Effects of Viscum coloratum (Kom.) Nakai f. Lutescens Kitag polysaccharide on fertility, longevity and antioxidant capacity of Drosophila melanogaster

Abstract

The purpose of this study was to investigate the effects of Viscum coloratum (Kom.) Nakai f. Lutescens Kitag polysaccharide on fertility, longevity and antioxidant capacity of Drosophila melanogaster. The Drosophila melanogaster were randomly divided into five groups. Four generations of Drosophila melanogaster were cultured continuously in basic medium with or without polysaccharides (400, 200, 100 and 50 mg/L). The changes in fertility, lifespan and male-to-female ratio from F₁ to F₄ generation. The survival time under acute oxidative damage caused by paraquat and hydrogen peroxide (H₂O₂) and changes in superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were determined as well. High dose of V. coloratum polysaccharide could significantly improve the fertility and lifespans of Drosophila melanogaster from F₁ to F₄ generation, prolong the survival time under acute oxidative damage. Moreover, the MDA content was decreased sharply, while the activities of SOD and CAT were increased in high group. However, the male-to-female ratio was overall close to 1:1 for F1 to F4 generations among the four groups. V. coloratum polysaccharide improved the antioxidant capacity and vitality of Drosophila melanogaster and promoted their reproductive capacity in a dose-dependent manner.

Keywords:
Viscum coloratum (Kom.) Nakai f. Lutescens Kitag polysaccharide; Drosophila melanogaster; fertility; longevity; antioxidant capacity

1 Introduction

Viscum coloratum (Kom.) Nakai, a spermatophyte, is mainly parasitic on mulberry, poplar and other big trees, which is widely distributed in China. It serves as a precious traditional Chinese medicine (TCM) in China's medical prescriptions, which is mainly used for expelling rheumatism, improving one's health and reinforcing liver and kidney. In recent years, it has also been reported that V. coloratum (Kom.) Nakai has protection on cough, frostbite, stomach trouble, lumbocrural pain and threatened abortion (Yoo et al., 2019). In the clinic of TCM, V. coloratum (Kom.) Nakai f. lutescens Kitag is equivalent to V. coloratum (Kom.) Nakai ^[3]. Researches on V. coloratum (Kom.) Nakai medicines and related health products started late in China, which has recently attracted people's attention(Chen et al., 2020).

The main bioactive component in the fruit of V. coloratum (Kom.) Nakai f. lutescens Kitag is polysaccharide (V. coloratum polysaccharide), which is mainly composed of highly esterified galactose polymers (Szurpnicka et al., 2019), neutral polysaccharides and acidic polysaccharides (Stein et al., 1999). In recent years, through the optimization of extraction process(Jun & Yi, 2007) and the study of rheological properties, the research on V. coloratum polysaccharide has gradually turned to functional food and pharmacological research. V. coloratum polysaccharide has been used in the treatment of immunocompromised mice and mice with acute alcoholic liver injury, and the immune function and liver injury were recovered well (Ye et al., 2017).

Drosophila melanogaster (D. melanogaster) has the characteristics of simple breeding, rapid reproduction and clear genetic background (Rubin, 1988), which is mostly used in the research of drug and food detection(Jana et al., 2016; Brandt & Vilcinskas, 2013; Lamas et al., 2016). However, the effects of V. coloratum polysaccharide on fertility, longevity and antioxidant capacity of Drosophila melanogaster have not been reported. Therefore, in this study, with D. melanogaster as subject, the effects of V. coloratum polysaccharide on fertility, longevity and antioxidant capacity were investigated to provide a theoretical basis for the development of health products and medicines of V. coloratum polysaccharide.

