Herbaceous Phyllanthus species extracts promotes longevity in Drosophila
melanogaster
Manasa N and Ashadevi JS*
Department of Zoology, Yuvaraja’s College (Autonomous), University of Mysore, Mysore570005, Karnataka, India
Abstract
Dietary antioxidants have been associated with various health benefits including ageing
and age related diseases. Antioxidant rich diet can neutralize the reactive oxygen species and
protect against free radical damage. Many antioxidants are involved in delaying the ageing
process and prolong the lifespan. Phyllanthus species are said to be one of the potent
rejuvenative herbs in Ayurvedic medicine. The present study has undertaken to investigate the
influence of two herbaceous Phyllanthus species extracts namely, P. amarus (PAE) and P.
debilis (PDE) in two doses (1 mg and 10 mg/ml) on Drosophila melanogaster (Oregan-K strain)
lifespan along with the pre-adult parameters. Stress resistance ability in Phyllanthus extracts
treated flies were carried out under starvation and stress inducing molecule (acrylamide)
exposure. Our results revealed that both PAE and PDE extends the life span, prolongs the egg to
adult rate of development and increases the resistance ability in D. melanogaster. Both the
extracts have no effect on viability of the flies. Further, the studies revealed that dose-II of PAE
fed flies significantly increased the mean lifespan by 75 % when compared to other treated
groups. In addition, the resistance ability for starvation and stress induced toxicity by the
supplementation of PAE showed a significant difference with PDE along with the control group.
1
Further phytochemical analysis on these Phyllanthus species might throw a light on the
compounds responsible for their biological activities.
Key words: Phyllanthus amarus; P. debilis; Drosophila melanogaster; Longevity; Viability;
Starvation resistance; Acrylamide
*
Corresponding author
Name: Ashadevi JS
Communication address: Dr. Ashadevi JS, Assistant Professor, Department of Zoology,
Yuvaraja’s College (Autonomous), University of Mysore, Mysore-570005, India
Contact: 09448258374
Email: jsaycm15@gmail.com
2
INTRODUCTION
Ageing is an unavoidable, universal, biological phenomenon in all multicellular organisms.
Dietary antioxidants have been associated with various health benefits including ageing and age
related diseases. Ageing of an organism can be detected by longevity, which is an important
fitness component governed by genetic and environmental components (Partridge, 2005).
In recent years, the increase in longevity has been achieved by supplementing antioxidant
rich pharmaceuticals and medicinal plants. Antioxidant rich diets can neutralize the reactive
oxygen species and protect against free radical damage. Many antioxidants are involved in
delaying the ageing process and prolong the lifespan. It has been reported that the antioxidant
activity of plants might be due to their phenolic compounds that have capacity to scavenge the
free radicals (Huda-Fanjan, 2009). One such medicinal plant having high antioxidant property
and high phenolic compound is Phyllanthus belongs to the family Phyllanthaceae (Harish and
Shivanandappa, 2006; Kumaran and Karunakaran, 2007). Herbaceous Phyllanthus species are
potent rejuvenative herbs in Ayurvedic medicine. Major phytochemicals present in these herbs
are alkaloids, flavonoids, lignans, phenolics, tannins, steroids and triterpenes (Nahar, 2012). It
possesses anti-inflammatory action, with free radical scavenging and protects molecular
inflammation (Kassuya, 2006). It is also possess hepatoprotective, anticancer, antiviral,
antihepatitis, antidiabetic and immunomodulatory activities (Thyagarajan, 1988 & 2002).
Most of the studies on ageing were conducted using long-lived animals like rodents.
Since many invertebrates have proved enormous utility in defining the ageing process,
researchers have begun to search for pharmacological agents that can extend longevity in
invertebrates. A fruit fly, Drosophila melanogaster is one of the ideal model organisms to study
the ageing process, as it can be maintained at extremely low cost, easy to culture and have a short
3
life span (Miva, 2004). However, a very few studies on ageing and lifespan has been made using
Drosophila (Bahadorani and Hilliker, 2008; Perricone, 2006; Saratikov and Krasnov, 1987).
Puri (2002) has analyzed the longevity of D. melanogaster using yellow flowered mountain
plant. The extension of longevity on D. melanogaster has been reported using Rhodiola rosea
roots and blue berry extracts (Jafari, 2007). Further, it has been proved that the antioxidative
property of the plant extracts is the possible mechanism for the life span extension in D.
melanogaster (Peng, 2012).
