Phytochemical Screening and High Performance TLC Analysis of Some Cucurbits
M. Moshahid A. Rizvi
Fruit extracts of Lagenaria siceraria (Molina) Standl., Luffa cylindrica (Linn) Roem. and Cucurbita maxima (Duchesne) Lam. was carried out for the phytochemical screening and High Performance TLC analysis to ascertain relative relationship between active compounds. Present study revealed the presence of alkaloids, flavonoids, saponins and steroids in moderate concentration. High Performance TLC on Silica gel 60 F254 TLC aluminium sheet was used to separate the active compound present in the extracts. TLC sheet was scanned at 200 nm and revealed that the retention factor (Rf), 0.05 and 0.26 of active compounds were common in all three vegetables. Another two active compounds of Rf 0.40 and 0.63 were common in Lagenaria siceraria and Luffa cylindrica. However, Rf 0.20 was common in Lagenaria siceraria and Cucumis maxima. Lagenaria siceraria showed highest peaks, having 44.4% paired affinity with Luffa cylindrica and 28.6% with Cucumis maxima, respectively.
to cite this article:
M. Irshad, Irfan Ahmad, H.C. Goel and M. Moshahid A. Rizvi, 2010. Phytochemical Screening and High Performance TLC Analysis of Some Cucurbits. Research Journal of Phytochemistry, 4: 242-247.
Received: April 20, 2010;
Accepted: June 15, 2010;
Published: July 17, 2010
Cucurbits are dietary vegetable belongs to family Cucurbitaceae, which is primarily
comprised species consumed as food worldwide. Cucurbits are an excellent fruit
in nature having composition of all the essential constituents required for
good health of humans (Rahman, 2003; Duke,
1999; Chopra et al., 1992; Pawar
et al., 1985). Phytochemicals screening of dietary vegetables and
fruits are essential, because it have been used for the maintenance of health
and prevention of chronic diseases (Aggarwal and Shishodia,
2006). It contains large range of secondary metabolites, belongs to different
chemical families and possesses distinct biological activities (Rizvi
et al., 2009). The attraction of pharmaceutical companies and researchers
for elucidation of bioefficacies and provide knowledge for the advancement of
phytomedicine. For example; some cucurbits like Lagenaria siceraria (Molina)
Standl. and Luffa cylindrica (Linn) Roem have a Ribosome inactivating
protein (RIPs), which is well known for the array of biological activities and
possess anti-viral, anti-tumor and immunomodulatory activities (Islam
et al., 1990; Ng et al., 1992; Gao
et al., 1994; Wang and Ng, 2000). Sapogenins
isolated from the Luffa cylindrica, showed immunomodulatory activity
(Khajuria et al., 2007). Cucurbitacins predominantly
found in the cucurbitaceae family, having great interest because of the wide
range of biological activities like antioxidant, anti-inflamatory and inhibit
the proliferation of cancer cells (Tannin-Spitz et al.,
2007). Moreover, due to the prevalence of chronic degenerative diseases
worldwide, the availability information of phytomedicine will help individuals
to make choices in the consumption of foods that could protect them from such
chronic diseases. Present study made a phytochemical screening and High Performance
TLC fingerprinting for the comparative analysis of compounds in commonly used
three cucurbits vegetables.
MATERIALS AND METHODS
The cucurbits vegetables, Lagenaria siceraria (Molina) Standley (Bottle
gourd), Luffa cylindrica (Linn.) Roem (Sponge gourd) and Cucurbita
maxima (Duchesne) Lam (Pumpkin) were purchased from local market in month
of February. It was identified by the Departmental Botanist and vouchers of
specimen were stored in GBL, Department of Biosciences, Jamia Millia Islamia,
New Delhi. This study was conducted from 2008 to 2009.
ilica gel 60 F254 TLC aluminium sheet (20x20 cm), HPLC water, Methanol,
n-Butanol, Acetic acid, Formic acid, Ammonia hydroxide, Lead acetate, Glacial
acetic acids, Mercuric chloride, Potassium iodide, Sublime iodide, NaOH, Sulphuric
acids were purchase from Merck.
