Dietary Intake of Curcuma longa and Emblica officinalis Increases Life Span in Drosophila melanogaster

Intake of food and nutrition plays a major role in affecting aging process and longevity. However, the precise mechanisms underlying the ageing process are still unclear. To this respect, diet has been considered to be a determinant of ageing process. In order to better illustrate this, we used Drosophila melanogaster as a model and fed them orally with different concentrations of two commonly used Indian medicinal plant products, Curcuma longa (rhizome) and Emblica officinalis (fruit). The results revealed significant increase in life span of Drosophila flies on exposure to both the plant products, more efficiently by C. Longa than by E. officinalis. In order to understand whether the increase in lifespan was due to high-antioxidant properties of these medicinal plants, we performed enzymatic assays to assess the SOD and catalase activities in case of both treated and control Drosophila flies. Interestingly, the results support the free radical theory of aging as both these plant derivatives show high reactive oxygen species (ROS) scavenging activities.


Introduction
Life style diseases are wholly or partially attributed to diet and have become major health concern [1][2][3][4]. Long term effects of diet may cause various diseases like diabetes and cardiovascular diseases and can also decrease longevity [5][6][7]. It has also been well documented that mother's or the childhood nutrition can potentially have long term health impacts on an individual [8,9]. In addition, studies have shown that manipulation of dietary intake by increasing or restricting certain food components affects the aging process and longevity [10][11][12][13].
India has been blessed with many medicinal plants and these are in use as traditional medicine in "Ayurveda" since hundreds of years [14,15]. Many such plant products are included in daily diets especially in the form of spices and other food supplements and are known to be rich in antioxidant, anti-inflammatory, antibacterial, anticancerous, and antidiabetic properties [16]. However, detail about their molecular action and dose specificity is not yet known, attracting researchers to explore more in this field [17,18].
Understanding the biology of aging and life span extension is one of the prime areas of research and well supported by various theories. Oxygen-free radicals or reactive oxygen species (ROS) which cause aging are one of the principal theories of ageing in recent years [19]. The primary assumption of this theory is that normal antioxidant defense levels are not sufficient, so that some ROS escape elimination causing molecular damage, some of which are irreparable and accumulate with age. If ROS causes aging, then enhanced defense against ROS should reduce oxidative stress, slow down aging process, and ultimately extend the lifespan [20].
To this respect, Drosophila has been used extensively in aging and longevity studies due to its short life span and easy growing and handling properties in the laboratory [21][22][23]. The genome similarity and presence of highly conserved metabolic pathways with eukaryotes including human make Drosophila one of the best model organisms for investigating various genetic and metabolic pathways to avail the ground level understanding on various gene-environment interactions and their relation to aging process [24,25].
The present study aims at understanding the effects of food supplement on longevity using Drosophila melanogaster as the model organism and its impact on body weight. During this study, the food supplements used were the fruit (fresh crude extract) of Emblica officinalis (common Indian name "amla") and rhizome (powder) of Curcuma longa (common Indian name "haldi, " in English "turmeric"). These two are very familiar plant derivatives in India and are considered to contain medicinal properties. For example, curcumin is a major component of turmeric known to exhibit antiinflammatory, antitumor, and antioxidant properties [19]. Similarly, gallic acid, gallotannin, ellagic acid, and corilagin are some of the important constituents of amla and are also reported to possess several medicinal properties, for example, cardioprotective, gastroprotective, chemopreventive, anti-inflammatory, antimutagenic and also free radical scavenging, and antioxidant [26,27].

Drosophila Species Used and Experimental Conditions.
A mass culture of Drosophila melanogaster was used in this study which was being maintained in the laboratory since 2009 on standard maize-yeast-molasses media consisting of maize flour (17 gms), molasses (16 gms), baker's yeast (5 gms), agar (2 gms), nipagine (2 gms dissolved in 2 mL of ethanol), and water in a total volume of 300 mL and maintained at constant temperature and humidity in the laboratory.
For the control lines, the food media used were without addition of any supplement. While in case of treatments, the regular food media were mixed with either turmeric powder in different concentrations (added per 100 mL of media) or with amla at two different concentrations (20 mL and 30 mL per 100 mL of food media), the concentrations of turmeric in culture media were ascertained from earlier study [21,28] and from preexperimental optimization conducted in our laboratory. Since the food medium becomes dry at higher concentration of turmeric powder, in order to avoid dryness of the media, the maximum concentration used in this study was 0.7 g per 100 mL of media. Similarly, two concentrations of amla were determined after preliminary optimization to avoid media from being too watery. To maintain the purity, the turmeric powder and the amla juice were prepared on mixture grinding the sun-dried rhizome of C. longa and the fresh fruits of E. officinalis, respectively.