Based on the literature survey so far, there are no age related studies on D. melanogaster
using herbaceous Phyllanthus species extracts though it is considered as antioxidant rich plant.
The analysis of Drosophila ageing using herbaceous Phyllanthus species will be providing a
baseline data for further experiments in mammalian systems. In view of this, the present study
was chosen to carry out the effect of herbaceous Phyllanthus species that includes P. amarus and
P. debilis on life span and fitness parameters in D. melanogaster.
MATERIALS AND METHODS
Preparation of plant extracts
P. amarus and P. debilis were collected from paddy fields near Mysore, Karnataka, India.
The aerial parts of the plants were dried and were made into fine powder in the laboratory. The
powder was subjected to ethanolic extraction using a Soxhlet apparatus. The extraction was
carried out for 12-16 hrs. The crude extract thus obtained was concentrated using a flash
evaporator. The collected, dried extract was used for further analysis. All the studies were
analyzed in two concentrations namely doses I (1 mg/ml) and dose II (10 mg/ml).
4
Fly strain and culture
Oregon- K strain of D. melanogaster was obtained from Drosophila Stock Center,
Department of Studies in Zoology, University of Mysore, Mysore, Karnataka, India. Isofemale
lines were established from the obtained stock and flies were maintained in ‘Wheat cream agar
media’ with yeast granule. The experimental cultures were maintained at 22 ± 1º C with relative
humidity of 60 - 70% and further flies were multiplied by the subculture. Yeast supplemented
flies were considered as a control group. For all the analyses, extract supplemented flies were
considered as treated groups. Ten µl of extracts were added to 10 ml of culture media.
Longevity studies
Longevity was carried out in unmated male flies of D. melanogaster using standard
protocol (Yoon, 1990). Newly emerged male flies were segregated and isolated from the
synchronized culture bottles under steriozoom with mild anesthetic conditions. They were
released into 9 × 3 cm individual culture vials containing equal quantities of extract
supplemented media of P. amarus (PAE) and P. debilis (PDE). Flies were transferred to fresh
culture vials once in every three days without being etherized and survival rate was recorded in
each vial. Longevity was measured in two doses of each PAE and PDE supplemented groups.
Hundred such replicates were maintained in each group.
Rate of Development
Pre adult rate of developmental experiments were carried out as per the procedure of
Ushakumari and Ranganath (1986). Synchronized eggs were used to determine the rate of
development and 50 eggs were collected by standard Delcour’s technique (Ramachandra and
Ranganath, 1988). The eggs were placed into each culture vial, which were supplemented with
5
PAE and PDE and eggs were allowed for further development. Every day the number of flies
emerged in each vial was recorded for a period of 15 days. From this, mean developmental time
(in days) was calculated. Twelve such replicates were maintained for each group.
Viability
For viability study, the same cultures were used which were employed for the rate of
development. The total number of flies emerged in each vial was recorded and the percentage of
egg to adult viability was calculated.
Starvation Resistance
Starvation resistance test was carried out as per the method of Huey (2004). Tests were
conducted in PAE and PDE supplemented flies of D. melanogaster. Newly emerged male flies
were isolated and distributed in groups of 25 males per vial of size 9 × 3 cm containing equal
amounts of standard culture media with the extracts and flies were aged for 10 days. Then flies
were transferred to empty vials, which were plugged with water soaked sponge. The vials were
observed for every six hours once until all flies reached mortality and calculated the resistance
ability (hours) in all the analyzed groups.
Oxidative stress (OS) resistance test
The OS resistance test was carried out according to method of Prasad and Muralidhara
(2012) using Acrylamide (ACR)-an oxidative stress-inducing molecule (procured from Sisco
Research Laboratories, Mumbai, India). To know the potentiality of the PAE and PDE for
resistance under oxidative stress condition, flies were exposed to ACR at different concentrations
6
ranging from 10, 20, 30, 40 and 50 mM to fix the lethal dosage (LC50). Based on LC50 values, 20
mM of acrylamide was chosen for stress resistance test.
For conducting the OS resistance test, male flies were segregated from synchronized
culture bottles, transformed into culture vials containing the PAE and PDE and aged them for 10
days. Then flies were exposed to 20mM ACR in 5% sucrose solution till all the flies attains
mortality and the rate of survival was recorded with an interval of six hours in control and extract
supplemented groups. Hundred flies were maintained in each group.
Statistical Analysis
Data of all the experiments were expressed as mean ± standard error except viability.
