Geroprotective effects of × Sorbaronia mitschurinii fruit extract on Drosophila melanogaster

Abstract

BACKGROUND:

×Sorbaronia mitschurinii (also known as Aronia mitschurinii) is an intergeneric hybrid of×Sorbaronia fallax and Aronia melanocarpa. ×S. mitschurinii berry is a rich source of phytochemicals such as flavonoids and anthocyanins, which have a broad range of health benefits and a great geroprotective potential.

OBJECTIVES:

The goal of the present study was to investigate the geroprotective effects of Sorbaronia berry ethanolic extract (SBE) in Drosophila melanogaster and whether these effects depend on the concentration of SBE, duration of treatment, age, and sex.

METHODS:

SBE was used to supplement a diet of Drosophila imagoes throughout life, during 2 weeks after the imago hatching, and from 4 to 6 weeks of age. The relationship between the SBE effects on lifespan, stress resistance and the expression of stress response genes were examined.

RESULTS:

SBE treatment at 1-2 and 4-6 weeks of life increased the lifespan, while treatment throughout life reduced lifespan. SBE treatment increased Drosophila resistance to oxidative stress and starvation, but not to hyperthermia. A statistically significant effect of SBE treatment on the expression level of per, keap1, hif1, hsp27, hsp68, hsp83, and sirt1 genes in flies of both sexes and on sod1 expression only in female flies was demonstrated.

CONCLUSIONS:

The observed relationship between SBE concentration and lifespan effects suggests that the life-extending effect of SBE may be associated with the mechanism of hormesis.

Keywords

Sorbaronia berry extract lifespan geroprotector aging

Abbreviations

SBE

Sorbaronia Berry Extract

HPLC

High Performance Liquid Chromatography

JNK

jun-N-terminal Kinase

Hsp

heat shock protein

qRT-PCR

quantitative reverse transcription polymerase chain reaction

FLIC

Fly Liquid –Food interaction Counter

1 Introduction

Aging can be defined as a deleterious process of progressive decline in physiological integrity, and organ dysfunctions that lead to an increased risk of mortality [1]. This age-related decline of physiological functions spreads at all levels of biological organization: molecular, cellular, tissue, and organismic [2 –4]. The pharmacological agents which decrease the rate of aging and extend lifespan are referred to as “geroprotectors” [5 –7]. There are over 400 geroprotective compounds that can increase the longevity of model organisms (such as the nematode, Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the house mouse Mus musculus) that have been found to date [8, 9]. A significant number of geroprotective compounds are represented by substances of plant origin.

×Sorbaronia mitschurinii (also known as Aronia mitschurinii) was created purposefully by the Russian pomologist, Ivan Michurin, in the early 20th century [10, 11]. ×S. mitschurinii is now considered to be an intergeneric hybrid of the species belonging to the Rosaceae family: ×Sorbaronia fallax (A. melanocarpa×Sorbus aucuparia) and A. melanocarpa [12, 13]. In North America ×S. mitschurinii is known as cultivars of A. melanocarpa, including Viking, Nero, and Aron [11, 14]. ×S. mitschurinii and A. melanocarpa possess a similar phenotype and often may be differentiated only by cultivar names. Both species have similar morphology with glossy and glabrous flowers, leaves, stems, and black fruits, but ×S. mitschurinii is characterized by wider leaf blades, more robust stems, and larger fruits than the wild populations of A. melanocarpa [11, 15].

The black chokeberry is known all over the world due to its high content of various polyphenols, phenolic acids, flavonoids, anthocyanins, in addition to vitamins, macro- and microelements [14 , 16–25]. In past centuries, both in North America and in Russia, natural products (berries, leaves, stems) were used in traditional medicine [14 , 26]. Decoctions, juices and infusions of black chokeberry were shown to have anti-inflammatory, antiviral and immunomodulatory effects, which provide many health benefits and are effective for chronic disease prevention [14 , 26].

Recently, the acetone extract of A. melanocarpa fruits has been shown to increase the median lifespan and locomotor activity in Drosophila [27]. The life-extending effect was associated with an increased expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) genes and suppression of the methuselah (MTH) gene [27]. It was also shown that the lifelong use of the polyphenol-rich extracts from blueberry [28], cloudberry [29], apple polyphenols [30], cranberry [31] have positive effects on D. melanogaster lifespan.

The aim of this work was to investigate the geroprotective effects induced by different concentrations of Sorbaronia berry ethanolic extract (SBE) at various ages and different durations of treatment. We supplemented SBE to the diet of Drosophila adult flies throughout life, during 2 weeks after the imago hatching, and from 4 to 6 weeks of age, which correspond to young and average ages, respectively. We studied the effects of SBE treatment on the lifespan, healthspan (locomotor activity, intestinal integrity), stress resistance, and expression level of stress response genes.