2 Materials and Methods

2.1 Experimental materials and main instruments

The fruit of V. coloratum (Kom.) Nakai f. lutescens kitag were collected in Liangdang County, (Longnan, China) in July 2018. The commercial kits of Superoxide dismutase (SOD) (lot number: 20160822), catalase (CAT) (lot number: 20170509) and MDA (lot number: 20170729) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Yeast extract was purchased from Kmaels Biotechnology Co., Ltd (Shanghai, China). Other instruments used in this study were as follows: thermostatic biochemical incubator (Ningbo Dongnan Instrument Co., Ltd., Ningbo, China), high temperature sterilizer (Shanghai Sanshen Medical Appliance Co., Ltd., Shanghai, China), ultra clean bench (Shandong Biobase Scientific Instrument Co., Ltd., Jinan, China), L5 UV-visible spectrophotometer (INESA Analytical Instrument Co., Ltd., Basil, Switzerland), and high-speed refrigerated centrifuge (Beijing Era Beili Centrifuge Co., Ltd., Beijing, China).

2.2 Extraction of V. coloratum polysaccharides

The method of extracting polysaccharides from the fruit of V. coloratum (Kom.) Nakai f. lutescens kitag mainly referred to the method described in previous studies (Huang & Huang, 2020). The fruit were dried at 50 °C and pulverized. After refluxing and degreasing in anhydrous ethanol, it was dried at 50 °C. Then, 100 g of the dried material was added to 1000 ml of distilled water and heated at 40 °C for 2 h to dissolve it. The supernatant obtained by vacuum filtration was concentrated under reduced pressure. The extract was mixed with Sevag reagent in a volume ratio of 1:5, and shaken and centrifugation to remove the denatured protein, and this process was repeated 3 times. The obtained crude polysaccharide was dissolved in distilled water at 40 °C, followed by stirring and slowly adding Fehling's reagent simultaneously. After precipitation of the copper complex, the mixture was stood for 1 h, washed with distilled water for 3 times, and centrifuged to obtain the complex. Distilled water at 4 °C was added into the complex, and then 0.5 mol/L HCl was added dropwise to dissolve all the compound. Next, the crude polysaccharide was precipitated with 95% ethanol, followed by centrifugation. The obtained floccule was washed with ethanol, acetone and diethyl ether, and freeze-dried into a white polysaccharide powder. Finally, the purity was detected by High performance liquid chromatography - gel permeation chromatography (HPLC-GPC).

2.3 Determination of fertility

Wild-type D. melanogaster, provided by the Genetics Laboratory of Northwest Normal University, cultured in a constant temperature and humidity incubator at 25 °C and 65% relative humidity. The basic medium for culturing D. melanogaster, including 250 ml of distilled water, 28 g of corn flour, 22 g of sucrose, 2.5 g of agar, 2 ml of propionic acid and 2.5 g of fresh yeast, was prepared by heating. As previously described (Peng et al., 2009), V. coloratum polysaccharide at concentrations of 50, 100, 200 and 400 mg/L were added into the basic medium as low, medium and high dose of V. coloratum polysaccharide groups, and the basic medium was set as the control group. Specifically, in constant-temperature incubator, 10 pairs of unmated male and female D. melanogaster were placed into the medium, and cultured for 7 d. The parents were removed. After the offspring (F₁) appeared, the number of male and female was counted. After analyzing the recorded data, 10 pairs of unmated F₁ male and female were taken into a new medium for subculture, respectively. After cultured for 7 d, the grandchildren (F₂) were propagated. The F₂ generation was further subcultured to reproduce the F₃ generation, and the F₃ generation was further subcultured to reproduce the F₄ generation by the same way. Each treatment was performed for three replicates. The number of D. melanogaster of each generation was counted, and the male-to-female ratio was calculated for each generation to investigate the effect of V. coloratum polysaccharide on the fertility ^[20-21].

2.4 Determination of longevity

The average and maximum lifespans were measured. Unmated female and male were collected for isolated culture. Referring to the method of Ye et al. ^[20-21], male and female were inoculated with 10 bottles in the medium containing 50 mg/L, 100 mg/L, 200 mg/L or 400 mg/L, respectively. They were observed and recorded daily, and the number of dead individuals was recorded until the last D. melanogaster died. The data were analyzed to calculate the lifespans according to following equations. Average lifespan=sum of lifespans /total number of D. melanogaster; and maximum lifespan=sum of the survival time of the last four D. melanogaster that died/4. Briefly, individuals of F₁-F₄ generations were taken from those in fertility test for the determination of lifespan, and the method was performed as above. The differences in mortality were compared.