Viability experiment data was expressed as percent viability. To know the significant difference
among the analyzed groups in all experiments, the obtained data was subjected to statistical
analyses using the SPSS software (version 20.0). The survival curve for longevity and starvation
resistance was obtained from Kaplan-Meier analysis. In each parameter, groups were compared
by one-way analysis of variance (ANOVA) followed by Tukey’s test. Values of p<0.05 were
considered as statistically significant. To know the relation between longevity and starvation
resistance test, Pearson’s correlation coefficient was applied.
7
RESULTS
Longevity studies
The mean lifespan of extract supplemented flies were shown in table 1. The mortality in
case of the control group was initiated at 42 days. The maximum lifespan was 76 days with the
mean lifespan of 60.84 ± 0.98 days. The mortality in PAE supplemented groups began at 64 days
in dose-I treatment and at 69 days in dose-II treatment. The maximum lifespan in dose-I of PAE
group was found to be 117 days with the mean lifespan of 95.82 ± 1.33, whereas in a dose-II
group, it was 116 days and mean lifespan was 105.75 ± 0.80 (Figure 1A). However, the mortality
in both the dose treatments of PDE supplemented group was initiated at 63 days. The fly
survived in maximum was 114 days in dose-I treatment, 127 days in dose-II treatment with the
mean lifespan of 90.97 ± 1.46 and 97.54 ± 1.57 days respectively (Figure 1B). The data on
statistical analyses reveal that all the five analyzed groups showed significant difference. Both
the doses of PAE and PDE treated flies showed highly significant difference with the control
group with the p<0.05. Among treatment groups dose-II of PAE showed significant differences
with other extract supplemented groups.
Table 1: Effect of PAE and PDE on lifespan extension in D. melanogaster
Groups
Control
PAE-I
PAE-II
PDE-I
PDE-II
Mean lifespan of D. melanogaster
Minimum
Maximum
Mean ± SE
(days)
(days)
42
76
60.84a ± 0.98
64
69
63
63
117
116
114
127
95.82b ± 1.33
105.75c ± 0.80
90.97d ± 1.46
97.54e ± 1.57
F value
185.097
df (4, 495)
The strains with the different letter in the parenthesis are significantly different at the 5 % level according to DMRT
8
A
B
Figure 1: Survival curve of D. melanogaster flies supplemented with PAE (A) and PDE (B)
along with the control group.
Rate of development
The patterns of rate of development from egg to adult of all the analyzed groups were
graphically represented in figure 2. It was observed that the adults of the control group were
started to emerge first where it started to eclosing on the 11th day and continued up to the 17th
day, and the peak of emergence was on the 13th day with the mean emergence of 13.51 ± 0.04
day. The adult from the groups of both PAE-I and II dose treatments were started eclosing on
13th day and continued up to 23rd and 24th day respectively with the peak emergence on 16th day
(Figure 2A). The mean emergence of PAE-I and II doses were found to be 16.87 ± 0.07 day and
16.40 ± 0.07 day respectively. However, the dose-I and II treatment of PDE groups started
emerging on 12th and 13th day respectively, the emergence was extended up to 23rd day. The peak
of emergence in dose-I and II were found to be on 17th and 16th day, with the mean emergence of
17.35 ± 0.08 day and 17.01 ± 0.06 day respectively (Figure 2B). The statistical analysis revealed
9
that irrespective of dose, the extract supplemented groups of both the species showed significant
300
300
250
250
% Emergance
% Emergance
difference with the control group.
200
150
100
50
200
150
100
0
0
11 12 13 14 15 16 17 18 19 20 21 22 23 24
11 12 13 14 15 16 17 18 19 20 21 22 23 24
Days
Days
Con
A
50
PAE I
PAE II
B
Con
PDE I
PDE II
Figure 2: Pattern of development in D. melanogaster flies supplemented with PAE (A) and
PDE (B) along with the control group at 22 ± 1 ºC.
Viability
The data on viability of the extract supplemented groups were graphically complied
in the figure 3. The percentage of total flies emerged in control group was found to be
81.5 %, whereas in dose-I and II of PAE showed 81.32 % and 77.33 % respectively.
Similarly, the viability in dose-I and II of PDE were found to be 79.5 % and 82.16 %
respectively. Among the analyzed groups, the adult viability was found to be least in PAE
dose-II, while the highest in PDE dose-II. Statistical analysis revealed that there is no
significant difference among control and extract supplemented groups.
10
% Viability
83
82
81
80
79
78
77
76
75
74
CONTROL
DOSE-I
DOSE-II
PAE
PDE
Groups
Figure 3: Viability in D. melanogaster flies supplemented with PAE and PDE
along with the control group at 22 ± 1 ºC.
