The effects of phytocompounds on the lifespan expansion of Drosophila melanogaster

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The effects of phytocompounds on the lifespan expansion of Drosophila melanogaster


Abstract— Phytocompounds, namely resveratrol and spermidine, have recently been shown to modulate lifespan extension in Caenorhabditis elegans, Drosophila melanogaster, mice etc, partly via dietary-restriction mimetic mechanism and partly via SIRT1 activity. The life prolonging effects of such phytocompounds depend largely on the dietary composition. Hence, we hypothesize that phytocompounds, which are structural analogues of resveratrol and spermidine, would also exhibit lifespan extension capabilities. In our study, we investigate the effects of phytocompounds on gender-specific lifespan of Drosophila melanogaster fed with a standard diet and a high-fat diet.

Index Terms— Phytocompounds, Lifespan, Dietary Restriction, Dietary Compositions, SIRT1

Introduction

Recent research shows tremendous progress in betterment of human life and extension of the same. The human life span can easily be extended beyond what nature has intended by various means. Recent research shows apparent progress made in life expansion of mice.

In our project we are checking the life expansion capacity of phyto-compounds using Drosophila melanogaster (both wild and mutant variety). We chose fruit flies over yeast and rodents as they are superior to yeast and easy to rear than rodents. Their size being small with simple diet helps in inexpensive maintenance of large number of flies in simple laboratory conditions. A short life span with large polytene choromosomes make gene mapping accurate in Drosophila melanogaster. Inducing mutation is really easy and a large variety of mutant strains are available for standardizing various assays. The experiments done on developmental mutants in fruit flies have helped to understand the birth defects of human.

Aging, the complicated mechanism shows damage in cellular homeostasis due to accumulation of misfolded, oxidized and cross linked molecules. Life extension is implicated in various pathways like silent information regular 2 (SIR2), insulin and insulin like growth factor signalling and the target of rapamycin (TOR). Autophagy has recently been reported to extend life span in invertebrates. Hence the study of life expansion can serve as the first line of insight into the up or down regulation of SIR2, TOR or autophagy signalling cascade.

Phytocompounds have been purported to initiate a variety of beneficial effects in human. Though the exact molecular mechanism is not known on how these compounds will lead to life extension based on reported literature we aim to extend the life span of Drosophila melanogaster both wild type and mutant variety of W118 and CSBZ. Most robust non genetic approach of life expansion in model organisms is considered as DR (Dietary Restriction). Phytocompounds acting as DR mimetic can be considered as their plausible mode of action in life expansion. Out of the three prominent suggested candidates of motorman, rapamyacin and reveratrol we would compare our chosen compounds with that of the standard concentration of 200μm of resveratrol as our positive control.

Of Naringenin was chosen as the test compound due to its wide array of positive implications in mammalian health. Naringenin, a bioactive flavonoid with potent antioxidant and anti-inflammatory activities, is the predominant flavanone in grapefruit (Fuhr et al. 1993). Naringenin inhibits the humancytochrome P450isoformCYP1A3, which can changepharmacokineticsin a human (ororthologous) host of several popular drugs in an adverse manner, even resulting in carcinogens of otherwise harmless substances (Fuhr et al. 1993). Naringenin also reduceshepatitis Cvirus production by infectedhepatocytes(liver cells) incell culture. This highlights naringenin's ability to inhibit the secretion of very-low-densitylipoproteinby the cells (Namhias et al. 2008). Naringenin exhibits anti-obesity functions (Mulvihill et al. 2009). By suppressing HMG-CoA reductase, naringenin lowers the plasma and hepaticcholesterolconcentrations and ACAT in rats fed with a high-cholesterol diet (Lee et al. 1999)

Figure: Structure of naringenin. Adapted from Fuhr et al, 1993

I. MATERIALS AND METHODS

A. Media

For our experiments we used four types of media to standardize the apt composition required to culture the wild type strain in our laboratory conditions. The four media were named as Media 1, Media 2, Media 3 and Media 4, respectively. The composition and method of preparation for the different media are given below.