2 Material and methods

2.1 Reagents

Experiments were performed using the following reagents, which were purchased from the respective suppliers: Clay –Terrakot (Chelyabinsk, Russia); Hydrochloric acid –Sigmatec (Khimki, Russia); Ethanol (95%) –RFC (Moscow, Russia); Acetonitrile and 10%formic acid (HPLC grade) –Component-reactive (Moscow, Russia); Sorbent diasorb-130-C16T –BioChemMack S&T (Moscow, Russia); Blue food dye (Brilliant Blue FCF) –Roha Dyechem Ltd. (Mumbai, India); Nipagin (methyl 4-hydroxybenzoate), propionic acid, and paraquat (methyl viologen dichloride hydrate) –Merck KGaA (Darmstadt, Germany); Aurum Total RNA Mini kit and iScript cDNA Synthesis Kit –Bio-Rad, (Hercules, CA, USA); Quant-iT RNA Assay Kit –Life Technologies Corporation, (Eugene, OR, USA); qPCRmix-HS SYBR –Evrogen (Moscow, Russia). Deionized water was obtained using the RIOs-DI3 UV water purification system which was supplied by Merck KGaA (Darmstadt, Germany).

2.2 Plant Material

Sorbaronia berries were harvested in the autumn (August –September 2019) in the Botanical Garden (Scientific Collection of Living Plants, No. 507428) of the Institute of Biology of the Komi Science Center of the Ural Branch of the Russian Academy of Sciences (Syktyvkar, Russia), on the territory of the Komi Republic (Northwest Russia). The berries were pre-frozen at a temperature of –20° C for further preparation of the extract.

2.3 Extraction

We used an ethanolic extraction method to maximize the preservation of the anthocyanin fraction of the chokeberry fruit [32]. To prepare the extract, the fruits were crushed and centrifuged to obtain a supernatant. This mass was mixed with clay, and centrifuged again. The clay was prepared by mixing dry clay powder with 0.1 M hydrochloric acid. The resulting liquid was poured off and mixed with the extractant: 1%solution of concentrated hydrochloric acid in 95%ethanol. The resulting solution was centrifuged and then the ethanol and hydrochloric acid from the extract were evaporated on an IR-1M vacuum rotary evaporator (Khimlaborpribor, Russia) at 40° C to dry the residue. The experimental concentrations of SBE were prepared from the ethanol extract obtained by dilution in 95%ethanol. Since anthocyanins occur in plants in the form of salts with sugar moieties, the extraction was carried out with an acid solution, which gives the maximum yield. Phenolic compounds of plants are poorly soluble under these conditions and their yield is insignificant. Polyphenolic compounds are insoluble and can precipitate under the conditions described.

2.4 High-performance liquid chromatography-mass spectrometry (HPLC-MS)

SBE samples were analyzed on a Thermo Finnigan Surveyor HPLC system equipped with a diode array detector (200–600 nm) and a mass selective detector (Thermo Fisher Scientific Inc., USA). Detection was carried out at a wavelength of 520 nm, an eluent feed rate of 1 ml/min, analysis time of 40 minutes, in isocratic mode. A solution of acetonitrile and 10%formic acid (7 : 93, v/v) in water was used as eluent. A chromatography column 4×250 mm with a sorbent Diasorb-130-C16T (particle size: 7μm) was used. For sample preparation, 1 mg of the extract was dissolved in 10 ml of deionized water, after which it was applied to a prepared cartridge with the Hypersep C18 sorbent. The cartridges were washed off with 1 ml of eluent followed by a wash with 10 ml of deionized water. Mass spectra were obtained using a Thermo Finnigan LCQ Fleet liquid chromatograph (Thermo Fisher Scientific Inc., USA) in combination with electrospray ionization at a kinetic energy of 40 eV.

The compounds which were isolated from the extract in their pure form (delphinidin glucoside, delphinidin rutinoside, and cyanidin glucoside) were used as standards for the HPLC-MS, as well as for quantitative analysis. The structures of these compounds were confirmed by IR, UV, and NMR spectroscopy, additionally by molecular ions in MS. Then the column was calibrated using delphinidin glucoside, a calibration curve was plotted and the ratio of each component in the extract was estimated quantitatively.

