|Year : 2015 | Volume
| Issue : 42 | Page : 356-359
Identification of longevity, fertility and growth-promoting properties of pomegranate in Caenorhabditis elegans
Hasan Kiliçgün1, Nazli Arda2, Evren Önay Uçar2
1 Department of Nutrition and Dietetic, School of Health, Erzincan University, 24100, Erzincan, Turkey
2 Department of Molecular Biology and Genetics, Istanbul University Faculty of Science, Istanbul, Turkey
|Date of Submission||02-Nov-2013|
|Date of Acceptance||12-Sep-2014|
|Date of Web Publication||12-Mar-2015|
Department of Nutrition and Dietetic, School of Health, Erzincan University, 24100, Erzincan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Pomegranate (Punica granatum L.) is commonly consumed as fresh fruit and fruit juice. It is also used in the production of jam, wine, food coloring agent, and flavor enhancer. Objective: The aim of this study was to identify the possible longevity, fertility and growth promoting properties of different ethanolic extract concentrations of pomegranate in Caenorhabditis elegans, which is increasingly popular and has proven to be a very useful experimental model organism for aging studies as well as for testing antioxidants and other compounds for effects on longevity. Materials and Methods: In this study, five experimental groups (20, 10, 5, 2.5 and 1.25 mg pomegranate extract/mL and one control group) were used to determine the most effective dose of pomegranate in terms of longevity, fertility and growth parameters. Results: It was seen that, pomegranate extracts up to the concentration of 5 mg/mL, had the potential to promote for the longevity, formation of new generations, fertility of new generations and growth properties of C. elegans although higher concentrations significantly reduced these parameters. Conclusion: these findings indicated that pomegranate could be used as a supplement to enhance longevity, fertility and growth rate for the other living organisms and human beings, but the dose should be carefully adjusted to avoid adverse effects.
Keywords: Caenorhabditis elegans , fertility, growth, longevity, pomegranate
|How to cite this article:|
Kiliçgün H, Arda N, Uçar EÖ. Identification of longevity, fertility and growth-promoting properties of pomegranate in Caenorhabditis elegans. Phcog Mag 2015;11:356-9
|How to cite this URL:|
Kiliçgün H, Arda N, Uçar EÖ. Identification of longevity, fertility and growth-promoting properties of pomegranate in Caenorhabditis elegans. Phcog Mag [serial online] 2015 [cited 2021 Apr 16];11:356-9. Available from: http://www.phcog.com/text.asp?2015/11/42/356/153089
| Introduction|| |
Accumulation of oxidative damage upon the macromolecules increases progressively by the aging process. Various fruits and plants may provide protection differently against oxidative stress because they differ in many aspects, including the contents of vitamins, minerals, and fibers as well as their antioxidant capacities. Overexpression of antioxidant enzymes or supplementation of some antioxidants appears to be effective in extending the longevity in some nematodes and Drosophila strains and even in mouse models. ,,,,,,, The pomegranate (Punica granatum L.) is one of the oldest edible fruits, widely grown in many tropical, subtropical countries and commonly consumed both as fresh fruit and fruit juice. Since pomegranate is a rich source of anthocyanins, ellagic tannins and other phenolic compounds, which are already proved to have antioxidant and antitumoral activity; it is also used in the production of jam, wine, liqueur, food coloring agent, and flavor enhancer. ,, In this study, the nematode Caenorhabditis elegans was used as a model organism. The key attributes of C. elegans as an experimental system for biological studies are its simplicity, easy cultivation in the laboratory, short life cycle, transparency, suitability for genetic analysis, and small genome size as well as its utilization to test antioxidants and other compounds for their effects on longevity when compared with other animals including mammals. Because the nematode C. elegans has proven to be a very useful experimental model for the studies on longevity, C. elegans has been included in various natural substances and commercial health food supplements studies to evaluate possible longevity effects of natural substances. ,,, The aim of this dose-dependent study was to identify the possible longevity, fertility and growth promoting properties of different concentrations of pomegranate ethanolic extract in C. elegans, and to evaluate its potential to be used as an adjuvant in aging and reproduction.