A.1. Media 1 Composition

Materials

Amount to be added

Distilled Water

100 mL

Corn Flour

6g

D-Glucose

2g

Granulated Sugar

4g

Agar

1g

Yeast extract

1.2g

Propionic Acid

375µL

Preparation

To prepare Media 1 corn flour was dissolved in cold distilled water and then the total volume was made to 100 mL. The solution was boiled and mixed nicely to remove air bubbles and prevent clump formation. D-glucose and granulated sugar was added and the solution was boiled again. Yeast extract was added and the solution was boiled again to allow proper mixing. Agar was added and dissolved nicely by boiling the solution. After the solution had cooled, propionic acid was added. The media was then quickly transferred into 50mL Falcon tubes. Care was taken to ensure that the media did not stick to the walls of the bottle while it was being transferred.

A.2. Media 2 Composition

Materials

Amount to be added

Distilled Water

100mL

Sugar

10g

Yeast extract

10g

Agar

1.5g

Preparation

To prepare Media 2 sugar was dissolved in distilled water and total volume was made to 100mL. Yeast was then added to the solution after it had been boiled. After yeast had been dissolved, the solution was boiled and agar powder was added to it. It was then allowed to cool, and propionic acid was then added to it. The media was then quickly dispensed into 50mL Falcon tubes. Care was taken that the media did not stick to the walls.

A.3. Media 3 Composition

Materials

Amount to be added

Distilled Water

100mL

Yeast extract

10g

D-glucose

7.5g

Agar

0.8g

Corn flour

5.5g

Phosphoric acid

500µL

Propionic acid

500µL

Preparation

To prepare Media 3 yeast extract was added to distilled water and mixed till it dissolved. The solution was then boiled. D-glucose, agar and corn flour were then added to the solution and mixed by stirring. Heating and stirring was done continuously till the media acquired the desired consistency. The media was then allowed to cool and propionic acid (85%) and Phosphoric acid (8.5%) were then added to the media and mixed properly. The media was then dispensed into Falcon tubes and care was taken to ensure it did not spill or stick to the walls.

A.4 . Media 4 Composition

Materials

Amount to be added

Distilled Water

100mL

Jaggery

10g

Rawa

10g

Agar

1g

Propionic acid

750µL

Preparation

To prepare Media 4 jaggery was taken and added to warm water and mixed thoroughly. Total volume was made up to 100mL. The solution was heated with constant stirring followed by the addition of rawa and agar. After the media had attained desired consistency, heating was stopped and the media was allowed to cool. Propionic acid was then added to it and mixed properly. The media was then quickly transferred to clean 50mL Falcon tubes. Care was taken to ensure that the media did not stick to the walls.

B. Fly maintenance

For the purpose of our studies, wild type flies were reared on masticated banana kept in moist condition. Once larva appeared on this feed, age matched third instar larvae were transferred to the media 4. Fly stock was maintained in a 12 hour day/night cycle at 25°C. Newly eclosed flies were used for all the assays.

(a)

(b)

Figure: (a) Stock Maintenance, (b) Treatment groups

C. Assays

1. Longevity assay

To measure fly longevity, newly enclosed adult flies were collected over 24hrs and transferred to individual vials containing fresh food over layered with 75µl of polyphenol-yeast solution with a final concentration of 200µM resveratrol and 200µM and 400µM naringenin respectively. Control vial composed of only yeast paste without polyphenol. Flies were housed at a density of ten flies per vial. Flies were transferred to fresh vial every 3 days and number of death recorded at each transfer. Triplicate vials were maintained for each compound.

2. Measurement of fly fecundity

To measure fly fecundity, fresh food with a final concentration of resveratrol (200µM) and naringenin (200µM, 400µM) respectively, were transferred to Petri-plates. Newly eclosed flies were collected over 24 hours and housed at a density of one male and two female per Petri-plate. Flies were transferred to fresh food with respective compounds every 2 days. The number of eggs laid by individual females was recorded for a period of 7 days.

3. CAFE (Capillary Feeding) assay

Vials were set up according to procedure put forth by Ja et al. with slight modifications. Flies were fed with naringenin (200 and 400µM) and resveratrol (200µM) for two weeks and then transferred to CAFE vials containing 20µl of liquid food. Rate of feeding was recorded for a period of 24 hours. Duplicate set-up was maintained for each group.