2.5 Drosophila rearing and maintaining

D. melanogaster wild type Canton-S, which is a standard laboratory strain to study lifespan-extending interventions [33], was obtained from the Bloomington Stock Center at Indiana University (#64349, Bloomington, USA). To obtain control and experimental flies, 10 pairs of male and female parents were placed into vials with the nutrient medium for 24 h oviposition. Progeny flies were collected within 24 h after eclosion, anaesthetized using CO₂, sorted by sex (males and non-virgin females), and placed into vials (30 flies per vial) containing the nutrient medium with the investigated concentrations of SBE or the control medium without SBE.

The flies were maintained at 25°C and 60%relative humidity under a 12 h: 12 h light/dark cycle in a constant climate chamber, Binder KBF720-ICH (Binder, Germany). The food media on which the flies lived contained water –1000 ml, corn flour –92 g, dry yeast –32.1 g, agar-agar –5.2 g, glucose –136.9 g to which 5 ml of a 10%solution of nipagin in ethanol, and 5 ml of propionic acid were added to inhibit microbial and fungal growth in the food.

2.6 Treatment with Sorbaronia berry extract

SBE solution (30μl per vial) in 95%ethanol at concentrations of 0.01; 0.1; 1.0; 2.5; 5.0, and 10 mg/ml were directly added to the surface of the fresh medium. The 30μl of 95%ethanol was added to the medium surface of the control vials. Vials were dried under a fan.

2.7 Ages of SBE treatment

We treated flies with SBE at different adult flies‘ ages. The first group of flies was treated with the extract throughout their whole life. The second group was exposed to SBE treatment for 2 weeks after the imago hatching (designated as “2 weeks of SBE treatment”) and the third group was treated with SBE from 4 to 6 weeks of age (designated as “4–6 weeks of SBE treatment”). The second and third groups, before and after the addition of the SBE, respectively, were fed with a control medium without SBE. The experiments were carried out in 3 biological replicates.

2.8 Lifespan analysis

Within 24 h after imago hatching, flies (males and non-virgin females) were separated by sex using CO₂ anesthesia and transferred in control or experimental vials (30 flies per vial). Dead flies were counted daily, and the remaining live flies were transferred to vials with fresh medium twice a week. The survival curves were plotted and the mean lifespan was calculated.

2.9 Stress resistance analysis

The newly eclosed flies were mated for 24 h after which males and non-virgin females were collected and treated with SBE at concentrations of 0.1, 1.0, and 5.0 mg/ml for 14 days and 33 days. The control groups (0 mg/ml) were treated with 95%ethanol. Flies were transferred to vials with fresh medium twice a week. To study the resistance to oxidative stress, flies were exposed to a medium composed of 2%agar, 5%sucrose, and 20 mM paraquat. During starvation, the flies were kept on a 2%agar medium. Hyperthermia was induced by continuous exposure of the flies to 35 °C. Dead flies were identified using the DAM2 Drosophila Activity Monitor (TriKinetics Inc., USA) by the complete absence of movement of individual flies, kept in small glass tubes. Stressful conditions were applied until the death of flies without transferring to fresh tubes.

The most important advantage of this approach is that stress resistance can be determined with a relatively fewer number of individuals. Only 8 individuals can be used in one assay [34]. We used 16–32 flies in 2–4 replicates (a total of 32–112 individuals) in each experimental variant. The resistance to oxidative stress was analyzed in 2 replicates, including 2 replicates for male and female flies at the ages of 14 days and 2 replicates for males and females at the age of 33 days. The resistance to starvation was analyzed in either 2 or 3–4 replicates, including 3–4 replicates for flies at the age of 14 days and 2 replicates for flies at the age of 33 days. The resistance to hyperthermia was analyzed in either 2 or 4 replicates, including 2 replicates for flies at the age of 14 days and 2 replicates for flies at the age of 33 days.

2.10 Analysis of locomotor activity

The age-dependent changes in spontaneous locomotor activity were measured using the LAM25 Locomotor Activity Monitor (TriKinetics Inc., USA) under standard 12 h lights-on, 12 h lights-off conditions. The data from 10 flies in 4 vials as replicates were collected during 24 h and represented an average total daily locomotor activity. Males and non-virgin females were analyzed separately, from the age of 1 to 9 weeks. The experimental flies were contained on the nutrient medium with SBE in concentrations 0.1, 1.0, and 5.0 mg/ml, 96%ethanol was applied to control vials (0 mg/ml) over the medium. Flies were transferred to new vials containing fresh medium twice a week.

2.11 Analysis of intestinal integrity

The flies from both control and experimental cohorts were tested at the ages of 2, 6, and 8 weeks. The experimental flies were treated with SBE at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0, 10 mg/ml and the control group (0 mg/ml) was treated with 96%ethanol. Flies were transferred to vials with fresh medium twice a week. Males and non-virgin females were analyzed separately.