2.5 Determination of survival time under acute injury

The grouping way was the same as described in “Determination of fruit flies' longevity”. A 20 mmol/L paraquat solution and a 9% H₂O₂ solution were prepared with a 6% glucose solution for future use. The collected D. melanogaster (regardless of male and female) were isolated and starved for 2 h, and then transferred to culture tubes. Next, 2 ml of paraquat solution and H₂O₂ solution were dropped on a filter paper strip until the filter paper strip was complete wet, which was then placed in one of the culture tubes. The number of died D. melanogaster was observed every 4 h until all were died. The average survival time, half survival time and maximum survival time in each group were recorded (Li et al., 2007; Harman, 1981).

2.6 Determination of antioxidant activity

The method used in this test was performed as previous described with some modification ^[20]. First, 50 individuals cultured for 30 d were transferred to a blank culture tube containing wet filter paper, and they were anesthetized with diethyl ether. After weighing, they were put into a mortar that had been pre-cooled at 4 °C, and then pre-cooled normal saline was added at a ratio of 49:1 (μl/mg) for full grinding. The mixture was centrifuged at 3 500 rpm/min for 15 min. The supernatant was taken to further experiment. Superoxide dismutase (SOD) activity, catalase (CAT) activity and malondialdehyde (MDA) content in the supernatant were determined by SOD kit, CAT kit, MDA kit and protein kit. The reagents used in the determination must be prepared strictly in accordance with the instructions, and the determination methods must also strictly follow the conditions recommended by the manufacturer.

2.7 Statistical analysis

All data were expressed as $\overline{x}$±$s$. SPSS 22.0 software (International Business Machines, corp., Armonk, NY, USA) was used for statistical analysis. The difference was performed by One-way analysis of variance (ANOVA) for the significance between control and treatment groups. P<0.05 indicated a significant difference and P<0.01 indicated an extremely significant difference.

3 Results and Discussion

3.1 Characteristics of V. coloratum polysaccharide

V. coloratum polysaccharide is a white powder obtained by enzymolysis with complex enzyme, followed by separation and extraction, decolorization and vacuum freeze-drying. It has been determined that the yield of V. coloratum polysaccharide was 19.83%. The obtained V. coloratum polysaccharide was prepared into a solution with a concentration of 0.01%, and its content was determined by UV spectrophotometry. It showed no characteristic absorption of proteins and nucleic acids at 260 and 280 nm, respectively. However, one crack-free peak appeared by using HPLC-GPC, indicating that V. coloratum polysaccharide was a kind of polysaccharide, and its purity was 99.95%.

3.2 Effects of V. coloratum polysaccharide on fertility of D. melanogaster

The results of different concentration of V. coloratum polysaccharide on the fertility, total number and male-to-female ratio of F1 to F4 generations of D. melanogaster were shown in Table 1 -3. With the increased concentration, the number of female and male in the F₁-F₄ generations were all increased. When the concentration was 50 mg/L, both the number of female and male in each of the F₁-F₄ generations and the total number of all generations were significantly higher than those in control group (P<0.01). When the concentration was 400 mg/L, both the number of female and male in each of the F₁-F₄ generations and the total number of all generations were 200% of those in control group. When the concentration was between 50 and 400 mg/L, the male-to-female sex ratio did not change significantly in the F₁-F₄ generations, and the overall ratio was close to 1:1. Therefore, the above results suggested that V. coloratum polysaccharide had a significant promotion effect on the fertility of fruit flies.

Table 1
Effects of V. coloratum polysaccharide in different concentrations on generational fertility of D. melanogaster ($\overline{x}$±$s$, flies).