| Materials and methods|| |
The C. elegans wild-type (N2) strain and its food source Escherichia coli OP50 strain were obtained from Caenorhabditis Genetic Center at the University of Minesota, (USA). C. elegans cultivation media supplements were purchased from MERCK (Germany). The fruits of pomegranate (P. granatum L.) were purchased from local markets from its wild habitat in Irlύganlύ Denizli province and unpeeled fruits were washed, cut into small pieces, lyophilized and ground to obtain fine powder. The powder was used for the preparation of the extract. The 10 g fine powder was soaked in 500 ml of ethanol for 24 h and stirred continuously. The mixture was filtered through a Whatman No. 1 filter paper. The filtrate was vacuum-dried in rotary vacuum evaporator at 40°C. The extract was lyophilized and stored at 4°C for further use. Pomegranate extract (2 g) was mixed with 100 mL nematode growth medium (NGM) and diluted to the proper concentrations before it was used for the experiments.
Quantization of constitutive egg-laying was performed according to the standard protocol described by Michael Koelle.  Briefly, 25 late (L4) nematodes were picked from an unstarved plate to a fresh plate. 36 h later, 20 animals were picked to a fresh plate. Especially in that period, it was ensured that no eggs were transferred. The animals were set at 20°C for exactly 30 min. Nematodes spawn average of 25-100 eggs in petri dishes in this period. The number of eggs was determined by ×20 objective microscopy at the end of 30 min. To help scanning across the plate systematically, parallel black lines drawn helped to place the plate inside a plate lid with it. The study was repeated daily intervals. The standard deviation between days was about 20%. The life span analysis experiments were performed according to the standard protocol described by Sutphin and Kaeberlein,  except for the concentrated OP50 bacteria, which were killed by incubating at 65°C for 30 min. Different concentrations of pomegranate were added to both NGM and lawn of bacteria to allow complete exposure of animals. In this study, one control and five experimental groups, containing 20, 10, 5, 2.5 and 1.25 mg pomegranate extract per milliliter of NGM were used. The worms were grown at 20°C, observed and counted daily. To prevent any statistical mistake, the escaping animals from the petri dishes were excluded from the replacement three petri dishes, which were under the same conditions with experimental groups. Furthermore, to determine the most effective dose of pomegranate on longevity, fertility and growth rate of worms, the whole process was carried out in three times for each experiment. There were not any differences between three times experiment results. On the other hand, One-way Analysis of variance, followed by Scheffe's test were performed to determine statistical differences between the groups with the aid of Statistical Package for the Social Sciences (SPSS) software version 11.0 (SPSS, Chicago, IL, USA). Statistical significance was defined as P < 0.05 for all tests and error bars were placed for all figures.
| Results and discussion|| |
Antioxidant substances are often considered as a promising strategy for modulating aging and extending longevity. Since, antioxidant studies in mammals are expensive and give ambiguous results, the interest for the determination of longevity-extending efficacy of antioxidants in C. elegans is becoming increasingly popular. The main advantages of the use of this model organism are the normal adult longevity, which is 14-20 days and if growth conditions are favorable, the nematode develops rapidly from fertilized eggs through four larval stages (L1-L4) to become an adult hermaphrodite within 3 days. , When the rates of the formation of the new generation between control and experimental groups were compared, it was observed that 2.5 and 5 mg/mL pomegranate extract in the growth medium induced the formation of the new generation. However, the formation of new generation potential of pomegranate significantly reduced at higher (10 and 20 mg/mL) concentrations [Figure 1]. There was no significant difference between the control group and the concentration of 1.25 mg/mL in terms of the forming new generation. These results suggested that the same extract, depending on its dose might also act as an inhibitor or as an activator on the same parameter. When the literature was examined in detail, we could not see detailed studies concerning dose properties of pomegranate about the forming new generation in C. elegans. This could be the first study providing information on this topic. On the other hand, there were two studies done in recent years with pomegranate in male rats. These studies showed that pomegranate had beneficial effects on male fertility, concentrations of spermatids, spermatocytes and spermatogonia. ,
|Figure 1: Comparing the rate of formation of new generation (from cracked eggs of Caenorhabditis elegans) of the control group and different concentrations of pomegranate extract|
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The effects of different concentrations of pomegranate on growth of the nematodes from the new generation were shown in [Figure 2]. The growth rates differed according to the different concentrations of pomegranate. Worms in a medium containing 2.5 or 5 mg/mL pomegranate extract grew more rapidly than the control. In contrast, higher concentrations of pomegranate (10 and 20 mg/mL) decreased growth rates of worms. These negative effects of the high concentrations of pomegranate on the growth of worms can be explained with high polyphenol content of pomegranate.  Because it is known that polyphenol may have prooxidant activity depending on the dose as well.  There were no differences between the control group and the concentration of 1.25 mg/mL in terms of growth rate of worms.