Starvation resistance
Survival curves for starvation resistance of extracts supplemented flies were plotted in the
figure 4. Under starvation, the mortality in control groups was started at 12th hour, the maximum
resistance ability was prolonged till 42nd hour. The mean starvation resistance ability was found
to be 34.32 ± 0.73 hour. The mortality in PAE dose-I initiated at 12th hour and extended till 54th
hour, with the mean starvation resistance ability was found to be 36.3 ± 0.90 hour. In dose-II of
PAE supplemented flies mortality was started at 18th hour and the ended at 60th hour with the
mean value of 37.14 ± 0.77 hour (Figure 4A). However, in PDE-I and II doses mortality was
initiated at 18th hour and extended till 54th hour, with the mean resistance ability was found to be
39.96 ± 0.79 hour and 40.14 ± 0.71 hour respectively (Figure 4B). The statistical result revealed
that all the extract-supplemented groups are significantly difference with the control group.
11
A
B
Figure 4: Survival curve of D. melanogaster on Starvation resistance by the
supplementation of PAE (A) and PDE (B).
Correlation analysis between longevity and starvation resistance
Correlation studies were performed between longevity and starvation resistance in both
the PAE and PDE extracts supplemented groups along with control group. The obtained results
were plotted in the figure 5 as scatter plots. The results revealed that there is a strong positive
correlation exists between the two parameters in all the analyzed groups with the maximum ‘r’
value of 0.961 in PAE dose-I and with the minimum ‘r’ value of 0.813 in dose-II of PAE. In case
of PDE-I and II doses the ‘r’ value was found to be 0.935 and 0.954 (P<0.01) respectively.
12
Starvation Resistance
60
CON
50
40
30
20
10
0
0
20
40
60
80
Longevity
70
PAE I
Starvation Resistance
Starvation Resistance
70
60
50
40
30
20
10
50
40
30
20
60
80
100
Longevity
120
60
70
70
80 90 100 110 120
Longevity
70
Starvation Resistance
Starvation Resistance
PAE II
60
PDE I
60
50
40
30
20
10
0
PDE II
60
50
40
30
20
10
0
0
50
100
Longevity
150
0
50
100
Longevity
150
Figure 5: Scatter plots showing correlation between lifespan and starvation resistance in
flies supplemented with PAE and PDE.
13
Oxidative Stress resistance test
The result obtained from oxidative stress resistance test was shown in figure 6. It
revealed that mortality in the control group was initiated from 12th hour and prolonged until 48th
hour, with mean resistance of 30.96 ± 0.82 hour. In case of PAE-I and II doses, mortality was
initiated from 18th hour and ended at 56th hour, with the mean resistance ability of 33.4 ± 0.71
hour and 34.48 ± 0.67 hour respectively. Similarly, in PDE dose–I and II showed the mean
value of 30.80 ± 0.78 hour and 32.1 ± 0.89 hour, as the mortality in both the doses of PDE
started at 12th hour and completed at 56th hour. Both the doses of PAE and dose–II of PDE
treated groups showed better ACR resistance when compared to control group.
120
% Survival
100
80
Con
PAE I
60
PAE II
40
PDE I
PDE II
20
0
0
6
12
18 24 30 36
Mortality (Hours)
42
48
56
Figure 6: Effect of PAE and PDE on ACR-induced mortality in D. melanogaster flies.
14
DISCUSSION
Longevity is the total duration of life span of an individual. It is a good indicator of
viability of organisms (Medina, 1996; Strickberger, 1962). Adult diet manipulation has been the
principle means of longevity extension in fruit flies (Min, 2007). In the present study, a short life
spanned D. melanogaster has been selected as a model organism to know the effect of
herbaceous Phyllanthus species extracts on life span and fitness parameters. Experiments were
carried out in unmated male flies by supplementing extracts of P. amarus (PAE) and P. debilis
(PDE) through adult feeding. Unmated male flies were used for the experiments because of less
hormonal effect in these male flies (Demerce, 1994).
Jafari (2007) have shown that the root extract of Rhodiola rosea can extend the longevity
in D. melanogaster. Antioxidants are considered as longevity promoters (Suckow and Suckow,
2006; Krishna and Watson, 2013). The antioxidant activity of blueberry extracts prolongs the
mean lifespan in D. melanogaster and decreased the paraquat-induced mortality rate (Peng,
2012). The high antioxidant rich plants Curcuma longa and Emblica officinalis increases the
life span significantly in D. melanogaster (Rawal, 2014).