Polysaccharide concentration (mg/L)	F1		F2		F3		F4
	♀	♂	♀	♂	♀	♂	♀	♂
CK (0)	298±3.13aA	299±2.62aA	274±1.53aA	270±1.72aA	244±1.92aA	233±0.62aA	235±3.13aA	228±2.62aA
50	309±2.06aA	316±2.80aA	295±1.43aA	288±2.18aA	259±1.84aA	253±1.53aA	251±2.06aA	244±2.81aA
100	331±2.61bA	334±2.65bA	324±1.47bA	314±1.55bA	302±1.53bB	296±1.68bB	296±2.61bB	293±2.65bB
200	437±2.44cB	440±2.28cB	431±1.65cB	428±1.37cB	407±2.43cC	402±0.53cC	409±2.44cC	404±2.28cC
400	487±4.18dC	491±3.25dC	480±3.58dC	475±2.45dC	497±2.81dD	491±2.14dD	474±4.18dD	477±3.25dD

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Table 2
Effects of V. coloratum polysaccharide in different concentrations on total number in different generations of D. melanogaster ($\overline{x}$±$s$, flies).

Polysaccharide concentration (mg/L)	F1	F2	F3	F4	Total
					♀	♂	♀+♂
CK (0)	597±3.70aA	544±3.63aA	477±3.21aA	463±3.67aA	1051±3.27aA	1030±3.37aA	2081±7.80 aA
50	625±3.21bA	583±3.01 bA	412±3.62bA	495±3.61bA	1114±3.67bB	1101±4.77 bB	2215±7.58 bB
100	665±3.61cB	638±3.19cB	598±3.57cB	589±3.65cB	1253±5.08cB	1237±5.21cC	2490±7.81cC
200	877±2.80dC	859±2.60dC	809±3.50dC	813±3.62dC	1684±4.50dC	1674±4.54dD	3358±8.50dD
400	978±2.97eD	955±3.20 eD	988±2.99 eD	951±2.81eD	1938±2.84eD	1934±4.81eE	3872±7.70eE

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Table 3
Effects of V. coloratum polysaccharide in different concentrations on sex ratio in different generations of D. melanogaster.

Polysaccharide concentration (mg/L)	F1	F2	F3	F4	Mean
CK (0)	0.997	1.014	1.047	1.030	1.022
50	0.978	1.024	1.024	1.029	1.014
100	0.991	1.031	1.020	1.010	1.013
200	0.993	1.007	1.012	1.012	1.006
400	0.992	1.011	1.012	0.994	1.002

3.3 Effects of V. coloratum polysaccharide on longevity of D. melanogaster

Table 4 and Table 5 showed the results of different concentrations of V. coloratum polysaccharide on the generational lifespan and accumulated lifespan of D. melanogaster, respectively. With the increased concentration, the lifespans of female and male in the F₁-F₄ generations both increased. When the concentration was 50 mg/L, the accumulated lifespans of female and male, the total lifespan of female and male and the longest lifespans of female and male in the F₁-F₄ generation were significantly increased than those in control group, respectively (P<0.05). When the concentration was 100, 200 and 400 mg/L, the accumulated lifespans, the total lifespan and the longest lifespans in the F₁-F₄ generations were extremely significant increase than those in control group (P<0.01). Those results showed that V. coloratum polysaccharide had a significant effect on prolonging the lifespans of D. melanogaster.

Table 4
Effects of V. coloratum polysaccharide in different concentrations on generational lifespan of D. melanogaster ($\overline{x}$±$s$, d).