The Smurf assay was used for intestinal barrier permeability estimation [35]. Control and experimental cohorts were kept for 16 hours on a food medium containing 2.5%(mass/volume) non-absorbable blue food dye after that the blue-dyed flies (“Smurf” flies) with impaired gut integrity were counted.

2.12 Analysis of food choice (FLIC assay)

Time-dependent changes in food preference were analyzed using flies at the ages of 7 and 45 days old from the control and SBE-treated groups. Flies were transferred to vials with fresh medium twice a week. Males and non-virgin females were analyzed separately. A fly liquid–food interaction counter (FLIC) system (Sable Systems, USA) was used to analyze feeding behavior as described [36]. For food choice assay, each channel of the Drosophila Feeding Monitor (DFM) was loaded with either 5%sucrose (–SBE control) or 30μl/ml SBE in 5%sucrose (+SBE experiment). Before the assay, flies were starved in a Drosophila vial without medium for 2 h 30 min. The assay was performed for 3 h using 6 flies. FLIC Monitor software (downloaded from flidea.tech) was used to collect raw data from the DFM. Feeding Preference Index (PI) values from the FLIC system were calculated as the difference in total feeding time between the control and experimental foods divided by total feeding time for both foods [36]. The PI ranged from 1 (complete preference for –SBE control food) to –1 (complete preference for + SBE experimental food) with a value of 0 indicating no food preference. The PI value was calculated for each individual fly and presented as the mean PIs for the experimental groups.

2.13 Quantitative reverse transcription PCR

Gene expression was measured by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) with a reverse transcription step. RNA was isolated using an Aurum Total RNA Mini kit according to the manufacturer’s instructions. RNA concentration was measured using a Quant-iT RNA Assay Kit according to the manufacturer’s instructions. cDNA was synthesized according to the iScript cDNA Synthesis Kit from the resulting RNA solution. The reaction mixture for the PCR reaction was prepared based on qPCR mix-HS SYBR and primers (Supplementary Table 1). The primer design was performed using the QuantPrime online tool [37]. The polymerase chain reaction was carried out using primers (Supplementary Table 1) in a CFX96 amplifier (Bio-Rad, USA) using the following program: 1) 95°C for 30 s, 2) 95°C for 10 s, 3) 60°C for 30 s, 4) steps 2–3 were repeated 49 times 5) DNA melting step.

The expression of the studied genes was calculated relative to the expression of the housekeeping genes Tubulin, eEF1 α2, RpL32 using the CFX Manager 3.1 software (Bio-Rad, USA). Differences that were considered significant changes (increases or decreases) in expression were with p < 0.05 according to the Student’s t-test and crossed the Regulation Threshold. The level of mRNA expression was determined using qRT-PCR in the experimental flies, which were previously contained at nutrient medium with SBE at the same concentrations as in the lifespan experiments (0.1, 1.0, and 5.0 mg/ml) at the ages of 14 and 33 days. Flies were transferred to vials with fresh medium twice a week. For each experimental variant, 20 males or 10 females were used. The experiments were carried out in 3 biological replicates and 3 technical replicates. The delta-delta CT method was used for quantification [38].

2.14 Statistical analysis

To compare the statistical differences in survival functions, median lifespan and maximum lifespan between control and experimental groups, the modified Kolmogorov-Smirnov test [39], Fisher exact test [40], and Wang-Allison test [41] were used, respectively. When statistically significant differences were obtained for survival functions, the median and maximum lifespans, a multiple comparison test (Holm - Bonferroni test) was performed with a significance level less than 0.05 [42, 43]. The analysis of variance (ANOVA) test was used to compare differences in locomotor activity, food preference, stress resistance and gene expression levels between control and the SBE treated flies. Post-hoc pairwise comparisons between control and treated variants were performed using Tukey’s Honestly Significant Difference (HSD) tests.

Statistical analyses of the data were performed using STATISTICA software, version 6.1 (StatSoft, USA), R, version 2.15.1 (The R Foundation) and OASIS 2 (Online Application for Survival Analysis 2) [44]. R code for simple FLIC analyses version 4.0 was used to analyze the signal data from FLIC DFMs (https://github.com/PletcherLab/FLIC_R_Code) [36]. Holm-Bonferroni sequential corrected p-values were calculated with an EXCEL calculator [45].