The fruits were cleaned with distilled water and juice was extracted by
Juicer (Morphy Richards). The extracts were filtered through muslin cloth and
centrifuged at 5000 rpm for 15 min. It was lyophilized into powder and stored
in vacuum desiccators for further analysis.
The lyophilized powder of fruit extracts of cucurbits vegetables were screened
for phytochemical constituents using standard procedures (Harborne,
1993; Sofowora, 1993; Trease and
Evans, 2002; Kumar et al., 2009; Olawale-Abulude,
2007). The experiments were performed in triplicates and solvents were used
High Performance TLC Study
Chromatography was performed on silica gel 60 F254 TLC aluminium
sheet (20x20 cm). Concentration of samples (10 mg mL-1) were applied
as 6 mm wide bands using Camag Linomat 5 automated TLC applicator with nitrogen
flow providing delivery speed of 150 ηL sec-1 through the syringe.
After completion of sample application, the plate was developed in a Camag Twin
through glass tank presaturated with mobile phase of n-Butanol: Acetic acid:
Water (4:1:5) for one hour. The TLC runs was performed under laboratory conditions.
After development, the plates was taken off and dried. The spots were quantified
using a Camag TLC Scanner model 3 equipped with Camag Wincats software. The
scanning procedure was programmed as, speed 20 mm sec-1, slit width
6.0x0.45 mm and wave length (λmax) 200 nm. The retention factors
(Rf) and % area was automated calculated by the Wincats software. Rf,
is defined as the distance travelled by the compound divided by the distance
travelled by the solvent.
Analysis of High Performance TLC Finger Printing
The High Performance TLC peaks were analyzed by the method adopted by Ellison
et al. (1962). It was followed to make the suitable comparisons in
the form of qualitative relationships of active compounds on the basis of their
biochemical affinities. Values of Paired Affinity (PA), Group Affinity (GA)
and Isolation Value (IV) were calculated as equation 1, 2 and 3, respectively.
The HPTLC fingerprints also have been successfully used to distinctively identify
the different phytochemical constituents on the basis of their Retention Factors
(Rf) (Sunita and Abhishek, 2008; Ferreira
et al., 2009).
RESULTS AND DISCUSSION
The result of the phytochemical screening is presented in Table
1, different standard test were performed and it was revealed the presence
of alkaloids, flavonoids, saponins and steroids in moderate concentration in
all three vegetables. However, trace amount of tannins, glycosides and anthraquinone
were noticed. It was concluded that the cucurbits vegetable have not only essential
nutrients, they also contains secondary metabolites of distinct groups. Among
the effective ingredients, flavonoids are one of the most popular compounds;
possess a variety of biological activities at nontoxic concentrations. The roles
of dietary flavonoids in cancer prevention have been widely discussed (Ren
et al., 2003; Aggarwal and Shishodia, 2006).
It have been reported effective in reducing blood lipid, as an anti-oxidative,
in assimilating cholesterol, inhibiting thrombosis, dilating the coronary artery,
etc. (Hollman and Katan, 1999; Di
Carlo et al., 1999). High Performance TLC on Silica gel 60 F254
TLC aluminium sheet was used to separate the active compound present in
the extracts. The TLC sheets was scanned at λmax 200 nm and
revealed 10 peaks of Lagenaria siceraria, 8 peaks of Luffa cylindrica
and 6 peaks of Cucumis maxima, respectively. The peaks were used for
comparative analysis and revealed the active compounds of Rf 0.05 and 0.26 found
in all three sps. However, active compounds of Rf 0.40 and 0.63 were common
in Lagenaria siceraria and Luffa cylindrica. Similarly, Rf 0.20
was common in Lagenaria siceraria and Cucumis maxima. It was
concluded that some phytochemicals in cucubits sps were commonly present in
all genus and some were found species specific. The concentration of active
compounds was assessed by the % peak area (Fig. 1). The concentration
of active compounds of Rf 0.05 and 0.26 were in order of Cucumis maxima>Luffa
|| Phytochemical analysis of cucurbits by different standard
|-: Negative (absent), +: Positive (slightly present), ++:
Positive (moderately present)
|| Common active compounds its concentration
|| Paired affinity, group affinity and isolation values of three
Active compounds of Rf 0.40 was found in highest concentration in Lagenaria
siceraria than the Luffa cylindrica. Hence, at Rf 0.63, Luffa
cylindrica contained highest concentration than the Lagenaria siceraria.
