Chinese Journal of Oceanology and Limnology Vol. 25 No. 4, P. 373-377, 2007 DOI: 10.1007/s00343-007-0373-5

Positive effect of porphyrans on the lifespan and vitality of Drosophila melanogaster*

ZHAO Tingting (赵婷婷)†,††, ZHANG Quanbin (张全斌)†,**, QI Huimin (綦慧敏)†,††, LI Zhien (李智恩) † (†Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071,China) (††Graduate School of the Chinese Academy of Sciences, Beijing 100039, China)

Received Apr. 9, 2007; revision accepted June 21, 2007

Abstract The effects of degraded porphyran (P1) and natural porphyran (P) on the lifespan and vitality of Drosophila melanogaster are studied. The porphyrans, added daily to the food medium at 0.2% and 1% concentrations, can significantly increase the lifespan in average of 55.79 and 58.23 d in 0.2% P1 diet females and 1% P1 diet males, extending by 12.29% and 8.60% over the corresponding controls, respectively. The effects of porphyrans on D. melanogaster in heat-stress condition were also examined, and found a remarkable increase in survival time. The results which are consistently associated with the use of porphyrans are related to their free radical scavenger action. Considerable increase in vitality demonstrated that vitalities of middle-aged fly (assessed by measuring their mating capacity) was observed after porphyrans addition. Therefore, porphyrans are effective in reducing the rate of aging, and P1 in low molecular weight is better than natural P.

Keyword: Drosophila melanogaster; porphyrans; lifespan; heat-stress; mating capacity

1 INTRODUCTION Although the underlying mechanisms of aging

remains unclear, the free radical theory of aging is currently the most acceptable. According to the theory, cells continuously produce free radicals and constant radical damage eventually kills the cells. When radicals kill or damage enough cells in an organism, the organism ages (Harman, 1956). Antioxidants can eliminate radicals, and the elevated levels of antioxidants can prevent cells from radicals’ damage and slow down the aging. For example, melatonin, the most potent hydroxyl radical scavenger (Tan et al., 1993), has been demonstrated to be an effective geroprotector for D. melanogaster (Bonilla et al., 2002; Izmaylov and Obukhova, 1999).

Porphyra, is believed in East Asia nations to be a vegetable that can slow down aging, and used as a drug in traditional Chinese medicine. Porphyrans, the sulfated polysaccharides from Porphyra haintenensis, are effective radical scavenger in vitro experiments (Zhang et al., 2003a; Zhao et al., 2006). During the last years, porphyrans have been reported to possess antihyperlipedemic, antitumor and immune-stimulating activities (Yashizawa et al.,

1993; Yashizawa et al., 1995; Zhou and Chen, 1990). In our previous study, we demonstrated that the porphyran fractions can effectively improve enzymatic and non-enzymatic antioxidant defense system, showing that porphyran could slow down the aging progress (Zhang et al., 2003b; Zhang et al., 2004).

Lifespan test is a most direct method for aging research, in which Drosophila melanogaster has been widely adopted because of its relatively short lifespan and the adult flies show clear signs of cellular senescence observed in mammals (Fleming et al., 1992). Previously, we gained a degraded porphyran (P1) by free radical scission (Zhao et al., 2006), which has similar in structures to the natural one and have a linear backbone of alternating 3-linked β-D-galactosyl units and 4-linked α-L-galactosyl 6-sulfate or 3, 6-anhydro-α-L- galactosyl units. In this study, lifespan of male and female D. melanogaster under P1 and natural porphyran (P) was studied, with additional investigation on the mating capacity for

* Supported by the Innovative Key Project of the Chinese Academy of Sciences (KZCX-YW-209). ** Corresponding author. qbzhang@ms.qdio.ac.cn

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understanding the vitality.

2 MATERIALS AND METHODS 2.1 Stocks and cultures

All the fruit flies were maintained at 25±1°C with a 12:12 h light/dark cycle and fed with standard diet as described previously: 0.3 g of alga-alga 5 g of corn flour, 1.5 g of yeast, 1.25 mL of 100% ethanol, 5 mL of a brown sugar solution (100 g of sugar in 100 mL of distilled water), 0.65 g of methyl p-hydroxybenzoate and 43.75 mL of water (Bonilla et al., 2002). Adult flies were housed in groups of 25 per standard shell vial with gauze on one side.

Virgins were collected by sorting males and females within 10 h of eclosion, using slight CO2 and ice anaesthesia, a procedure that ensurs the sorting before mating (Miyatake, 1999).

Natural porphyrans were polysaccharides from Porphyra haitanensis T. J. Chang et B. F. Zheng. P1, molecular weight 35 kD, is a degraded products by H2O2 and ascorbate (Zhao, et al., 2006). Sulfate and 3, 6-anhydro-galactose contents are 16%–20% and 10%–12% for P and P1, respectively.