3 Results

3.1 Chemical analysis of berry extracts

To determine the active substances in the ethanol extract of chokeberry, an HPLC method was carried out at a wavelength of 520 nm, an eluent flow rate of 1 ml/min, and an analysis time of 40 minutes in an isocratic mode (Fig. 1). The chromatogram (Fig. 1) shows the main peaks: at 10.54 min –delphinidin glucoside, 13.38 min –delphinidin rutinoside, 16.64 min –cyanidin glucoside, which indicates that these substances are the main ones in the analyzed extract. Delphinidin and cyanidin are anthocyanidins, they are found in many bright, blue-red fruits and berries, such as Manitoba berries, Saskatoon berries [46], and eggplant [47]. Cyanidin is the most common in raspberries, strawberries [46], and black chokeberry [17], sour cherry [48] in apple skin [49], and açai [50].

Fig. 1

High performance liquid chromatography sample “Sorbaronia”: (1) 10.54 min –delphinidin glucoside; (2) 13.38 min –delphinidin ruthenoside; (3) 16.64 min –cyanidin glucoside.

Like other fruits and berries, Sorbaronia fruits contain a wide range of biologically active polyphenols such as phenolic acids (neochlorogenic, chlorogenic and caffeic acids), flavonoids (quercetin-3-O-rutinoside, quercetin-3-O-galactoside and quercetin-3-O-glucoside). They form anthocyanins (cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, cyanidin-3-O-xyloside and cyanidin-3-O-glucoside) and proanthocyanidins ((–) –epicatechin) in various proportions [51, 52]. This largely depends on the time of collection, on soil factors, climatic conditions, in addition to the extraction method (water, acetone, ethanol, water-ethanol) [16 –19].

3.2 Lifespan

Aging is associated with multiple physiological disorders. While reproductive potential and locomotor activity decrease, inflammatory reactions and pathological processes increase [2, 53]. Thereby the effect of geroprotective intervention may depend on the physiological state and age of the organism. To reveal the connection between the geroprotective effect and the age of treatment we studied the lifespan effects of SBE when adult flies were treated at different ages (continuously throughout life, 2 weeks after the imago hatching, and from 4 to 6 weeks of age). We take into account the age-related physiological changes in the imago fitness traits, including reproductive maturation (the first days post eclosion), reproductive activity (at about 3 weeks of life), decrease in reproductive activity and further death [53].

The SBE that was constantly supplied with food throughout life at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0, and 10.0 mg/ml, decreased the median (up to 4.2%in males and up to 3%in females) and the maximum (up to 8.6%in males and up to 5.3%in females) lifespan (Figs. 2A, B; Supplementary Table 2).

Fig. 2

The effect of SBE on longevity. Survival curves (A, C, E), mean and maximum lifespan (B, D, F) of male flies, which were treated with SBE continuously throughout life (A, B), 2 weeks after the imago hatching (C, D), and from 4 to 6 weeks of age (E, F). The gray background shows the ages of SBE treatment. The results of the three biological replicates are pooled. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 –Bonferroni-adjusted p-values.

Fig. 3

The effect of SBE on longevity. Survival curves (A, C, E) and mean lifespan (B, D, F) of female flies, which were treated with SBE continuously throughout life (A, B), 2 weeks after the imago hatching (C, D), and from 4 to 6 weeks of age (E, F). The gray background shows the ages of SBE treatment. The results of the three biological replicates are pooled. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 –Bonferroni-adjusted p-values.

SBE treatment at 1–2 weeks of adult life, at a concentration of 2.5 mg/ml slightly increased the median lifespan (by 3%) in males; however, it decreased median lifespan in females up to 9%at concentrations of 0.01, 0.1, 1.0, and 10.0 mg/ml. The maximum lifespan of females also decreased up to 5%at concentrations of 0.01 and 10.0 mg/ml (Figs. 2C, D; Supplementary Table 3).

SBE treatment at 4–6 weeks of adult life increased the median lifespan in males by 5%at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0 mg/ml and by 4%at a concentration of 10 mg/ml. The maximum lifespan of males has increased by 9%at concentrations of 0.1 and 5.0 mg/ml. SBE treatment did not induce statistically significant effects (p > 0.05) on the median lifespan of females, while the maximum lifespan of females was increased by 3%at all studied concentrations except 10.0 mg/ml (Figs. 2E, F; Supplementary Table 4).

Thus, while SBE treatment throughout life has a negative effect on male’s and female’s lifespan, SBE treatment at the first 2 weeks of life (reproductive activity period) in addition to the age of 4-6 weeks (post-reproductive period) has the lifespan increasing effect in male but not in female flies.