The peaks were also used for the evaluation of Paired Affinity (PA), Group Affinity
(GA) and Isolation Value (IV) (Table 2). Highest PA value
44.4% was found between Lagenaria siceraria and Luffa cylindrical
and lowest PA value 28.6% was found between Luffa cylindrica and Cucumis
maxima. The PA value between Lagenaria sciceraria and Cucumis
maxima was 37.5%. These values predict the close relationship of Lagenaria
siceraria with Luffa cylindrica than the Cucumis maxima. The
relationships were also confirmed through the group affinity and isolation values
of these cucurbits vegetables (Table 2). These finding supported
that some of the active compound like sapogenins, cucurbitacin glucosides and
cucurbitacines are commonly present in the cucurbits species (Khajuria
et al., 2007; Tannin-Spitz, 2007; Chen
et al., 2005). Therefore, further needed to isolate the separated
compound to elucidate the chemical structure and its bioefficacies.
The commonly used cucurbits vegetables, Bottle gourd, Sponge gourd and Pumpkin have not only essential nutrients, they also contain secondary metabolites such as alkaloids, flavonoids, glycosides, steroids and saponins. The essences of these metabolites are beneficial for maintenance of human health and chronic degenerative diseases. HPTLC fingerprinting revealed the presence of some common compound presence in the all three cucurbits vegetables and some are restricted to the species. There is further need of structural elucidation and bioefficacies of these active compounds.
Authors highly acknowledge the LSRB, DRDO for financial support and CIF of Jamia Hamdard, New Delhi for HPTLC Instrumentation facility.
Aggarwal, B.B. and S. Shishodia, 2006.
Molecular targets of dietary agents for prevention and therapy of cancer. Biochem. Pharmacol., 71: 1397-1421.CrossRef | PubMed | Direct Link |
Chen, J.C., M.H. Chiu, R.L. Nie, G.A. Cordell and S.X. Qiu, 2005.
Cucurbitacins and cucurbitane glycosides: Structures and biological activities. Nat. Prod. Rep., 22: 386-399.CrossRef | PubMed |
Chopra, R.N., I.C. Chopra and B.S. Verma, 1992.
Supplement to Glossary of Indian Medicinal Plants. CSIR, New Delhi, pp: 51
Di Carlo, G., N. Mascolo, A.A. Izzo and F. Capasso, 1999.
Flavonoids: Old and new aspects of a class of natural therapeutic drugs. Life Sci., 65: 337-353.CrossRef | PubMed | Direct Link |
Duke, J.A., 1999.
Handbook of Phytochemical and Constituents of GRAS Herbs and other Economic Plants. CRC Press, Boca Raton, FL., USA., pp: 98-119
Ellison, W.L., R.E. Alston and B.L. Turner, 1962.
Methods of presentation of crude biochemical data for systematic purposes with particular reference to the genus Bahia (Compositae). Am. J. Bot., 49: 599-604.Direct Link |
Ferreira, J.L.P., A.C.F. Amaral, R.B. de Araujo, J.R. Carvalho and C.E.B. Proenca et al
Pharmacognostical comparison of three species of Himatanthus
. Int. J. Bot., 5: 171-175.CrossRef | Direct Link |
Gao, W., J. Ling, X. Zhong, W. Liu, R.Z. Yang, H. Coa and Z. Zhang, 1994.
