 21-27.
27. Kinkela, T., Bezard, J. 1993. Lipids in some Congolese Foods. Sciences des Aliments, 13 (3),
567-575.
28. Kinkela, T. 1990. Utilisation des cereales et des oleagineux locaux au Congo. La Poste recolte en
afrique. Actes des seminaire international tenue a Abidjan, Cote d’Ivoire. AUPELF- UREF. p. 203.
29. Knight, J.A., Anderson, S., Rawle, J.M. 1972. Chemical basis of the sulfophospho vanillin
reaction for estimating total serum lipids. Clin. Chem, 18(3), 199-202.
30. Koch, E. 1995. Pharmacology and modes of action of extracts of Palmetto fruits (Sabal Fructus),
stinging nettle roots (Urticae Radix) and pumpkin seed (Cucurbitae Peponis Semen) in the
treatment of benign prostatic hyperplasia. In, Loew D, Rietbrock N,eds. Phytopharmaka in
Forschung und klinischer Anwendung. Darmstadt, Verlag Dietrich Steinkopf. p. 57–79.
31. Kolodziej, H., Kiderlen, A.F. 2005. Antileishmanial activity and immune modulatory effects of
tannins and related compounds on Leishmania parasitised RAW 264.7 cells. Phytochemistry,
66(17), 2056–2071.
32. Lazos, E.S. 1986. Nutritional, Fatty Acid and Oil Characteristics of pumpkin and Melon seeds.
Journal of Food Science, 51, 1382-1383.
33. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with folin
phenol reagent. J. Biol Chem, 193, 265-275.
34. Lu Liu, S.W., Jiang, S.B.S., Wu, S.G. 2004. Tannins inhibits HIV-1 activity by targeting
gp-41.Acta Pharmacol, 25, 213-8.
35. Mahmoud, T., Mohammad, Z.N., Makoto, T. and Setsuko, K. 2008. Acoustic Technology for High
Performance Disruption and Extraction of Plant Proteins. Journal of Proteome Research, 7,
3035–3041.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
351
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
36. Marles, R.J., Farnsworth, N.R. 1995. Antidiabetic plants and their active constituents. Phyto
Medicine, 2, 137-189.
37. Mercy, B.A., Elie, F., Clerge, T., Martin, F., Felicite, M.T. 2005. Nutritive value of some
Cucurbitaceae oilseeds from different regions in Cameroon. Afr. J. Biotechnol, 4(11), 1329-1334.
38. Miller, G.L. 1972. Use of DNS reagent for the determination of glucose. Anal. Chem, 31, 426-428.
39. Murkovic, M., Hillebrand, A., Winkler, J., Leitner, E., Pfannhauser, W. 1996. Variability of Fatty
Acid content in Pumpkin seeds. (Cucurbita pepo L.) Zeistchriff fuer lebensmittel Untersuchung
und forschung, 203 (3), 216 – 219.
40. Ojiako, O.A. and Igwe, C.U. 2007. Nutritional and Antinutritional Compositions of Cleome
rutidosperma, Lagenaria siceraria and Cucurbita maxima seeds from Nigeria. J. Med Food, 10,
735-738.
41. Okaka, J.C., Akobundu, E.N.T. and Okaka, A.N.C. 1992. Human Nutrition, an integrated approach,
Enugu State, University of Science and Technology, Enugu, Nigeri. 3-6 13, 109-110.
42. Okigbo, R.N., Anuagasi, C.L., Amadi, J.E., Ukpabi, U.J. 2009. Potential inhibitory effects of some
African tuberous plant extracts on Escherichia coli, Staphylococcus aureus and Candida albicans.
Inter. J. Intergr. Biol, 6(2), 91-99.
43. Oseni, O.A. and Okoye, V.I. 2013. Studies of Phytochemical and Antioxidant properties of the
Fruit of Watermelon (Citrullus lanatus). J Pharm Biomed Sci, 27(27), 508-514.
44. Prieto. P., Pinda, M. and Agvilar, M. 1999. Spectrophotometric quantitation of antioxidant
capacity through the formation of a phosphomolybdenum complex, specific application to the
determination of vitamin E. Anal Biochem, 269, 337-341.
45. Robinson, R.W., Walters, D.S.D. 1997. Cucurbits. (Crop Production Science in Horticulture n°.6).
New York Cab International. 226.
46. Romana, S. 2014. Optimization of Genomic DNA Extraction Protocol for Molecular Profiling of
Banana Plantain (Musa Species). European Scientific Journal, 10(33), 243-249.
47. Schiebel, S.G., Friederich, M. 1998. Phytotherapy of BPH with pumpkin seeds a multicenter
clinical trial. Zeits Phytother, 19, 71–76.
48. Sodipo, O.A., Akiniyi, J.A., Ogunbamosu, J.U. 2000. Studies on certain characteristics of extracts
of bark of Pansinystalia macruceras (K schemp) pierre Exbeille. Global J. Pure Appl, Sci. 6,
83-87.
49. Sofowora, A. 1982. Medicinal plants and traditional medicine in Africa. John Wiley & Sons Ltd,
Chichester , p. 179.
50. Souza, S.M.C., Aquino, C.M., Milach, J., Bandeira, A.C., Nobre, M.E.P., Viana, G.S.B. 2007.
Antiinflammatory and antiulcer properties of tannins from Myracrodruon urundeuva Allemão
(Anacardiaceae) in Rodents. Phytother. Res, 21, 220- 225.
51. Sreevidya, N. and Mehrotra, S. 2003. Spectrophotometric method for estimation of alkaloids
precipitable with Dragendorff’s reagent in plant materials. J AOAC Int, 86, 1124–1127.
52. Subin, M.P. and Steffy, F. 2013. Phytotoxic Effects of Cadmium on Seed Germination, Early
Seedling Growth and Antioxidant Enzyme Activities in Cucurbita maxima Duchesne. International
Research Journal of Biological Sciences, 2(9), 40-47.
53. Suphakarn, V.S., Yarnnon, C., Ngunboonsri, P. 1987. The effect of pumpkin seeds on
oxalcrystalluria and urinary compositions of children in hyperendemic area. Am J Clin Nutr, 45,
115–121.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
352
Evaluation of biochemical and phytochemical parameters in germinating and nongerminating seeds of Cucurbita maxima
54. Suphiphat, V., Morjaroen, N., Pukboonme, I. 1993. The effect of pumpkin seeds snack on
inhibitors and promoters of urolithiasis in Thai adolescents. J Med Assoc Thai, 76, 487–493.
55. Thayumanavan, B. and Sadasivam, S. 1984. Physicochemical basis for the preferential uses of
certain Rice varieties. Qual. Plant Foods Hum. Nutr, 34, 253.
56. Younis, Y.M., Ghirmay, S., al-Shihry, S.S. 2000. African Cucurbita pepo L. Properties of seed
and variability in fatty acid composition of the seed oil. Phytochemistry, 54(1), 71-75.
Kiran Kumar M et al.,
Int. Journal of Applied Sciences and Engineering Research, Vol. 5, No. 3, 2016
353