Polysaccharide concentration (mg/L)	F1		F2		F3		F4
	♀	♂	♀	♂	♀	♂	♀	♂
CK (0)	50.89±0.36aA	51.61±0.46aA	56.94±0.44aA	51.82±0.26aA	54.79±0.26aA	54.90±0.70aA	50.54±0.64aA	54.65±0.94aA
50	51.88±0.58aA	53.92±0.30aA	57.62±0.72aA	54.54±0.28aA	56.81±0.35aA	52.10±0.34aA	53.64±0.32aA	59.90±0.43aA
100	70.91±0.32bB	70.76±0.28bB	69.81±0.43bB	66.89±0.32bB	70.94±0.26bB	63.85±0.42bB	65.91±0.34bB	63.88±0.34bB
200	74.63±0.40cB	76.04±0.48cB	78.01±0.72cB	78.85±0.70cB	73.94±0.19cB	70.95±0.32cB	73.84±0.41cB	78.96±0.32cB
400	79.73±0.72dC	78.98±0.52dC	81.01±0.32dC	81.88±1.00dC	82.91±0.30dC	79.11±0.32dC	80.18±0.43dC	83.24±0.63dC

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Table 5
Effects of V. coloratum polysaccharide in different concentrations on accumulated lifespan of fruit fly generations ($\overline{x}$±$s$, d).

Polysaccharide concentration (mg/L)	Accumulated lifespan of (F1- F4)		Average lifespan of (F1- F4)		The longest lifespan of (F1- F4)
	♀	♂	♀	♂	♀	♂
CK(0)	213.16±4.11aA	213.01±4.79aA	53.29±0.72aA	53.25±0.65aA	57.81±0.55aA	58.74±0.39aA
50	219.95±3.79bA	220.46±2.30bA	54.99±0.68 aA	55.12±0.39aA	64.40±0.45bB	66.76±0.28bB
100	277.54±3.09cB	265.38±2.82cB	69.39±0.91bB	66.35±0.79bB	72.80±0.49cC	73.73±0.43cC
200	300.42±2.45dC	304.80±0.75dC	75.11±0.67cC	76.20±0.50 cC	80.72±0.76dD	77.39±0.47dD
400	323.83±2.63dD	323.21±0.36dD	80.96±0.30dD	80.80±0.49dD	85.52±0.83eE	84.38±0.89eE

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

3.4 Effects of V. coloratum polysaccharide on survival time of D. melanogaster under acute injury

Table 6 and Table 7 showed the effects of different concentrations of V. coloratum polysaccharide on the survival time of D. melanogaster under acute injury. When treated with hydrogen peroxide and paraquat, the survival time in the F₁-F₄ generations was significantly prolonged after intervention with 200-400 mg/L V. coloratum polysaccharide in comparison with the control group (P<0.05). When the concentration was 400 mg/L, the average survival time in the F₁-F₄ generations treated with hydrogen peroxide and paraquat were increased by about 6 h in comparison with the control group, the half survival time were also increased by about 4 h in comparison with the control group, and the longest survival time were increased by about 5 h in comparison with the control group. The above results showed that V. coloratum polysaccharide had significant effects on prolonging the survival time of D. melanogaster with acute injury.

Table 6
Effects of V. coloratum polysaccharide in different concentrations on the survival time in paraquat treatment of D. melanogaster ($\overline{x}$±$s$, h).

Polysaccharide concentration (mg/L)	F1			F1			F3			F4
	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time
CK (0)	25.12±1.04aA	23.12±0.84aA	38.17±0.76aA	24.92±1.14aA	23.39±0.54aA	38.49±0.96aA	25.32±1.64aA	23.86±0.77aA	38.21±0.19aA	25.41±1.09aA	23.36±0.97aA	38.11±0.14aA
50	27.44±1.76aA	24.54±0.91aA	39.32±0.59aA	27.64±0.26bB	24.94±0.31aA	39.92±0.99aA	27.86±0.39bB	25.04±0.86aA	40.03±0.91aA	28.16±0.77bB	25.64±0.38aA	40.63±0.29aA
100	28.28±1.74bA	25.25±0.24bA	39.36±0.66aA	28.78±0.14bB	25.55±0.26bA	40.56±0.96bA	29.15±0.63cB	25.63±0.56aA	40.96±0.66bA	29.75±0.58bB	25.83±0.57aA	41.69±0.87bA
200	30.25±1.07cA	26.18±0.55bA	40.25±1.09bA	30.65±0.27cB	26.54±0.39bA	40.95±0.89bA	31.55±0.87dC	26.89±0.46bA	41.15±0.94bA	31.65±0.83cC	27.19±0.34bA	41.65±0.44bA
400	31.12±0.86cA	27.39±0.38cA	40.86±0.96bA	31.29±0.39cB	27.61±0.68cB	41.69±0.76cB	31.59±0.59dC	28.32±0.49cB	41.85±0.51cB	31.87±0.39cC	28.82±0.89cB	42.15±0.91cB

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Table 7
Effects of V. coloratum polysaccharide in different concentrations on the survival time in hydrogen peroxide treatment of D. melanogaster ($\overline{x}$±$s$, h).