Setting up of the Experiments.
Since maximum emergence of Drosophila flies takes place during the morning hours, freshly emerged flies were collected between 9 a.m. and 11 a.m. In order to keep the flies stress free, three male and three female Drosophila flies were kept in each food vial in case of both control and treated experiments. The three Drosophila couples were transferred to fresh food vials containing respective culture media every five days to avoid the effect of media contamination on the flies and not to mix-up with their offspring. For each experimental condition and control, 36 vials were set up with six flies (three males and three females) in each (total 216 Drosophila flies) and were kept inside a BOD incubator with constant temperature (24 ± 1 ∘ C) and other environmental conditions. = ∑ / , where is the mean lifespan, is the lifespan of th fly, and is the total number of flies in the sample) was calculated in each case. Maximum and minimum days of life span in case of each experimental condition were noted down to study the range variation.

Body
Weight. The weight of single fly was recorded at 10, 20, and 30 days interval during their lifespan in both sexes separately in case of both control and treated flies.

Preparation of Crude Extract.
The Drosophila fly crude protein was prepared at 4 ∘ C to prevent the enzymes from being degraded. Six flies were homogenized in the lysis buffer which contained 50 mM phosphate buffer and 0.1 mM EDTA in a total volume of 800 L. The lysate was then centrifuged at 10,000 g for 10 minutes and the supernatant forms the crude extract [29].

Superoxide Dismutase Activity Test by NBT Assay Method.
The test mixture, consisting of 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 75 M NBT, 2 M riboflavin, and 50 L protein sample in a total volume of 2.5 mL, was placed below a 15 W light source and the reaction was allowed to run for 30 min, during which time the color developed. The reaction was stopped by switching-off the light and the tubes were covered with a black paper [30][31][32]. The absorbance was observed at different time points at 560 nm in a spectrophotometer to determine the enzymatic activity. The rate of increase in absorbance units ( ) per minute for the negative control and for the test samples is determined by the formula given as (1) and the percentage inhibition for each sample is calculated using the formula given in (2):

