Wild-type w¹¹¹⁸ flies (Drosophila melanogaster) were obtained from the Bloomington Drosophila Stock Centre (Bloomington, IN, USA), and reared on a standard cornmeal-yeast medium at 25°C and 60% humidity under a 12/12-h light/dark cycle.

The dried stigmas of C. sativus L. were obtained from the Qinghai-Tibet plateau of China in 2014 and were identified by Professor Xiuhua Wang (Northeast Forestry University, China). Aqueous extracts were obtained as previously described (21). C. sativus L. (2 g) were immersed in 100 ml deionized water overnight at 25°C. The aqueous extraction was boiled for 3 h, and the extraction process was repeated twice. Total extracts were mixed and concentrated to 50 ml. Drosophila that were fed a standard cornmeal-yeast medium were used as the control group, and those fed the standard medium containing 1% extract of C. sativus L. were used as the experimental group.

Crocin I (Chengdu Must Bio-Technology Co., Ltd., Chengdu, China) was used as a reference standard for the quality control of the C. sativus L. extracts. High-performance liquid chromatography (HPLC) analysis was performed as previously described (22). Briefly, the HPLC system (LC-20AT, Shimadzu Corporation, Kyoto, Japan) consisted of a quaternary pump, C18 gravity column (Alltima C18; 250×4.6 mm, 5 µm) and HPD-20A detector. The mobile phase was 15% methanol in water at a flow rate of 1.0 ml/min and detector wavelength of 275 nm.

The lifespan of the Drosophila was determined at 25°C and 60% humidity with a 12/12-h light/dark cycle. Newly enclosed adult flies were collected within a 24-h period, and the flies were allowed to mate for 48 h. A total of 30 adult flies were cultured and transferred to new vials containing fresh food every 2–3 days. The surviving flies were counted every day, and each experiment was repeated three times.

The 3-to 5-day old adult flies (15 males and 15 females) were starved for 2 h prior to being transferred to a vial containing 5 layers of filter paper hydrated with 5% sucrose (w/v) with toxic compounds, including 0.6% SDS (w/v; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 6 mM paraquat (Sigma-Aldrich; Merck Millipore) or 4% DSS (w/v; MP Biomedicals, LLC, Santa Ana, CA, USA). Sucrose (5%) solution with no additives was used as the positive control for all experiments. Filter papers were changed every day, and the survival rate was monitored daily, as mentioned above.

Adult Drosophila were orally exposed to 0.6% SDS and incubated at 25°C for 96 h. A total of 10–15 dissected fly intestines were stained with 7-AAD (5 µg/ml in PBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 30 min at room temperature, fixed for 30 min in PBS with 4% paraformaldehyde, and then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, as previously described (21). The intestines were mounted using mounting medium [70% glycerol in PBS containing 2.5% 1,4-diazabicyclo[2,2,2]octane (DABCO); Sigma-Aldrich; Merck Millipore), and images were captured using an Axioskop 2 Plus microscope (Carl Zeiss AG, Oberkochen, Germany). The presented data are from three independent experiments.

Adult females were orally exposed to 1% SDS and incubated at 25°C for 48 h. A total of 10–15 intestines were dissected in ice-cold PBS and incubated in dihydroethidium (DHE; 5 µM in PBS; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at room temperature, then rinsed 4 times in PBS for 5 min each, fixed in PBS with 4% paraformaldehyde for 20 min, and then stained with DAPI for 10 min. Intestines were then rinsed 4 times with PBS and mounted using mounting medium (70% glycerol in PBS containing 2.5% DABCO; Sigma-Aldrich, Merck Millipore), then analyzed with an Axioskop 2 Plus microscope (Carl Zeiss AG). The data presented are from three independent experiments.

Statistical analysis was performed using a two-tailed unpaired Student's t-test with Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.001 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard error.