Luffin-S- a small novel ribosome inactivating protein from Luffa cylindrical
: Characterization and mechanism studies. FEBS Lett., 347: 257-260.
Harborne, J.B., 1993.
Phytochemistry. Academic Press, London, pp: 89-131
Hollman, P.C.H. and M.B. Katan, 1999.
Dietary flavonoids: Intake, health effects and bioavailability. Food Chem. Toxicol., 37: 937-942.CrossRef | PubMed | Direct Link |
Islam, M.R., H. Nshida and G. Funatsu, 1990.
Complete amino acid sequence of luffin-a, a ribosome-inactivating protein from the seeds of sponge gourd (Luffa cylindrica
). Agric. Biol. Chem., 54: 2967-2978.PubMed |
Khajuria, A., A. Gupta, S. Garia and B.P. Wakhloo, 2007.
Immunomodulatory effects of two sapogenins 1 and 2 isolated from Luffa cylindrica
in Balb/C mice. Bioorganic Med. Chem. Lett., 17: 1608-1612.PubMed |
Rizvi, M.M.A., M. Irshad, E.H. Gamal and B.Y. Salaem, 2009.
Bioefficacies of Cassia fistula
: An Indian labrum. Afr. J. Pharm. Pharmacol., 3: 287-292.Direct Link |
Tannin-Spitz, T., M. Bergman and S. Grossman, 2007.
Cucurbitacin glucosides: Antioxidant and free-radical scavenging activities. Biochem. Biophys. Res. Commun., 364: 181-186.PubMed |
Ng, T.B., W.Y. Chan and H.W. Yeung, 1992.
Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants. Gen. Pharmacol., 23: 579-590.CrossRef |
Olawale-Abulude, F., 2007.
Phytochemical screnning and mineral contents of leaves of some Nigerian woody plants. Res. J. Phytochem., 1: 33-39.CrossRef | Direct Link |
Pawar, V.D., D.A. Patil, D.M. Khedkar and U.M. Ingle, 1985.
Studies on drying and rehydration of pumpkin (Cucurbita maxima
). Indian Food Packer, 39: 58-66.
Rahman, A.S.H., 2003.
Bottle gourd (Lagenaria siceraria
) a vegetable for good health. Nat. Prod. Radiance, 2: 249-256.Direct Link |
Ren, W., Z. Qiao, H. Wang, L. Zhu and L. Zhang, 2003.
Flavonoids: Promising anticancer agents. Med. Res. Rev., 23: 519-534.CrossRef | PubMed | Direct Link |
Sofowora, A., 1993.
Screening Plants for Bioactive Agents: Medicinal Plants and Traditional Medicine in Africa. 2nd Edn., Spectrum Books Ltd., Ibadan, Nigeria, ISBN-13: 9782462195, pp: 134-156
Sunita, S. and S. Abhishek, 2008.
A comparative evaluation of phytochemical fingerprints of Asteracantha longifolia
Nees. Using HPTLC. Asian J. Plant Sci., 7: 611-614.CrossRef | Direct Link |
Trease, G.E. and W.C. Evans, 2002.
Pharmacology. 15th Edn., Saunders Publishers, London, pp: 42-44
Wang, H.X. and T.B. Ng, 2000.
Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria
) seeds. Life Sci., 67: 2631-2638.PubMed |
Kumar, A., R. Ilavarasan, T. Jayachandran, M. Decaraman, P. Aravindhan, N. Padmanabhan and M.R.V. Krishnan, 2009.
Phytochemicals investigation on a tropical plant, Syzygium cumini
from Kattuppalayam, Erode district, Tamil Nadu, South India. Pak. J. Nutr., 8: 83-85.CrossRef | Direct Link |