Polysaccharide concentration (mg/L)	F1			F2			F3			F4
	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time	Average survival time	Half survival time	The longest survival time
CK (0)	26.00±1.07aA	23.89±0.96aA	38.48±0.87aA	25.19±1.26aA	23.69±0.65aA	38.79±0.36aA	25.64±1.04aA	24.03±0.64aA	38.36±0.39aA	25.56±0.69aA	23.86±0.67aA	38.61±0.74aA
50	29.32±1.79aA	25.16±1.01aA	39.63±0.70aA	28.01±0.37bB	25.24±0.45aA	40.22±0.39aA	28.16±0.33bB	25.22±0.56aA	40.25±0.84aA	28.33±0.67bB	26.13±0.38aA	41.13±0.89aA
100	29.16±1.56bA	25.99±0.36bA	39.67±0.77aA	29.15±0.28bB	25.83±0.35bA	40.87±0.84bA	29.48±0.74cB	25.80±0.44aA	41.14±0.96bA	29.95±0.48bB	26.33±0.52aA	42.19±0.67bA
200	31.13±1.07cA	26.90±0.67bA	40.59±1.01bA	30.92±0.47cB	26.86±0.62bA	41.25±0.19bA	31.88±0.39dC	27.06±0.18bA	41.65±0.64bA	31.85±0.33cC	27.69±0.64bA	42.35±0.49bA
400	32.02±0.86cA	28.09±0.38cA	41.16±0.86bA	31.57±0.52cB	27.94±0.61cB	41.91±0.71cB	31.96±0.94dC	28.58±0.99cB	42.35±0.81cB	32.07±0.69cC	29.32±0.69cB	43.95±1.11cB

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

3.5 Effects of different concentrations of V. coloratum polysaccharide on anti-oxidase activity and malondialdehyde content of D. melanogaster

With the increased concentration of V. coloratum polysaccharide, the SOD activity in male and female in the F₁-F₄ generations showed a trend of first decreasing and then increasing (Table 8). When the concentration was 50 mg/L, the SOD activity in the F₁-F₄ generations was the lowest (P<0.05). When the concentrations were gradually increased from 100 to 400 mg/L, the SOD activity in the F₁-F₄ generations gradually increased (P<0.05, P<0.01).

Table 8
Effects of V. coloratum polysaccharide in different concentrations on superoxide dismutase activity of D. melanogaster ($\overline{x}$±$s$, U/mg•protein).

Polysaccharide concentration (mg/L)	F1		F2		F3		F4
	♀	♂	♀	♂	♀	♂	♀	♂
CK (0)	31.42±0.34bA	35.92±0.41bA	32.40±0.38bA	35.90±0.45bA	30.91±0.70bA	35.31±0.67bA	31.20±0.80bA	34.75±0.74bA
50	25.20±0.28aA	28.70±0.54aA	27.43±0.41aA	30.93±0.91aA	27.37±0.60aA	30.93±0.67aA	27.34±0.48aA	30.58±0.47aA
100	36.51±0.47cB	40.01±0.54cB	38.73±0.51cB	42.23±0.62cB	37.41±0.58cB	40.93±0.50cB	37.30±0.56cB	40.63±0.44cB
200	41.28±0.92dB	44.78±0.81dB	43.20±0.40dB	47.73±0.58dB	43.31±0.80dB	47.78±0.86dB	45.25±0.38dB	48.47±0.91dB
400	46.21±0.87eB	49.86±0.98eB	48.53±0.41eB	51.68±1.03eB	48.43±0.81eB	52.05±0.70eB	48.44±0.45eB	50.39±0.60eB