HPLC analysis was used for the identification and quantification of Crocin I in the previously collected C. sativus L. aqueous extracts. The chromatogram of the standard was eluted at a retention time of 9.3 min, and it was determined that the C. sativus L. aqueous extracts that were collected previously contained 0.181% Crocin I (Fig. 1)

Aging is a multifaceted process associated with a gradual decline in physiological function, which leads to serious diseases, including cancer and diabetes (23). A previous study demonstrated that various traditional plants and their extracts contain high levels of phytochemicals, which are capable of extending survival and preventing or delaying age-associated diseases (24). Drosophila fed with the standard medium with 1% extract of C. sativus L. were used as the experimental group (Fig. 2). It was determined that significant increases in the maximum lifespan of female and male flies occurred in the group fed with the C. sativus L. extract compared with the control group (P<0.0001; Fig. 2A and C). In addition, the mean lifespan increased by 18.3% in females and 8.8% in males (Fig. 2B and D). These findings suggest that C. sativus L. extract may promote longevity and increase the mean lifespan, and that the magnitude of this increase may be associated with gender.

The intestinal epithelium is susceptible to pathogen damage, oxidative stress and toxic compounds. In order to determine the protective effect of C. sativus L. extract, adult flies from both culture conditions (the control and experimental groups) were orally treated with the inflammatory reagent, SDS or DSS. These chemicals may interfere with the normal function of the intestinal barrier and stimulate local and systemic inflammation. This may result in tissue damage and melanotic phenotypes in the adult Drosophila intestine (8,10). As presented in Fig. 3A and B, survival rates were significantly increased in the C. sativus L. feeding groups compared with the control group, following exposure to SDS or DSS for 6–7 days (P<0.001). The survival rate in the treatment groups fed with C. sativus L. extract were 35.5 and 67.7% for SDS and DSS, respectively, which were significantly higher compared with the survival rates of the control group (3.3 and 13.2%, respectively). Following ingestion of an ROS-producing agent, paraquat, for 4 days, the C. sativus L. feeding groups exhibited significantly higher survival rates (48.9%) compared with the control group (P<0.001; Fig. 3C). This survival rate was >36.7% compared with the control group (12.2%; Fig. 3C). These findings indicated that C. sativus L. extract may increase the Drosophila survival rate following oral infection with SDS, DSS and paraquat. Therefore, C. sativus L. may contribute to the resistance of infection due to pro-inflammatory reagents.

The ingestion of toxic compounds, such as SDS, has been identified to induce epithelial cell damage (8). To determine whether the reduced survival rate of adult flies resulted from increased cell death in response to SDS treatment, adult flies in the control and C. sativus L. feeding groups were treated with 0.6% SDS for 96 h. Subsequently cell viability of intestinal epithelial cells was detected using 7-AAD staining, which is a type of nucleic acid dye that is capable of passing through the plasma membrane and combining with the DNA of the dead cells. An increase in the number of dead cells was observed in the control group, whereas only a few dead cells were detected in the experimental group (Fig. 4A).

Various stresses may lead to the production of excessive ROS, which may then cause oxidative damage, and ultimately cell death. This depends on the maintenance of an equilibrium between ROS production and scavenging. Bandegi et al (25) have proposed that the C. sativus L. extract and its active constituent, Crocin I, may prevent chronic stress-induced oxidative stress damage to the brain, liver and kidneys in rats (25). The present study determined whether treatment with C. sativus L. eliminated the excessive ROS levels in the Drosophila intestine. DHE, a redox sensitive dye that intercalates into cellular DNA when oxidized (26), was used to quantify ROS levels. Adult flies were exposed to SDS for 48 h. DHE fluorescence was reduced in the posterior midgut of the C. sativus L.-fed group compared with the control (Fig. 4B). These findings demonstrated that C. sativus L. extract may maintain the host redox homeostasis following SDS exposure, therefore protecting against damage of intestinal epithelial cells.

A previous study revealed that SDS may induce melanotic tumors and morphological changes in the Drosophila intestine (21). Following treatment with 0.6% SDS for 4 days, the intestinal length of the control group was observably shorter than the C. sativus L.-fed group. Additionally, melanotic tumors were observed in the posterior midgut of the control group. No melanotic masses were observed in the C. sativus L.-fed group (Fig. 5). These findings indicated that increased epithelial cell death may induce morphological changes in the adult Drosophila intestine.