2.2 Lifespan test

10-h-old flies of both genders were transferred to glass vials. In each vial 5 mL of the control food or porphyran-containing food was added. Every day at 3–4 p.m., dead flies were counted and weighted. The culture medium was replaced three times a week. In preliminary experiments, porphyrans were used in concentrations of 0.01%–2%. 0.2% and 1% concentrations per unit of the nutrient medium were chosen as the ultimate optimal. Four times were performed.

2.3 Thermal stress test

Male flies were fed daily with porphyrans- containing food (0.2% or 1%) starting on the third day up to the seventh day of age. The flies were then transferred to vials containing 1 mL of a solution containing 1% agar in 5% sucrose and placed into an incubator at 36°C. The average survival time (in hours) was determined in both control and porphyrans fed flies. Three times were performed.

2.4 Mating capacity test

Virgins were cultured as described above in single gender groups with standard medium. For 30 days after, the virgins were transferred to vials containing 0.2% or 1% porphyrans in standard medium. For 10 days after, 15 virgin males were mixed with 10 virgin females in one mating chamber. Three mating chambers were used for each group. Mating status was recorded during 11, 22 and 33 min period at 25°C.

2.5 Statistical analysis

Survival data were analysed with non-parametric Rank Sum test with the Kaplan-Meier procedure. The significance of differences between experimental groups and controls was determined by one-way analysis.

3. RESULTS 3.1 Effect of porphyrans on lifespan

In the flies of both genders, the two porphyrans P1 and P could increase in the lifespan, 50% and 90% mortality (Table 1). The mean lifespan (MLS) of 53.6 and 49.7 d for control males and females, respectively. Males treated with 1% P1-diet had

Table 1 Effect of the three porphyrans on the mean lifespan, 50% and 90% of both genders Drosophila melanogaster

Lifespan (day) Group Mean Change (%) 50% mortality 90% mortality

Male Control

53.62±2.51

–

49.83±0.36

64.23±0.38

0.2% P1 54.50±2.12 1.63% 55.75±0.31* 63.04±0.33 1.0% P1 58.23±2.33* 8.60% 58.01±0.47* 71.71±0.29* 0.2% P 56.89±2.15* 6.10% 54.73±0.29* 71.70±0.29* 1.0% P 55.55±2.98 3.59% 54.37±0.38 68.32±0.47

Female Control 49.69±2.77 – 53.44±0.29 61.35±0.51 0.2% P1 55.79±0.41* 12.29% 55.37±0.14 65.34±0.14* 1.0% P1 54.33±3.21* 9.34% 55.53±0.36 63.02±0.91 0.2% P 49.60±1.12 -0.18% 48.76±0.38 64.72±0.29 1.0% P 54.13±1.94* 8.93% 54.05±0.41 67.06±0.23* Flies were housed in groups of 25 per standard shell vial with a 12:12 h light/dark cycle at 25±1°C and transferred to

fresh food three times one week. Values are mean ± SEM (in days) of four experiments. The difference compared to control is significant: *p<0.05.

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significantly longer mean lifespan (MLSP1=58.2 d) than controls or on 0.2% diet (MLSP1=54.5 d). However, no significant difference was notified in longevity in males treated with P on both concentrations. In contrast, females on 1% P1-diet lived shorter than that on 0.2% diet but longer than the controls (MLSP1, 1% =54.3 d, MLSP1, 0.2%=55.8 d). For P, the flies on 1% (MLSP, 1% =54.1 d) had significantly longer lifespan than either control or those on 0.2% diet (MLSP, 0.2% =49.6 d).

3.2 Effect of porphyrans on heat-stress

Males maintained on P1 and P containing diets survived longer than controls at 36 ºC (Fig.1).

Fig.1 Thermal stress test

Relative effect of porphyrans treatment on duration time. The files fed with porphyrans during 5 days when maintained at 36°C in vials containing 1 ml of a solution of 1% agar in 5% sucrose. Three times were performed. The difference compared to control is significant: *p<0.05, ** p<0.01

3.3 Effect of porphyrans on mating capacity

Mating capacity showed a remarkable reacton to the porphyrans-diet, with significant increase in mating couples as compared to controls (Table 2).

Table 2 Mating capacity test

Mating couple 11 min 22 min 33 min

P1, 0.2% 0 1 5 P1, 1% 1 1 2 P, 0.2% 0 0 1 P, 1% 0 1 2 Control 0 0 0

30-day old virgins fed with porphyrans for 10 days. Then 15 males combined with 10 females. Data represent the sum of three replicate mating-trials. For each group 75 flies were observed.