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Moreover, with the increase of concentration, the CAT activity in the F₁-F₄ generations showed an increased trend (Table 9). When the concentration was 50 mg/L, the CAT activity in the F₁-F₄ generations was significantly increased than those in control group (P<0.05). When the concentration were gradually increased from 100 to 400 mg/L, the CAT activity in the F₁-F₄ generations were extremely significant increased than those in control group (P<0.01).

Table 9
Effects of V. coloratum polysaccharide in different concentrations on catalase activity of D. melanogaster ($\overline{x}$±$s$, U/mg•protein).

Polysaccharide concentration (mg/L)	F1		F2		F3		F4
	♀	♂	♀	♂	♀	♂	♀	♂
CK(0)	7.73±0.32aA	8.61±0.89aA	7.61±0.47aA	7.83±0.68aA	7.65±0.75aA	8.70±0.91aA	8.70±0.96aA	10.00±1.12aA
50	9.64±0.42bA	10.88±0.41bA	9.81±0.53bA	11.05±0.74bA	9.47±0.84aA	10.77±0.90aA	9.14±0.79aA	10.87±0.65aA
100	10.17±0.42bA	11.41±0.35bA	10.67±0.52bA	11.91±0.66bA	10.15±0.55bA	11.45±1.17bA	10.54±0.86bA	11.90±1.01bA
200	11.27±0.56bA	12.51±0.60bA	11.37±0.87bA	12.59±0.95bA	11.38±0.86bA	12.62±0.89bA	11.16±0.41bA	12.56±0.73bA
400	13.37±0.79cA	14.60±0.90cA	15.25±0.96cA	15.49±0.66cA	14.44±0.89cA	15.55±1.06cA	15.28±0.61cA	15.48±0.82cA

• Different lowercase letters represent a significant difference (P<0.05), and uppercase letters stand for an extremely significant difference (P<0.01).

Furthermore, with the increase of concentration, the MDA contents in the F₁-F₄ generations all showed a trend of increasing first and then decreasing (Table 10). When the concentration was 50 mg/L, the MDA contents in the F₁-F₄ generations were lower than those in control group, and when it was higher than 400 mg/L, there were extremely significant differences from the control group (P<0.01). When the concentration was 500 mg/L, the MDA contents in t the F₁-F₄ generations were the lowest. It indicated that with the increased concentration, the effect of the V. coloratum polysaccharide on scavenging MDA was gradually enhanced, and the antioxidant effect was also gradually enhanced. The results showed that high dose of V. coloratum polysaccharide significantly increased the activity of SOD and CAT and reduced the content of MDA in D. melanogaster in the F₁-F₄ generations, and improved the antioxidant capacity and vitality. It was also found that the activities of SOD and CAT and the content of MDA in female were lower than those in male with different concentrations of V. coloratum polysaccharide, which also showed that the antioxidant effect of female was stronger than that of male.

Table 10
Effects of V. coloratum polysaccharide in different concentrations on malondialdehyde content of D. melanogaster ($\overline{x}$±$s$, nmol/mg).

Polysaccharide concentration (mg/L)	F1		F2		F2		F3
	♀	♂	♀	♂	♀	♂	♀	♂
CK (0)	1.18±0.06dB	1.36±0.03dC	1.17±0.05cB	1.35±0.04dC	1.20±0.04dC	1.45±0.04dC	1.44±0.03dC	1.71±0.06dC
50	0.90±0.07bA	1.10±0.05bA	1.01±0.07bA	1.09±0.05bA	0.97±0.05bA	1.15±0.04bA	0.89±0.05bA	1.07±0.07bA
100	1.13±0.03cB	1.21±0.03cB	1.16±0.09cB	1.19±0.07cB	1.02±0.06cA	1.20±0.04cA	0.99±0.07cA	1.17±0.06cB
200	0.94±0.08bA	1.02±0.06bA	0.99±0.05bA	1.05±0.05bA	0.91±0.07bA	1.11±0.09bA	0.84±0.05bA	1.02±0.07bA
400	0.84±0.07aA	0.91±0.09aA	0.87±0.04aA	0.96±0.04aA	0.79±0.04aA	0.88±0.06aA	0.75±0.03aA	0.89±0.07aA