|Figure 2: Comparing the growth rate of the nematodes from new genaration in control group and concentrations of pomegranate|
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The hatching promoting effect of pomegranate on new nematodes generation increased significantly until concentrations of 5 mg/mL and also that promoting effects were observed both 3 and 4 days. But, pomegranate did not show an increasing effect on hatching promoting properties at higher concentrations (10 and 20 mg/mL). Furthermore, a significant decrease in the hatching and growth of worms was observed at higher concentrations [Figure 3] and [Figure 4]. These results may be explained with internal hatching, which is the retention of the eggs in the body. This rarely occurs in adverse environmental conditions, such as starvation and exposure to toxic compounds and bacteria. Therefore, internal hatching provides physical protection and transport for small larvae. ,, From these studies; we could conclude that hatching of worms could be inhibited by the high concentrations of pomegranate.
|Figure 3: Comparing the fertility (hatching) promoting properties of new nematodes generation of control group and different concentrations of pomegranate extract|
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|Figure 4: Comparing the fertility (hatching) promoting properties of new nematodes generation of control group and different concentrations of pomegranate|
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The longevity effects of different concentrations of pomegranate were observed day by day. After 23 days experimental period, it was seen that the longevity promoting effect of pomegranate increased with 1.25, 2.5 and especially 5 mg/mL concentrations. However, the same longevity promoting effect was not observed at 10, 20 mg/mL concentrations of pomegranate [Figure 5]. This effect might be attributed to dose-dependent toxicity of pomegranate. As a matter of fact, some studies reported that at higher doses, antioxidant substances could behave as inhibitor for longevity. In one of these studies, Psoralea corylifolia showed some in vitro antioxidant capacity and at low doses, caused significant longevity extension. However, P. corylifolia was also an inhibitor of the proteasome, cell division and mitochondrial function and at higher doses shows clear dose-dependent toxicity and reduction in lifespan of C. elegans. ,, These studies were compatible with our study in that the same plant extract could have both longevity promoting effect and reducing effect, depending on its concentration. However, as far as we knew, there was no detailed study concerning dose properties and the longevity effects of pomegranate. This may be the first study providing information on this topic. Thus, it might be suitable to use this pomegranate concentration (of 5.0 mg/mL) at tissue level when used as a supplement for therapeutic regimens and longevity.
|Figure 5: The longevity promoting properties of different concentrations of pomegranate extract in Caenorhabditis elegans|
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| Conclusion|| |
As a result, it was expected that this study could lead to a better understanding of the importance antioxidant substances for the dose-response studies and these findings also showed that the study of the edible antioxidant foods remains as a significant field to explore in terms of fertility, growth and longevity researches.
| References|| |
Sohal RS, Mockett RJ, Orr WC. Mechanisms of aging: An appraisal of the oxidative stress hypothesis. Free Radic Biol Med 2002;33:575-86.
Bokov A, Chaudhuri A, Richardson A. The role of oxidative damage and stress in aging. Mech Ageing Dev 2004;125:811-26.
Guo C, Wei J, Yang J, Xu J, Pang W, Jiang Y. Pomegranate juice is potentially better than apple juice in improving antioxidant function in elderly subjects. Nutr Res 2008;28:72-7.
Gulcin I, Kirecci E, Akkemik E Topal F, Hisar O. Antioxidant, antibacterial, and anticandidal activities of an aquatic plant: Duckweed (Lemna minor
). Turk J Biol 2010;34:175-88.
Erel ÞB, Reznýcek G, Þenol SG, Yavasogulu NU, Konyalýoglu S, Zeybek AU. Antimicrobial and antioxidant properties of Artemisia
L. species from western Anatolia. Turk J Biol 2012;36:75-84.
Orhan DD, Özçelik B, Hoºbaº S, Vural M. Assessment of antioxidant, antibacterial, antimycobacterial, and antifungal activities of some plants used as folk remedies in Turkey against dermatophytes and yeast-like fungi. Turk J Biol 2012;36:672-86.