Figure: CAFE set up for measurement of fly feeding rate

4. Measurement of fly physical activity

Flies were fed with naringenin (200 and 400µM) and resveratrol (200µM) for two weeks prior to performing negative geotaxis assay. Ten flies from every treatment were transferred into empty vials and tapped three times to initiate negative geotaxis reaction. Number of flies at a distance of 4cm from bottom was noted down at the end of 4s. The assay was performed 5 times for every treatment group. Flies were allowed to rest for 1 min between each climb.

5. Stress Assays: Heat or cold shock, Starvation Assay

For each of the assays, flies were fed with naringenin (200 and 400µM) and resveratrol (200µM) for two weeks. Ten flies from each of the treatments were transferred to separate vials for the heat or cold shock treatment and the starvation assay. Non treated flies served as control.

5a. Heat Shock Procedure

Flies were exposed to heat shock by dipping the vials in water bath maintained at 38.5ºC and the fly mortality was noted every 10 minutes until all the flies were dead.

5b. Cold Shock Procedure

Flies were exposed to cold temperatures (0ºC) for 8 h, and then returned to room temperature. The recovery time of the flies from each treatment was noted down.

5c. Starvation Assay

Ten flies from each treatment were kept in empty vials without food, supplied with water for moisture. Death of flies was noted down every one hour until all flies in each vial were dead.

6. Lipid and Carbohydrate Metabolism

Materials required

Three whole Drosophila flies each from treatment group fed with naringenin (200 and 400µM), resveratrol (200µM) and control, 2% Sodium sulphate solution, Chloroform/Methanol solution (1:1), distilled water.

Extraction

Three whole flies were homogenized in 0.2ml of 0.2% sodium sulphate solution. The homogenate was washed with 0.8mL of chloroform/methanol (1:1) solution. The washes were centrifuged at 4000rpm for 8 minutes at 4ºC. Supernatant from each of the tubes were transferred to clean centrifuge tubes and 0.6mL of distilled water was added. The tubes were re-centrifuged at 4000rpm for 8 minutes at 4ºC. The top fraction (water methanol) was collected for carbohydrate analysis and the bottom fraction (chloroform) was collected for lipid analysis.

6a. Total Lipid Estimation

Materials required

Lipids samples at different concentrations, concentrated Sulphuric acid, freshly prepared vanillin reagent (20mL of 85% phosphoric acid, 5mL of hot water and 0.3g of vanillin powder).

Procedure

Different concentrations of samples were added and volume made up with chloroform. The tubes were heated in boiling water bath at 100ºC till all the chloroform had evaporated leaving behind the lipid. 0.1mL of concentrated sulphuric acid was added to each of the tubes and heated for a further 10 minutes in boiling water bath. The tubes were cooled and 2.4mL of freshly prepared vanillin reagent was added and mixed well. Absorbance was read at 490nm in UV spectrophotometer.

6b. Total carbohydrate estimation

Materials required

Extracted carbohydrate samples, 4% phenol solution, concentrated sulphuric acid.

Procedure

Different concentrations of carbohydrates were taken in glass tubes and volume was made up with distilled water. 500µL of concentrated sulphuric acid was added and mixed. Absorbance was taken at 490nm in UV spectrophotometer.

Results

Figure 1: Media Standardization: Survival of flies in different media

Figure 2: Fly fecundity with varying concentrations of naringenin and resveratrol

Figure 3: Percentage of fly feeding after treatment with different concentrations of resveratrol and naringenin

Figure 4: Negative geotaxis of flies at (a) week 1 and (b) week 3 of treatment with different concentrations of resveratrol and naringenin

Figure 5: Tolerance to heat shock in flies fed with different concentrations of resveratrol and naringenin

Figure 6: Recovery time after exposure of flies to cold shock

Figure 7: Comparison of extent of survival of flies after exposure to starvation stress

Figure 8: (a) Standard curve for lipid estimation, (b) Levels of lipids in flies treated with different concentrations of resveratrol and naringenin

Figure 9: (a) Standard curve for carbohydrate estimation, (b) Levels of carbohydrates in flies treated with different concentrations of resveratrol and naringenin.