3.3 Locomotor activity

Aging is associated with a decrease in the functionality of the whole organism and in particular the locomotor function [4]. Therefore, to reveal the potential efficiency of SBE to delay the functional aging of flies, we investigated the effect of the most effective geroprotective concentrations (0.1, 1.0, and 5 mg/ml) on the age-related changes of locomotor activity.

The two-way ANOVA test revealed statistically significant differences in locomotor activity between male and female flies at different ages in control and experimental variants (p < 0.01, source of variation: Age) (Fig. 4; Supplementary Table 5). The ANOVA test revealed no statistically significant difference between the control and SBE treated flies (p > 0.05, source of variation: Conditions) (Supplementary Table 5). It should be noted that no harmful effects of SBE on locomotor activity as an indicator of health were found in flies of both sexes.

Fig. 4

Effects of the SBE on the age-related changes of average daily locomotor activity in Drosophila males (A) and females (B).

3.4 The Smurf assays

The age-related increase in permeability of the intestinal barrier is one of the markers of the aging process and an increased risk of mortality [35 , 55]. We have studied the effect of SBE on the Drosophila intestinal barrier permeability at the ages of 2, 6 and 8 weeks old (Supplementary Table 6). However, almost no “Smurf” flies were found in males and females at the ages of 2, 6 and 8 weeks old. These results suggest that SBE treatment at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0, and 10.0 mg/ml had no negative effect on the permeability of the intestinal barrier.

3.5 Food choice (FLIC assay)

The high levels of phenolic compounds in Aronia may contribute to its pronounced bitterness, sourness and astringency of SBE-containing food media [56, 57] and influence the feeding rate [36]. Both reduced or excessive food consumption may affect nutritional status, health, and finally the lifespan [58, 59]. To find out how SBE supplementation affects food consumption we studied the dietary preferences of flies at different ages using the FLIC system [36]. In addition, we studied the possible effect of habituation on chokeberry flavor by analyzing feeding behavior in flies that were kept either on control media or media with SBE before the test.

The results obtained reflect the lack of a strong preference (the mean PI varies in wide ranges) of the males and females for –SBE or +SBE food media (Fig. 5). Four-way ANOVA tests showed that there are no statistically significant differences in the feeding preference index between male and female flies of different ages in control and experimental variants, which were either pretreated or not with SBE (p > 0.01) (Fig. 5, Supplementary Table 7). At the same time the interaction between SBE concentration, age, and sex showed that feeding PI values may be affected differently in aging females and males. Overall these data confirm the results of a previous study that demonstrated no significant difference in food intake between flies from the control and those supplemented with 2.5 mg/ml Aronia acetone extract groups estimated by gustatory assay [27] demonstrating that the observed effects on lifespan and on healthspan are not associated with changes in food consumption.

Fig. 5

The effects of SBE supplementation on the food choice. SBE was supplemented in liquid food obtained by dilution of 30μl of 0.01 mg/ml, 2.5 mg/ml, and 10 mg/ml SBE in 1 ml of 5%sucrose solution. PI = 1 –complete preference for –SBE control food; PI = –1 –preference for +SBE experimental food; PI = 0 indicating no food preference. Four-way ANOVA was used to examine the influence the sex, age, SBE pretreatment, and SBE concentration factors on feeding preference index. Error bars denote 0.95 confidence intervals.

3.6 Stress resistance

The mechanisms of resistance to harmful stress factors and longevity are interrelated [60]. Oxidative stress, which leads to the disruption of redox homeostasis, is one of the important factors of aging, and the organism’s ability to defend against oxidative stress plays an important role in longevity [61]. The treatment of flies with paraquat, high temperature and starvation may be a cause of free radicals, which in the future can lead to pathological processes in the body and further death [62 –64]. In addition, decreased stress tolerance is one of the manifestations of aging [65, 66]. Therefore, we were interested in how the SBE treatment will affect the resistance of Drosophila to various stress factors (paraquat, hyperthermia and starvation) at the ages of 14 and 33 days old (see Fig. 6). In these assays we used the SBE at concentrations of 0.1, 1.0 and 5.0 mg/ml, which demonstrated the most effective geroprotective potential.

Fig. 6

Effects of the SBE treatment on the resistance of Drosophila males and females at the age of 14 and 33 days old to oxidative stress. Three-way ANOVA was used to test for the influence of SBE concentration, age, and sex on stress resistance. Error bars denote 0.95 confidence intervals.