In this study, we explored the effects of V. coloratum polysaccharide on the fertility, longevity and antioxidant capacity of D. melanogaster. The antioxidant activity was evaluated using the activity of SOD and CAT and the content of MDA as indicators, and the survival time under acute oxidative damage caused by paraquat and hydrogen peroxide was measured. The results showed that high dose of V. coloratum polysaccharide significantly improved the fertility and lifespans of the female and male in the F₁ -F₄ generation, reduced MDA content and increased SOD and CAT activities, and prolonged the survival time under acute oxidative damage caused by hydrogen peroxide and paraquat. Therefore, V. coloratum polysaccharide could improve the antioxidant capacity and vitality of D. melanogaster and promote their reproductive capacity. In addition, the antioxidant effect on female was stronger than male. The male to female ratio in the F₁-F₄ generations did not change much, and was overall close to 1: 1.

The aging of D. melanogaster is closely related to the levels of free radicals, antioxidant capacity and methylation in the body. Excessive free radicals react with unsaturated fatty acids in the cell membrane, which can destroy the structures of biological macromolecules, such as DNA and proteins, which in turn leads to the occurrence of diseases and even death ^[23]. Paraquat is a fast-acting contact-kill herbicide with strong toxicity. It can generate superoxide anion free radicals, which in turn caused oxidative damage to the body. Hydrogen peroxide is a kind of active oxygen that can generate hydroxyl radicals through chemical reactions. It can participate in the metabolic response of living cells to produce functional decline, which further causes oxidative damage to the body (Dinis-Oliveira et al., 2008).

V. coloratum polysaccharide is a macromolecular substance with a variety of biologically active functions. When the polysaccharide was fed to D. melanogaster, it significantly affected the SOD and CAT activities and MDA content in D. melanogaster, suggesting that V. coloratum polysaccharide has a specific relationship with fertility, lifespan and aging of D. melanogaster. MDA is the decomposition product of lipid peroxide produced from polyunsaturated fatty acids in the cell membrane under the action of free radicals when D. melanogaster is stimulated by external stress. It will cause body aging when polymerized with large molecules, such as proteins and peptides, thereby influencing the longevity of D. melanogaster.

Both SOD and CAT have certain antioxidant activities, which can remove free radicals in the body and delay aging by reducing lipid peroxidation. With high concentration of V. coloratum polysaccharide, the MDA content was decreased sharply, and the antioxidant activities of SOD and CAT were significantly increased. This may be due to the participation of V. coloratum polysaccharide in complex biochemical reactions. It can stimulate D. melanogaster to increase the activities of SOD and CAT, and eliminate the MDA content produced in the body, so as to enhance the resistance and indirectly promote fertility and longevity of D. melanogaster.

4 Conclusion

In conclusion, our results showed that V. coloratum polysaccharide improved the antioxidant capacity and vitality of D. melanogaster and promoted their reproductive capacity in a dose-dependent manner. This study will provide a theoretical basis for the development of V. coloratum polysaccharide into health functional foods and medicines that delay aging, prolong life and improve fertility.

Acknowledgement

This work was supported by Science and Technology Guidance project of Longnan City (No. 201609) and Gansu Province University Innovation Capacity Enhancement Program (No. 2019A-190).

• Practical Application: V. coloratum polysaccharide improved the antioxidant capacity and vitality of D. melanogaster and promoted their reproductive capacity in a dose-dependent manner

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Publication Dates

• Publication in this collection
  03 Sept 2021
• Date of issue
  2022

History

• Received
  20 Mar 2021
• Accepted
  04 Apr 2021