Kiliçgün H, Göksen G. Life span effects of Hypericum perforatum
extracts on Caenorhabditis elegans
under heat stress. Pharmacogn Mag 2012;8:325-8.
Kilicgun H, Goksen G, Olgun A, Ercisli S. Comparýson of thermotolerance effects of desferrioxamine and its concentrations in animal model caenorhabditis elegans
. Rom Biotechnol Lett 2012;17:7145-50.
Perez-Vicente A, Serrano P, Abellan P, Garcia-Viguera C. Influence of packaging material on pomegranate juice colour and bioactive compounds during storage. J Sci Food Agric 2004;84:639-44.
Yasoubi P, Barzegar M, Sahari MA, Azizi MH. Total phenolic contents and antioxidant activity of pomegranate (Punica granatum
L.) peel extracts. J Agric Sci Technol 2007;9:35-42.
Incedayi B, Tamer CE, Çopur ÖU. A research on the composition of pomegranate molasses. J Agric Fac Uludag Univ 2010;24:37-47.
Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, et al.
Extension of life-span with superoxide dismutase/catalase mimetics. Science 2000;289:1567-9.
Keaney M, Matthijssens F, Sharpe M, Vanfleteren J, Gems D. Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo
but do not retard aging in the nematode Caenorhabditis elegans
. Free Radic Biol Med 2004;37:239-50.
Lithgow GJ, Gill MS, Olsen A, Sampayo JN. Pharmacological intervention in invertebrate aging. Age (Dordr) 2005;27:213-23.
Gita S. There are Observable Metabolic Signature Patterns in C. Elegans
: Specifically for Different Life Stages Grown with and without the Added Antioxidants Vitamin C and Vitamin E? USA: Doctor of Philosophy Thesis of George Mason University; 2008.
Koelle MR, Horvitz HR. EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 1996;84:115-25.
Sutphin GL, Kaeberlein M. Measuring Caenorhabditis elegans
life span on solid media. J Vis Exp 2009.
Burnell AM, Houthoofd K, O'Hanlon K, Vanfleteren JR. Alternate metabolism during the dauer stage of the nematode Caenorhabditis elegans
. Exp Gerontol 2005;40:850-6.
Gruber J, Ng LF, Poovathingal SK, Halliwell B. Deceptively simple but simply deceptive - Caenorhabditis elegans
lifespan studies: Considerations for aging and antioxidant effects. FEBS Lett 2009;583:3377-87.
Atýlgan D, Parlaktas B, Uluocak N, Gencten Y, Erdemir F, Ozyurt H, et al.
Pomegranate (Punica granatum
) juice reduces oxidative injury and improves sperm concentration in a rat model of testicular torsion-detorsion. Exp Ther Med 2014;8:478-82.
Dkhil MA, Al-Quraishy S, Abdel Moneim AE. Effect of pomegranate (Punica granatum
L.) juice and methanolic peel extract on testis of male rats. Pak J Zool 2013;45:1343-9.
Gözlekçi S, Saraçoglu O, Onursal E, Ozgen M. Total phenolic distribution of juice, peel, and seed extracts of four pomegranate cultivars. Pharmacogn Mag 2011;7:161-4.
Chedea VS, Braicu C, Socaciu C. Antioxidant/prooxidant activity of a polyphenolic grape seed extract. Food Chem 2010;121:132-9.
Chen J, Caswell-Chen EP. Facultative vivipary is a life-history trait in Caenorhabditis elegans
. J Nematol 2004;36:107-13.
Calafato S, Swain S, Hughes S, Kille P, Stürzenbaum SR. Knock down of Caenorhabditis elegans
cutc-1 exacerbates the sensitivity toward high levels of copper. Toxicol Sci 2008;106:384-91.
Mosser T, Matic I, Leroy M. Bacterium-induced internal egg hatching frequency is predictive of life span in Caenorhabditis elegans
populations. Appl Environ Microbiol 2011;77:8189-92.
Tang SY, Gruber J, Wong KP, Halliwell B. Psoralea corylifolia
L. inhibits mitochondrial complex I and proteasome activities in SH-SY5Y cells. Ann N Y Acad Sci 2007;1100:486-96.
Pun PB, Gruber J, Tang SY, Schaffer S, Ong RL, Fong S, et al.
Ageing in nematodes: Do antioxidants extend lifespan in Caenorhabditis elegans
? Biogerontology 2010;11:17-30.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]