To investigate the effect of SBE on the resistance to different stresses we performed an ANOVA test for each stressful condition using SBE concentration, age, and sex as factors. It is worth noting that stress resistance is significantly dependent on age and sex and these factors (as well as all interactions of them) are always statistically significant (p < 0.001) (Figs. 6 –8, Supplementary Table 8). Three-way ANOVA tests detected that SBE treatment has a significant effect on the resistance of Drosophila to oxidative stress (p < 0.05) and starvation (p < 0.001) but not to hyperthermia. However, post-hoc analysis failed to reveal a statistically significant effect (p > 0.05) of SBE treatment on the resistance to oxidative stress in males, females, and at the same ages (14 and 33 days) (Fig. 6, Supplementary Table 9). At the same time, SBE treatment at a concentration of 5.0 mg/ml increased the resistance to starvation (p < 0.001) in females at the age of 14 days old (Fig. 7, Supplementary Table 9). Thus, the effect of SBE on resistance to stressful environmental conditions (oxidative stress, starvation, hyperthermia) is not significant in comparison with age-related changes and sex differences in stress resistance.

Fig. 7

Effects of the SBE treatment on the resistance of Drosophila males and females at the age of 14 and 33 days old to starvation. Three-way ANOVA was used to test for the influence of SBE concentration, age, and sex on stress resistance. Error bars denote 0.95 confidence intervals.

Fig. 8

Effects of the SBE treatment on the resistance of Drosophila males and females at the age of 14 and 33 days old to hyperthermia. Three-way ANOVA was used to test for the influence of SBE concentration, age, and sex on stress resistance. Error bars denote 0.95 confidence intervals.

3.7 Quantitative reverse transcription PCR

Changes in gene expression levels are associated with health and lifespan [67]. Plant extracts have a wide range of pharmacological effects, which at the molecular level are determined by the changes in the transcriptional activity of genes, including stress response genes. We have studied the changes in the expression level of cellular stress response genes, including circadian clock genes (Clk, per), oxidative stress resistance genes (Keap1, NRF2, Sod1, HIF1), heat shock proteins genes (Hsp27, Hsp68, Hsp83) and the longevity gene Sirt1. The mRNA expression level was identified by qRT-PCR in flies at the age of 14 and 33 days old (Supplementary Tables 10 and 11). The effects of SBE treatment at concentrations of 0.1, 1.0 and 5.0 mg/ml were analyzed (Fig. 9).

Fig. 9

Effects of SBE treatment on the relative expression level of stress response genes in male (A, C) and female (B, D) flies at the age 14 days (A, C) and 33 days (B, D). All diagrams represent the means of three biological replicates, each quantification is carried out in three technical replicates, the error bars show the standard error of the mean. Four-way ANOVA was used to test for the influence of SBE concentration, age, replicate and reference gene on the expression level of stress response genes. ^*p < 0.05. Significance was determined by four-way ANOVA followed by post hoc Tukey’s HSD tests. Error bars denote 0.95 confidence intervals.

To determine the effect of SBE concentration, fly age, and qRT-PCR conditions (each biological replicate and each reference gene) on the expression level of stress response genes we used a four-way ANOVA test with post hoc Tukey’s HSD tests for comparing treatments with a control variant (Supplementary Tables 12–15). The ANOVA test revealed a statistically significant effect of SBE treatments on the expression level of per, keap1, hif1, hsp27, hsp68, hsp83, sirt1 genes in flies of both sexes and on the sod1 expression in female flies only (Supplementary Tables 12 and 14). At the same time, the ANOVA test failed to reveal the effect of SBE on the expression of clk and nrf2. The interaction between SBE and age demonstrates that aging in females but not in males is associated with loss of statistically significant effects of SBE on the expression level of per, hif1, sod1, hsp83, and sirt1. For example, additional post-hoc analysis revealed that transcriptional activity of per gene decreased by 6 2 times (p < 0.05) in males at both ages, while the activity of per in females increased by 5–10 times (p < 0.05) at an age of 14 days only (Supplementary Tables 13 and 15). In addition, post-hoc multiple comparisons using Tukey’s HSD test revealed that the effect of SBE on the expression levels of most genes in males and females at an age of 14 days is highly variable depending on concentration. Tukey’s HSD also showed that SBE treatment at an age of 33 days in females affected the expression level of hsp27, but not other genes studied. However, in males, SBE induced a decrease at the level of expression of per, keap1, hif1, sirt1 and hsp27 at the age of 33 days. Taking into account these results it is difficult to link the changes in gene expression levels with the SBE effects on lifespan and stress resistance.

4 Discussion

The interest of society in improving the quality of life, reduction of age-related diseases and increase in longevity are growing every year [16 , 20]. Based on traditional medicine, we can assume that the supplementation of diet with natural products (berries, fruits and vegetables), can lead to a significant improvement in healthspan and a slowing down of the aging process [16 , 68–70].