4. DISCUSSION The most important results in our study is that D.

malanogaser of both genders showed a significant reaction to porphyrans. Oxidative stress is thought to be an important role in governing the lifespan of D. melanogaster (Fleming, et al., 1992). A large

amount of researches have been carried out on the relationship between lifespan and antioxidants (Brack et al., 1997; Ruddle et al., 1988). The present results also favor the free radical theory of aging. The life-extending effect of porphyrans could be a result of their radical scavenger capacity or reductive properties, or both (Zhang et al., 2003a; Zhao et al., 2006).

It is interesting to note that the longest lifespan extending by 12.29% was obtained in 0.2% P1 diet on females, whereas the largest effect was observed in 1% P1 diet on males. The different response of longevity to porphyrans in male and female D. malanogaster may be due to the difference in sensitivity of lifespan to porphyrans or in nutrient/energy demand and allocation/utilization. Results of other studies (Davies et al., 2005; Muller et al., 1997) also suggested differential sexual sensitivity to the substance. What was also interesting was that the concentration-effect relationship of P1 and P was different. The reasons of this discrepancy reamain unknown, but may be related to the difference in molecular weights. We have reported that P1 and P are similar in structures but different molecular weights (Zhao et al., 2006). In fact, P possesses over million Da, tens of times higher than that of P1, which makes it hard to be absorbed. This in turn could explain the concentration in effect. Alternatively, the mechanism of P influencing longevity is different from that of P1. The extending effect of P on lifespan could be carried out through the regulation of antioxidant defence systems. P can increase the activity of glutathione peroxidase, an antioxidative enzyme, which metabolizes potentially damaging molecules to non-toxic products. Furthermore, porphyrans stimulate other antioxidant enzymes including superoxide dismutase. In addition, porphyrans also inhibit lipid peroxidation and increase total antioxidant capacity (Zhang et al., 2003b; Zhang et al., 2004). It should be noted that the favourable effects of porphyrans on lifespan of the flies could not be explained as the outcome of caloric restriction since there is no change in the body-weight (data not shown).

Heat-stress test shows that the resistance was also positively affected by the two porphyrans. Drosophila was the first genetic system in which enhanced stress resistance was linked to the increase of lifespan (Service et al., 1985). In these flies, selection on stress resistance was used as a surrogate for selection on longevity (Rose, et al., 1992). In

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fact, the organism takes more advantage of antioxidant enzymes in stressful condition than in normal aging (Parkes, et al., 1998; Sun and Tower, 1999). Improved resistance to oxidative stress can increase the longevity, as shown by the up-regulation of SOD-catalase in longer-lived D. melanogaster (Dudas and Arking, 1995). Therefore, the favourable effect of porphyrans on heat stress should be attributed to their antioxidant property.

In mammalian systems, porphyrans have been reported as having a wide variety of protective and regulative effects: protecting rat liver from CCl4 toxicity, protecting rat from 60Coγ radiation, inhibiting S-180 sarcoma, antagonizing leukopenia and reduction the frequency of mieronuclci induced by eyclophosphamide (Zhou and Chen, 1989). Yashizawa and his assistants have reported that porphyrans could enhance the phagocytic and secretory activity of macrophages and induce the production of NO and cytokine (TNF-α, IL-1) (Yashizawa et al., 1993, 1995). We have not noticed the effect on biochemical and physiological activities. Therefore, we can only extrapolate from the activities presented in other systems. Whether the modulation that affects on immune systems also works on the flies remains unknown.

In addition, one could study whether a hormonal change can explain longevity increase in males animals, because recent data indicate that mutants deficient for ecdysone synthesis live longer with better heat stress resistance (Simon et al., 2003; Tatar et al., 2003). Can porphyrans decrease slightly ecdysone synthesis?

Aging follows a progressive intrinsic deteriora- tion in physiological status that culminates in death (Arking, 1998). Among them, mating capacity is an outstanding remark of vitality. In the study, both the porphyrans significantly improved the mating status in contrast to the controls that mated zero time during the test. We believed that porphyrans can improve the vitality of the flies.

5 CONCLUSION Dietary uptake of porphyrans can extend the

lifespan of male and female Drosophila melanogaster as well as their resistance to ambient temperature, and improve the vitality. Degraded porphyran is better than natural porphyran. Possible application of porphyrans in clinics for preventing human aging and aging related pathology can be explored. Multiple mechanisms are involved in the antiaging effect, of which antioxidant activity is one

of them. Currently, study on the metabolic rate of the male flies exposed to porphyrans is in schedule to test whether the polysaccharides can reduce the free radical damage to the tissues by lowering the metabolism. The effect of porphyrans on the reproduction of the female flies, in terms of the amount of eggs will also be tested. Moreover, other model species will be conducted to test the life prolonging effect by polysaccharides. It is interesting that the effect trend of natural on longevity is inverse to that of degraded porphyran, which needs more in-depth studies in the further.

6 ACKNOWLEDGEMENTS The authors are also grateful to Pro. Xu Zuhong

(Institute of Oceanology, CAS) for his helpful assistance in the experiment.

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