Results and Discussion
Nutrition plays a critical role in overall health of an organism. However, both under-and overnutrition may seriously impact long term health and life expectancy. Therefore, the study on dietary nutraceuticals has become challenging and fascinating demanding greater attention than before [3]. D. melanogaster is an excellent model organism to study aging due to its short generation time and lifespan [22]. In the present study, we have exposed D. melanogaster flies to food media supplemented with various concentrations of turmeric and amla to evaluate their effect on lifespan and body weight. Furthermore, any adverse effect on the physiology of the organism was also determined. When Drosophila fly food was supplemented with turmeric (0.25 g, 0.5 g, and 0.7 g per 100 mL of media), there was a marginal increase in lifespan at a lower concentration (0.25 g/100 mL). Interestingly, with increased concentration of turmeric in food medium, the life span also increased (Figures 1(a) and 1(b)) until a point of concentration, beyond which, increase in the lifespan was not that prominent. The observed effect might be due to the absorption threshold of turmeric, and higher concentrations may not get absorbed completely, thereby, having no further effects on the lifespan. Similarly, intake of amla (at two different concentrations) also increased the life span of D. melanogaster, but less effectively as compared to turmeric (Figures 1(a) and 1(b)). The average and the maximum life span for each control and exposed Drosophila flies were analysed and the survival curve shows day-wise percent of survival for each experimental conditions as well as control (Figures 1(a) and 1(b)). When the turmeric concentration of 0.5 g/100 mL was added as food supplement, the life span of flies could be increased to as high as 42-44 days in comparison to any other experimental conditions, indicating the action of these medicinal plant products in a dose-dependent fashion. The results of all the experimental and control conditions were compared with each other using one-way ANOVA and variation was found to be statistically significant (at < 0.001) in each case ( Table 1).
The present findings on the effect of these two medicinal plant products corroborate with the results of previous study to some extent. Although there is only one recent report available in literature showing positive effect of amla (in form of drug) on life span and reproductive fitness of Drosophila to certain extent [34], it has also been earlier reported that dietary supplements of curcumin (a component of turmeric) extends lifespan of D. melanogaster [21]. Since the mechanism by which curcumin enhances lifespan in Drosophila is unknown, in order to understand such mechanism, we performed the enzymatic assay of superoxide dismutase (SOD) and catalase enzymes. It is a well-known fact that the antioxidant enzymes, for example, SOD, catalase, and glutathione dependent enzymes, get induced as natural defense mechanisms against oxidative stress in biological systems [35]. For example, SOD, a group of metalloenzymes, facilitates the removal of hydrogen peroxide (H 2 O 2 ) on to molecular oxygen and water [35]. Overexpression of 4 BioMed Research International  enzymatically active bovine SOD in Drosophila flies has been shown to confer resistance to paraquat, an (O2−)-generating compound, and also increase the mean lifespan of several of the transgenic lines [36]. In another experiment, expression of Cu/ZnSOD overexpression extended the mean life span of flies up to 48%, while catalase had no significant effect [37], although it increased resistance towards exogenous hydrogen peroxide, paraquat and cold stress [35]. However, overexpression of catalase in mitochondria, nucleus, or peroxisomes has been shown to increase lifespan of mice [38,39]. CAT enzyme plays crucial roles as antioxidants and constitutes the primary defense system against the toxic effects of superoxide radicals (O2−) in organisms [40]. According to the most acceptable free radical theory of aging, the potentially toxicfree radicals, primarily the ROS, superoxide, and hydroxyl radicals generated during aerobic metabolism, are inactivated or scavenged by antioxidants before they can cause damage to lipids, proteins, or nucleic acids [40]. To test this hypothesis, the Drosophila fly protein samples of each experimental condition were tested for SOD and CAT activities. The results indicate that turmeric significantly increases the activity of total SOD at optimal concentrations, as determined by the nitro blue tetrazolium (NBT) reduction assay and native-PAGE (Figures 2(a) and 2(b)). Marginal increase on the activity of SOD was also observed in case of flies fed with amla supplemented food in 20 mL/100 mL concentration. Since increase in the life span seems to be correlated with the increase in the SOD activity, it can be argued that scavenging of the superoxide radicals might be a major factor in increasing the lifespan of Drosophila flies in the test conditions. Increased activity of CAT enzyme was also observed in flies supplemented with both turmeric and amla (Figure 2(c)), indicating antioxidants properties of these two medicinal plants. These two medicinal plants do not seem to have any adverse effect on the flies, as no significant change in the body weight with age of the medicinal plantsupplemented Drosophila flies could be found. Declining of the body weight after 30 days (also observed for the control set of flies, Figure 2(d)) justifies the above contention. However, amla and turmeric might not have been the sole factors in increasing the life span of the Drosophila flies as there might be other (unknown) enzymes and target factors involved on which turmeric and amla have an effect.
In conclusion, the results presented in this work support the hypothesis that intake of C. longa (curcumin) and E. officinalis (amla) increases life span in D. melanogaster. Very similar observations have been made in another experimental model organism, Caenorhabditis elegans [41]. The life span is significantly increased in D. melanogaster flies exposed to both turmeric and amla supplemented food, which is presumed to be due to their high antioxidant properties [28] as evidenced from both SOD and catalase enzymatic assay. However, the present observations reveal antioxidant properties and the ROS scavenging activities of amla to be lower than turmeric. In both cases, oxidative stress reducing ability was found to be dosage-specific. Additionally, it has been observed experimentally that the intake of both these antioxidants has no significant effect on body weight in Drosophila flies. The slight decline in the body weight in the later life (at 30 days) may be due to the old age of the flies. The present study therefore could demonstrate that turmeric and amla increase lifespan of Drosophila. However, further research is needed to underpin the exact component(s) with a role in increase of life span in Drosophila. Though few studies have been conducted with exposure to both plant products in certain disease conditioned mammals (diabetic, cancer, etc.) including human, no such reports are available on aging process and on its underlying mechanism [26,42]. Considering that Drosophila has served as a "model organism" for biomedical research, such knowledge could be well implemented for detailed understanding on the role of diet in ageing process in humans.