The present study is the first report to give a demonstration that SBE treatment in the first 2 weeks of life (fertility period) and at 4–6 weeks of age (post-reproductive period) for a limited time (two weeks), increases the lifespan of Drosophila males, while continuous SBE treatment throughout whole life leads to harmful effects on the lifespan of both females and males. These results are in agreement with the recent study of flavonoid effects on the Drosophila lifespan [71]. The long-term treatment with flavonoids did not change the lifespan of males and females, however, the short-term flavonoid consumption during middle age (30–40 days) had a positive effect on the lifespan of females [71]. Similarly, treatment of mouse models of various age-related pathologies with different doses of rapamycin at the age of 20–22 months old reduced the risk of age-related disorders (changes in the liver, heart, adrenal glands) [72] demonstrating a geroprotective effect.

At the same time, several studies have demonstrated that lifelong treatment with plant extracts may be beneficial for lifespan. For example, the treatment of D. melanogaster with 2.0 and 5.0 mg/ml of powder extract from bilberry throughout life increased the average lifespan by 10%[28]. An acetone extract of cloudberries (0.12 mg/ml) caused an increase in the median lifespan of female flies by 11%[29]. A polyphenol-rich apple extract increased the average fly lifespan by 10%[30]. An anthocyanin extract of cranberry (20 mg/ml) also increased the average lifespan of male flies by 10%[31].

The presence of both beneficial and harmful effects of SBE treatment may reflect the biphasic dose-response which is attributed to hormesis (the beneficial effects of low concentrations of toxic substances) [3 , 74]. It should be noted that the lifespan dose-response relationship for SBE treatment in males and females is depicted by an inverted U-shaped curve, which is also one of the most common features of hormesis [73 –75]. Hormesis is associated with the activation of protective cellular mechanisms when exposed to mild stress [76] to maintain homeostasis that provides long-term positive effects [75 , 78]. The hormesis inducers, so-called hormetins, can activate heat shock proteins and the proteasomal degradation of damaged proteins [79, 80], DNA repair [3], autophagy [81, 82], an antioxidant defense that can promote lifespan [83, 84]. Hormetins may be food components, including phenolic acids, flavonoids, terpenoids, vitamins, and trace elements that have a positive effect on the age-related quality of life [84 –86].

Despite the observed hormetic effects of SBE at the organism level, we failed to demonstrate its relationship with the expression level of the studied stress response genes. Apparently, the moderately positive effects of SBE on lifespan and stress resistance are not associated with significant changes in gene expression.

In addition, one of the feasible mechanisms of the geroprotective effect of SBE may be associated with the antimicrobial activity of black chokeberry [19, 87]. However, the antimicrobial preservatives (nipagin and propionic acid), which were added to the control and experimental food media, significantly reduce the possible contribution of the antimicrobial effect of SBE to longevity.

5 Conclusions

Therefore, the obtained results demonstrate that SBE has geroprotective potential which depends on the duration of treatment, age and sex of D. melanogaster individuals. In particular, it was found that SBE increases the mean lifespan in males in the case of short-term treatment at the first 2 weeks of life as well as at the age of 4–6 weeks, while constant SBE treatment throughout life has a negative effect on the flies’ lifespan. The observed dose-response relationship for SBE treatment and lifespan effects is depicted by a hormetic inverted U-shaped curve. In addition, SBE does not induce negative effects on Drosophila healthspan (locomotor activity and intestinal barrier permeability) at different ages and increases resistance to stress factors (oxidative stress and starvation). Despite the statistically significant effects of SBE on the expression level of aging-associated, stress-response genes, including circadian clock genes, oxidative stress resistance genes, heat shock proteins genes, we failed to reveal the relationship between the SBE effects on lifespan, stress resistance and levels of gene expression in Drosophila.

Footnotes

Acknowledgments

We are grateful to the Institute of Chemistry of the Komi Science Center (Syktyvkar, Russia) for assistance in the analysis of extract composition. We are grateful to the Botanical Garden of the Institute of Biology of the Komi Science Center of the Ural Branch of the Russian Academy of Sciences (Syktyvkar, Russia) for collecting Sorbaronia fruits (Scientific Collection of Living Plants, registration No. 507428).

Funding

This study was funded by a grant from the Russian Basic Research Foundation and the National Research Foundation of Korea according to the joint research project № 19-515-51001.

Declaration of Competing Interest

The authors declare no conflict of interest.

The supplementary material is available in the electronic version of this article: .

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