Further it has been reported that a
bioactive polyphenolinc compound curcumin extracted from Curcuma longa increases the life
span of D. melanogaster (Shen, 2013). The flavonoids and polyphenols of Cocoa increased the
mean life span in D. melanogaster (Bahadorani and Hilliker, 2008). Based on the obtained
result, the present study revealed that the longevity was increased in both PAE and PDE fed flies
when compared to control group. PAE fed groups showed an increase of longevity by 58.3% in
dose I treatment and 75% in dose II treatment. The longevity in PDE supplemented groups was
increased by 50% and 61.66% in dose I and dose II treatment respectively. Present investigation
15
supports the observation made by earlier researchers on the potentials of plant extracts as
longevity promoters (Peng, 2012; Rawal, 2014). The perusal of literatures confirmed the free
radical scavenging, antioxidant and other bioactive properties of Phyllanthus species. Further,
antioxidant compounds such as polyphenols and flavonoids are reported from these species
(Asha, 2004 & 2007; Hashim, 2013; Kumara, 2012; Kumaran and Karunakaran, 2007; Londhe,
2008; Naaz, 2007; Promyothin, 2007; Raphel and Kuttan, 2003). Thus, antioxidant compounds
present in the PAE and PDE could be playing a major role in extending the mean lifespan of D.
melanogaster.
The rate of development and viability are the key fitness parameters in Drosophila. The
duration of hatching from egg to adult is rate of development and the total number of flies
emerged from known densities of eggs is known as viability. Lint and Lints (1969) have reported
the relationship between rate of development and longevity. In the current study, rate of
development and adult viability were measured in PAE and PDE fed D. melanogaster flies to
know their association with longevity. The result indicated the prolonged rate of development
and unchanged viability in both the extract fed flies compared to control flies. This result
suggests that PAE and PDE has significant role only in the rate of development, not with the
viability of flies. There will be an association between longevity and pre-adult developmental
time in extract fed groups. The phytochemicals present in the Phyllanthus extracts could be
responsible for increase in longevity and rate of development.
One of the ubiquitous causes of stress is the shortage or suboptimal quality of food which
can be studied through various resistance test like heat, cold, starvation and oxidative stress. To
understand the resistance ability of Phyllanthus species under stress conditions, PAE and PDE
16
fed flies were exposed to starvation as well as oxidative stress with stress-inducing molecule and
observed the rate of survival. Starvation resistance can be defined as the resistance to food
deprivation (Wayne, 2006). In the present study, the survival rate of PAE and PDE fed flies were
significantly more and exhibited better starvation resistance compared to control flies. Among
the analyzed test groups, dose II of PDE showed high starvation resistance ability when
compared to other groups. Starvation resistance can influence the longevity. It has been reported
that the increased mean longevity of Drosophila are due to starvation resistance (Baldal, 2005;
Chippindale, 1996 and 1998; Harshman, 1999). Rose (1984) has shown that the increased
resistance to starvation was correlated with lifespan extension. The present result of correlation
analysis confirms the existence of positive correlation between lifespan extension and starvation
resistance in PAE and PDE supplemented flies (Figure 5). This result is in line with the
observation of Rose (1984). Numerous studies have demonstrated the effects of plant extracts in
reducing oxidative damage and promoting healthy ageing in invertebrate models (Dong, 2012;
Mani and Thulasimani, 2012). Similarly, in the present study OS with Phyllanthus extract treated
flies had better survival rate compared to OS induced control flies. The rate of survival was more
in PAE fed flies under stress induction. This result confirms that the analyzed Phyllanthus
species extracts have ability to reduce the oxidative damage.
In conclusion, present study summarizes that P. amarus and P. debilis ethanolic extracts
increases the life span, rate of development and starvation resistance ability in D. melanogaster.
They also show resistance against oxidative stress. The antioxidant compounds present in the
PAE and PDE might be causative factors for enhancing these parameters. There is a positive
correlation between longevity and starvation resistance. Therefore, we suggest that there is a
17
need for further comparative studies in this area to know which of the antioxidant compounds
present in the herbaceous Phyllanthus species has the ability to enhance life span and prevents
early ageing in humans.
ACKNOWLEDGEMENTS
The authors are thankful to the Principal, Yuvaraja’s College, University of Mysore,
Mysore for providing the facilities. The first author is thankful to Indian Council of Medical
Research, New Delhi for the financial assistance received in the form of Senior Research
Fellowship.
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