Discussion

Dietary restriction which involves reduced nutrient intake without malnutrition, is a proven intervention implicated in lifespan extension in yeasts, fruit flies, nematodes, rodents, primates etc. DR mimetics are an alternate pharmacological approach to achieve the beneficial effects of dietary restriction without reduced food intake. Resveratrol has been reported to be a strong and efficient DR mimetic thereby extending lifespan of invertebrates and metazoans (Fontana et al, 2010). Naringenin extended healthy lifespan of fruit flies via mechanisms similar to those mediated my resveratrol. Our results suggest that naringenin can be a potential candidate for DR mimetic. Naringenin has a wide array of positive implications in mammalian health. Naringenin inhibits the humancytochrome P450isoformCYP1A3, which can changepharmacokineticsin a human (ororthologous) host of several popular drugs in an adverse manner, even resulting in carcinogens of otherwise harmless substances. Naringenin also reduceshepatitis Cvirus production by infectedhepatocytes(liver cells) incell culture. This highlights naringenin's ability to inhibit the secretion of very-low-densitylipoproteinby the cells. Naringenin exhibits anti-obesity functions .By suppressing HMG-CoA reductase, naringenin lowers the plasma and hepaticcholesterolconcentrations and ACAT in rats fed with a high-cholesterol diet. Naringenin shares structural similarities with compounds like resveratrol and quercetin which have been reported to induce lifespan extension in metazoans.

With studies involving model organisms like Drosophila, standardizing optimal conditions for growth of flies is of absolute mandate. Before continuing with lifespan assays, we attempted to choose a particular media which would result in a high and healthy turnover of fly number. Media 4, which was primarily composed of semolina and jaggery, was found to maintain a high survival rate of flies (Fig 1). Media 3 was found to be least effective in yielding a high survival rate. Media 1 and 2 resulted in an average turnover or flies. The optimal concentration of all the vital nutrients in media 4 might be the probable reason for the flies to thrive high and healthy. Excessive concentration of carbohydrates in media 1, 2 and especially in media 3 proved to be detrimental to fly growth; again implicating the fact that excess nutrient might actually lead to decreased lifespan.

Probable DR mimetic drugs should not induce adverse effect on the capability of progeny expansion, which is a vital signature of healthy lifespan. Hence we checked for the effect of naringenin on fly fecundity. Naringenin at 200µM and 400µM enhanced fly fecundity compared to control group. The fecundity rate was almost equal to that of resveratrol group (Fig 2).

. Food intake is a crucial aspect of a healthy life. Therefore we checked for the effect of naringenin on food intake. Fly feeding rate was almost similar to that of resveratrol fed group (Fig 3).

Physical activity is affected upon administration of various drugs. Hence, we investigated the effect of naringenin on the physical activity of flies in terms of negative geotaxis. Flies exhibit reduced negative geotaxis with time. Therefore, we checked for the distance traversed by the flies at week 1 and week 3 following treatment with naringenin and resveratrol. We observed an expected reduction in negative geotaxis in all groups at week 3 compared to week 1 but the physical activity of naringenin and resveratrol fed flies were more than control group at week 3 (Fig 4a and 4b). This suggests that although there was a reduction of fly physical activity with time, naringenin was able to make to the flies more active compared to that of untreated group.

Long lived organisms exhibit enhanced resistance to environmental stresses. Also, DR mimetics that enhance lifespan are known to induce stress tolerance. Thus, naringenin was investigated for its effect on inducing resistance to stresses like heat shock, cold shock and starvation. Naringenin showed a higher survival percentage of flies exposed to heat shock as compared to control group (Fig 5). Also, naringenin fed flies exhibited fast recovery upon exposure to cold shock compared tocontrol group (Fig 6). Naringenin fed flies were more tolerant towards starvation as compared to both control and resveratrol fed group (Fig 7).

. Reduced levels of lipids and carbohydrates are hallmarks of dietary restriction whereby reduced nutrient level leads to disruption of the mTOR pathway. This in turn results in upregulation of autophagy which in turn is known to enhance lifespan. Hereby, we investigated for the total lipid and carbohydrate composition in naringenin fed flies compared to control and resveratrol group and as expected, the lipid and carbohydrate levels were found to be much lower in naringenin fed groups as compared to control group (Fig 8 and 9).

Hence, our results suggest the possible role of naringenin to be a potential DR mimetic.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.